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A Thesis for the Degree of Doctor of Philosophy
Genomic analysis of Cronobacter sakazakii
and molecular study of hfq and
CSK29544_02616 genes as virulence factors
Cronobacter sakazakii균 체 분 과 병원 인자
hfq CSK29544_02616 분자 연구
August, 2016
Seongok Kim
Department of Agricultural Biotechnology
College of Agriculture and Life Sciences
Seoul National University
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Genomic analysis of Cronobacter sakazakii
and molecular study of hfq and
CSK29544_02616 genes as virulence factors
Cronobacter sakazakii균 체 분 과 병원 인자
hfq CSK29544_02616 분자 연구
지도 상
이 논 농학 사학 논 출함
2016 8 월
울 학 학원
농생명공학부
사학 논 인 함
2016 8 월
원 장 ___________________(인)
부 원장 ___________________(인)
원 ___________________(인)
원 ___________________(인)
원 ___________________(인)
`
I
Abstract
Kim, Seong-ok
Department of Agricultural Biotechnology
The Graduate School
Seoul National University
Cronobacter sakazakii is considered to be an opportunistic pathogen
causing life-threatening diseases, including necrotizing enterocolitis,
septicemia, and meningitis particularly to infants with high fatality. However,
the mechanisms of its pathogenicity and virulence-associated factors remain
largely unknown. To understand them, complete genome of C. sakazakii
ATCC 29544, a type strain, was sequenced and analyzed using bioinformatics.
The complete genome of C. sakazakii ATCC 29544 is composed of a circular
chromosome (Genbank accession No. CP011047) and three plasmids,
including pCSK29544_p1 (Genbank accession No. CP011048),
pCSK29544_p2 (Genbank accession No. CP011049), and pCSK29544_p3
(Genbank accession No. CP011050). It has been known that C.sakazakii
infection mainly occurs via contaminated reconstituted infant formula milk. In
this regard, I tried to identify several gene clusters, predicted to be responsible
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II
for the survival of C.sakazakii in harsh environments, including dried infant
formula. Three gene clusters were found on chromosome, which may confer
advantages on C.sakazakii survival against extremely dried conditions. These
gene clusters include biosynthesis of capsular proteins (CSK29544_00281-
00284) as protectants from osmotic shock, cellulose (CSK29544_01124-
01127) to form biofilms, and nanKTAR (CSK29544_00587-590) to utilize
sialic acid for energy production. Moreover, the strain ATCC 29544 may have
arsenic resistance activity for survival in formula milk powder
(CSK29544_3p0051-0055), on the plasmid 3p. These gene clusters, in part,
may contribute to C.sakazakii survival under dried infant formula.
On human infection, C. sakazakii has invasins, OmpA
(CSK29544_03699) for BMEC adhesion and the ibeB-homologous cusC
(pCSK29544_3p0028) for the penetration of BMECs. For survival within the
host, the strain ATCC 29544 has the privileged iron acquisition system,
including siderophore biosynthesis system (iucABCD/iutA;
CSK29544_1p0024- CSK29544_1p 0028) and an ABC-type iron transport
system (eitCBAD; CSK29544_1p0056- CSK29544_1p 0059). The analysis of
complete genome sequences would shed light on the understanding of
virulence mechanisms by C. sakazakii.
As hfq has been reported as a critical virulence factor in many
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pathogenic bacteria, it was chosen to see its effects on C. sakazakii virulence.
Hfq is a global regulator conversed in many pathogens, but its contributions to
virulence vary from one species to another. Therefore, the role of Hfq in C.
sakazakii virulence was investigated for the first time. In the absence of hfq, C.
sakazakii was highly attenuated in dissemination in vivo, showed defects in
invasion into animal cells and survival within host cells, and exhibited low
resistance to hydrogen peroxide. Remarkably, the loss of hfq led to hyper-
motility on soft agar, which is contrary to what has been observed in other
pathogenic bacteria. The hyper-flagellated bacteria were likely to be
attributable to the increased transcription of genes associated with flagella
biosynthesis in a strain lacking hfq. Together, these data strongly suggest that
hfq plays important roles in the virulence of C. sakazakii by participating in
the regulation of multiple genes.
To identify new virulence factors required for invasiveness of C.
sakazakii ATCC 29544, transposon-mediated random mutant library was
constructed and screened for detection of mutants with reduced invasion
ability into human intestine epithelial Caco-2 cells compared with wild type.
One of the mutants showing the most reduced invasion ability but a
comparable growth with WT was found to have transposon insertion in the
CSK29544_02616, which is highly conserved in only Cronobacter species
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IV
and encodes an unidentified protein. The deletion mutant of
CSK29544_02616 was constructed and various virulence-associated traits,
including invasion to intestinal Caco-2 cell, in vivo animal study,
survival/phagocytosis within/into cultured-macrophages, motility, and biofilm
formation, was characterized. The lack of CSK29544_02616 showed
attenuations in invasiveness, in vivo colonization and dissemination,
phagocytosis and survival into/within macrophage-like cells. Based on the
observations, I questioned the function of CSK29544_02616 of C.sakazakii
ATCC 29544 in terms of virulence. As it is an unidentified protein, I
determined to perform ligand fishing assay to screen players which help the
function of CSK29544_02616 to be unveiled. I found that LpxA, the first
enzyme involved in lipid A biosynthesis, was shown to specifically interact
with CSK29544_02616. The amount of lipid A-core was decreased in
CSK29544_02616 mutation, whereas that of phospholipids was increased.
This altered ratio between Lipid A and phospholipids in outer membrane
influenced outer membrane assembly and changed cell surface to be more
hydrophobic, resulting in increased cell autoaggregation and biofilm
formation. Interestingly, the mutant was non-motile although it had flagella
and torque generation, indicating loss of CSK295444_02616 caused paralyzed
flagella. Taken together, CSK29544_02616 plays critical roles in the virulence
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V
of C. sakazakii by modulating LPS production. CSK29544_02616 would be a
good target for biocontrol of C. sakazakii in the way that the deletion of
CSK29544_02616 does not affect C. sakazakii growth, but influence its
virulence as an antibacterial agent.
In conclusion, understanding the pathogenesis of C. sakazakii ATCC
29544 by both genomic analysis and molecular studies of virulence factors
would be the cornerstone for the development of a new strategy to control C.
sakazakii pathogenesis.
Keywords: Cronobacter sakazakii, Complete genome sequences,
Pathogenesis, hfq, CSK29544_02616, LpxA
Student Number: 2010 – 23444
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VI
Contents
Abstract ................................................................................................................. I
Contents ............................................................................................................. VI
List of Figures.................................................................................................. XIII
List of Tables .................................................................................................XIIIV
Chapter I. Introduction ........................................................................................ １
I-1. Cronobacter sakazakii .......................................................................... ２
I-1-1. Clinical manifestations of C. sakazakii infections ..................... 4
I-1-2. Environmental reservior and mode of transmission .................. 5
I-2. Pathogenicity and virulence factors of C. sakazakii ................................. 7
I-2-1. Invasins ................................................................................... 7
I-2-2. Survival within host immune cells ........................................... 8
I-2-3. Stress tolerance ........................................................................ 9
I-3. Objectives of this study ......................................................................... 90
Chapter II. Complete genome sequence analysis of Cronobacter sakazakii .......... 12
II-1. Introduction ......................................................................................... 13
II-2. Materials and Methods......................................................................... 16
II-2-1. Bacterial strains, plasmids and growth conditions. ................ 16
II-2-2. Genome sequencing and assembly. ! 책갈피가 어 있지 습니다
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VII
II-2-3. Genome annotation . ! 책갈피가 어 있지 습니다.
II-2-4. Bioinformatics. ... ! 책갈피가 어 있지 습니다.
II-3. Results and Discussion ........................................................................ 19
II-3-1. Genome properties of C. sakazakii ATCC 29544T ................. 19
II-3-2. Pathogenicity and virulence factors. ...................................... 20
II-3-3. Comparative genome analysis............................................... 31
II-3-4. Comparative phylogenetic tree analysis ................................ 32
Chapter III. The hfq, a RNA chaperone, plays important roles in virulence and
stress adaptation in Cronobacter sakazakii ATCC 29544 ..................................... 36
III-1. Introduction ........................................................................................ 37
III-2. Materials and Methods ....................................................................... 41
III-2-1. Bacterial strains, plasmids, and culture conditions. ............... 41
III-2-2. Construction of a hfq deletion mutant using the Lambda-
Red recombination method.. ............................................................ 41
III-2-3. Complementation study ....................................................... 42
III-2-4. Gentamicin protection (invasion) assay ................................ 43
III-2-5. Motility assay.. .................................................................... 44
III-2-6. Bacterial survival assay in animal cells. ............................... 44
III-2-7. Bacterial resistance against hydrogen peroxide. ................... 45
III-2-8. qRT-PCR (quantitative real time) ......................................... 46
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VIII
III-2-9. Animal study in vivo............................................................ 46
III-2-10. TEM (transmission electron microscopy) analysis ............. 47
III-2-11. Western blot analysis ......................................................... 47
III-2-12. Primer extension analysis .................................................. 48
III-2-13. Statitical analysis............................................................... 50
III-3. Results ............................................................................................... 58
III-3-1. Construction and growth characteristics of the hfq mutant
in C. sakazakii. ................................................................................ 58
III-3-2. The loss of hfq attenuates colonization by C. sakazakii in
rat pups ........................................................................................... 63
III-3-3. Hfq is involved in the invasion of C. sakazakii into
human epithelial cells. ..................................................................... 67
III-3-4. Hfq is required for oxidative-stress resistance in C.
sakazakii ......................................................................................... 70
III-3-5. Loss of the hfq gene reduces intracellular survival of C.
sakazakii in macrophage-like cells. .................................................. 74
III-3-6. A C. sakazakii hfq strain is hypermotile due to higher
expression of flagella....................................................................... 77
III-4. Discussion .......................................................................................... 83
Chapter IV. CSK29544_02616, a newly identified protein, influences C.
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IX
sakazakii virulence by modulating LPS production.............................................. 91
IV-1. Introduction ........................................................................................ 92
IV-2. Materials and Methods ....................................................................... 96
IV-2-1. Strains, plasmids, and culture conditions.............................. 96
IV-2-2. Construction of random transposon mutant libraries and
screening. ........................................................................................ 96
IV-2-3. Determination of the transposon insertion sites. ................... 96
IV-2-4. Generation of ΔCSK29544_02616 deletion mutant by
using site-specific mutagenesis. ....................................................... 97
IV-2-5. Construction of plasmids. .................................................... 98
IV-2-6. Invasion assay. .................................................................... 98
IV-2-7. Survival assay...................................................................... 99
IV-2-8. In vivo animal study. .......................................................... 100
IV-2-9. Suspension-clearing and microscopic analysis.. ................. 101
IV-2-10. Overexpression and purification of the tagged-proteins .... 101
IV-2-11. Ligand fishing assay ........................................................ 102
IV-2-12. LC/MS/MS analysis ........................................................ 103
IV-2-13. Database search ............................................................... 104
IV-2-14. BACTH (Bacterial adenylate cyclase based two-hybrid
system) assay .............................................................................. 104
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X
IV-2-15. β-galactosidase assay ....................................................... 106
IV-2-16. In vitro GST pull-down assay .......................................... 106
IV-2-17. Western blot analysis ..................................................... 107
IV-2-18. LpxA enzyme assay ......................................................... 108
IV-2-18-1. Cell cultures ................................................................. 108
IV-2-18-2. Purification of LpxA .................................................... 109
IV-2-18-3. Purification of Holo-Acyl Carrier Protein ..................... 109
IV-2-18-4. Purification of V. harveyi AasS-Hisx6 ............................ 110
IV-2-18-5. Acylation of holo-ACP ................................................. 111
IV-2-18-6. Fluorescent enzyme assay for LpxA and AT.................. 111
IV-2-19. LPS extraction ................................................................. 112
IV-2-20. Outer membrane fraction . ............................................... 114
IV-2-21. Phospholipid quantitation . .............................................. 115
IV-2-22. Hydrophobicity assay . .................................................... 116
IV-2-23. Biofilm assay . ................................................................ 117
IV-2-24. RNA isolation and sequencing . ....................................... 117
IV-3. Results ............................................................................................. 128
IV-3-1. A gene discovered in an invasion-attenuated mutant.. ......... 128
IV-3-2. The CSK29544_02616 gene is required for C .sakazakii
invasion......................................................................................... 131
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XI
IV-3-3. The CSK29544_02616 gene contributes to C .sakazakii
virulence.. ..................................................................................... 133
IV-3-4. CSK29544_02616 directly interacts with LpxA. ................ 137
IV-3-5. CSK29544_02616 helps LpxA enzyme activity in a
synergistic manner.. ....................................................................... 146
IV-3-6. CSK29544_02616 is involved in modulating the ratio of
lipid A and phospholipid.. .............................................................. 156
IV-3-7. The altered balance between Lipid A and phospholipids
in CSK29544_02616 mutation influenced outer membrane
assembly........................................................................................ 158
IV-3-8. Reduction of LPS production in ΔCSK29544_02616
increased cell surface hydrophobicity and biofilm formation... ....... 163
IV-3-9. CSK29544_02616 abrogated flagella-mediated motility.. ... 166
IV-4. Discussion ........................................................................................ 170
References .................................................................................................... 17678
국 .......................................................................................................... 215
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XII
List of Figures
Fig. II-1. Genome map of a circular chromosome (A) and three plasmids (B). .... 20
Fig. II-2. Phylogenetic tree analysis using different methods ............................... 34
Fig. III-1. hfq mutant showed defectiveness of growth, but had no polar
effects ................................................................................................................. 59
Fig. III-2. The promoter regions of hfq were identified by primer extension
analysis ............................................................................................................... 61
Fig. III-3. The virulence of C. sakazakii was diminished due to the absence of
hfq in vivo ........................................................................................................... 65
Fig. III-4. Loss of Hfq impairs the invasion of epithelial cells. ............................ 68
Fig. III-5. Stress tolerance is attenuated in C. sakazakii in the absence of Hfq ..... 72
Fig. III-6. Lack of Hfq results in reduced intracellular survival of C. sakazakii
in macrophage-like cells...................................................................................... 75
Fig. III-7. Loss of Hfq causes enhanced motility ................................................. 79
Fig. III-8. hfq homolog in C. sakazakii ATCC 29544. ......................................... 89
Fig. IV-1. PCR confirmation of several mutants from invasion-screening .......... 130
Fig. IV-2. CSK29544_02616 is required for C. sakazakii invasion into
intestinal epithelial cells .................................................................................... 132
Fig. IV-3. CSK29544_02616 is required for the pathogenesis of C. sakazakii .... 135
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XIII
Fig. IV-4. N-terminally Hisx6 –tagged CSK29544_02616 was successfully
purified with its functionality ............................................................................ 140
Fig. IV-5. CSK29544_02616 was shown to be a binding parter of LpxA from
ligand fishing assay ........................................................................................... 141
Fig. IV-6. CSK29544_02616 specifically binds with LpxA in vivo .................... 142
Fig. IV-7. CSK29544_02616 specifically binds to LpxA in vitro ....................... 144
Fig. IV-8. Each substrate used for LpxA enzymatic assay was well purified ...... 149
Fig. IV-9. CSK29544_02616 increases the enzymatic activity of LpxA ............. 152
Fig. IV-10. lpxA mutant having lpxA/lpxA::kan was constructed ....................... 154
Fig. IV-11. CSK29544_02616 modulate the production of lipid A, resulting in
alteration of outermembrane assembly .............................................................. 160
Fig. IV-12. Increased lipid amount in outer membrane elevated
hydrophobicity .................................................................................................. 164
Fig. IV-13. The loss of CSK29544_02616 caused paralyzed flagella ................. 168
Fig. IV-14. C. sakazakii in the absence of CSK29544_02616 overexpressed
gene clusters encoding hook-basal body structures ............................................ 176
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XIV
List of Tables
Table II-1. Genome statistics .............................................................................. 23
Table II-2. Summary of genome: one chromosome and 3 plasmids ..................... 24
Table II-3. Number of genes associated with general COG functional
categories............................................................................................................ 25
Table III-1. Bacterial strains and plasmids used in Chapter III ............................ 51
Table III-2. Oligonucleotides used for the construction of strains and
plasmids used in Chapter III ................................................................................ 54
Table III-3. Primers for qRT-PCR analysis in Chapter III.................................... 57
Table IV-1. Bacterial strains and plasmids used in Chapter IV .......................... 119
Table IV-2. Oligonucleotides used for the construction of strains and plasmids
in Chapter IV .................................................................................................... 123
Table IV-3. LPS and phospholipid contents of WT and ΔCSK29544_02616 ..... 161
Table IV-4. The expression level of membrane protein from RNA sequencing
data ................................................................................................................... 162
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１
Chapter I.
Introduction
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２
I-1. Cronobacter sakazakii
Cronobacter genus (formerly Enterobacter) is a Gram-negative, rod-
shaped, peritrichous and yellow-pigmented facultative anaerobe belonging to
the Enterobacteriaceae family. The organism, initially referred to as “yellow-
pigmented cloacae,” was reclassified as “Enterobacter sakazakii” in 1980 on
the basis of differences in several evaluations, including DNA-DNA
hybridization, and biochemical reactions compared with Enterobacter cloacae
(Farmer et al., 1980b). According to Farmer’s original 16 biogroups,
Enterobacter has been reclassified into several Cronobacter spp. based on
geno-and phenotypic evaluations in 2008 (Farmer et al., 1980a; Iversen et al.,
2007a; Iversen et al., 2007b; Iversen et al., 2008). Recently, Cronobacter
genus consists of 7 species: Cronobacter sakazakii, Cronobacter malonaticus,
Cronobacter muytjensii, Cronobacter turicensis, Cronobacter dublinensis,
Cronobacter universalis, and Cronobacter condimenti (Iversen et al., 2007a;
Iversen et al., 2008; Joseph et al., 2012a). To date, 45 genome sequences of
C.sakazakii are available in GenBank database (6 complete and 39 incomplete)
(http://www.ncbi.nlm.nih.gov/genome/genomes/1170). As C. sakazakii
CMCC 45402 is no longer C. sakazakii, but C. malonaticus, thus, the exact
number of total genome sequences of C. sakazakii is 44 and that of complete
ones is 5 at this time. Recently, the understanding of C. sakazakii ecology has
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３
been urgently required, so that whole genome sequencing analysis and
multilocus sequence typing (MLST) using 7 housekeeping genes has been
established to identify the diversity of the Cronobacter genus from various
sources for the evolutionary relationships and environmental fitness of
Cronobacter species (Baldwin et al., 2009; Joseph & Forsythe, 2012).
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I-1-1. Clinical manifestations of C. sakazakii infections
Among seven species, Cronobacter sakazakii is a well-known
opportunistic food-borne pathogen causing bacteremia, meningitis and
necrotizing enterocolitis, particularly in low-birth-weight neonatal infants.
Although its incidence rate is quite low, the mortality is high ranging from 40
to 80% particularly to premature infants (Drudy et al., 2006; Mullane et al.,
2007; Willis & Robinson, 1988). For example, one of the most severe
outbreaks of C. sakazakii infection occurred in France in 1994 in a Neonatal
intensive Care Unit (NICU). The vehicles of C. sakazakii infections in this
outbreak turned out to be the feeding practices used in NICU (Caubilla-
Barron et al., 2007). More recently, from 2000 to 2008, there have been 120-
150 C. sakazakii infections to neonates, leading to meningitis, bacteremia, and
NEC. The mortality from those symptoms was reported to be 41.9%, <10%,
and 19%, respectively (Friedemann, 2009). It has been suggested that the
severity of C. sakazakii infection is somewhat related with the status of host
immune system, delineating low birth-weight neonates (i.e.<2.5 kg) and
infants <28 days of age are at heightened risk compared with more mature
infants (Caubilla-Barron et al., 2007; Himelright et al., 2002; van Acker et al.,
2001). The annual occurrence rate among the premature and low-birth weight
infants was reported to be 8.7 per 100,000 in the USA (Singh et al., 2015),
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one Cronobacter infection per 10,660 very low birth-weight neonates, and
one Cronobacter infection per 100,000 infants (Stoll et al., 2004). Due to the
severe perils of C. sakazakii infection, regardless of its low incidence, the
International Commission on Microbiological Specification for Foods has
classified C. sakazakii as a ‘severe hazard for restricted populations, life-
threatening or with substantial chronic sequelae over long duration’ (Foods,
2002). In addition, the World Health Organization and Food and Agriculture
Organization in 2008 issued a request to the scientific community for more
data on this organism (WHO/FAO). In December of 2011, the Food and Drug
Administration reported a series of neonatal disease outbreaks in Florida,
Illinois, Missouri and Oklahoma (FDA, 2011)
I-1-2. Environmental reservoir and mode of transmission
Cronobacter spp. are ubiquitous and have been isolated from a wide
range of environments, including water, soil, processed foods, powdered
infant formula (PIF), and facilities(Baumgartner et al., 2009; Dong et al., 2013;
Mozrova et al., 2014; Muller et al., 2013; Siqueira Santos et al., 2013).
Moreover, they have also been reported from clinical specimens, including
cerebrospinal fluid, intestinal and respiratory tracts, and skin wounds
(Gallagher & Ball, 1991). However, the most associated vehicle for
transmission of C. sakazakii is reconstituted PIF, derived from either intrinsic
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6
or extrinsic contaminant including under poor good manufacturing (GMP) or
reconstitution of PIF (Singh et al., 2015).
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7
I-2. Pathogenicity and virulence factors of C. sakazakii
Although the fatality of C. sakazakii infection as high as 40-80%
particularly to premature infants, the mechanisms of its virulence remain
largely unknown. Many reports have previously investigated various
virulence factors and/or traits, including outer membrane proteins (OmpA and
OmpX), enterotoxin, outer membrane protease (Cpa), slialic utilization
(nanAKT), iron acquisition system, biofilm formation, metalloprotease (zpx),
LPS, and transcriptional global regulators (hfq and LTTR)
I-2-1. Invasins
Regarding that C. sakazakii is an oral pathogen it must translocate
from intestinal lumen into the blood stream to make successful systemic
infections. As C. sakazakii invades into host in a receptor-mediated manner,
outer membrane proteins have been regarded as key players. It has been
reported that outer membrane protein A of Cronobacter is important for the
invasion of human intestinal epithelial INT-407 cells and human brain
microvascular endothelial cells (HBMECs) (Nair et al., 2009; Singamsetty et
al., 2008). Furthermore, outer membrane protein (OmpX) was also needed for
invasion. It was also demonstrated that C. sakazakii invade not only the apical
side, also the basolateral side of intestinal epithelial cells via OmpA and
OmpX by disrupting tight junctions (Kim et al., 2010).
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8
Lipopolysaccharide (LPS) is an immune stimulator recognized by
toll-like receptor 4 (TLR 4) and play an important role in breaking tight
junctions, leading to improved invasion of C. sakazakii (Kim & Loessner,
2008; Moriez et al., 2005)
I-2-2. Survival within host immune cells
After successful invasion into host cells, pathogenic bacteira may
encounter many immune cells and/or substances produced by them, barriers
eliminating pathogens. To survive within them, pathogens have their own
strategy to protect themselves. It has been demonstrated that C. sakazakii can
persist within macrophage-like cells up to 96 h by modulating that the ratio
between IL-10 and IL12 is high after 24 h (Townsend et al., 2007b). An outer
membrane protease, encoded by cpa, plays a key role in rendering serum
resistance by the cleavage of complement components. In addition, as iron is
essential for the bacterial growth, C. sakazakii has iron acquisition systems,
including siderophore production (iucABCD/iutA) and an ABC-type
transport system (eitCBAD) in the plasmid pESA3 (Franco et al., 2011a;
Jaradat et al., 2014). C. sakazakii has also the privileged gene clusters,
which encode sialic acid utilization. This sialic acid is found in human milk
and in infant formula in the form of sialyloligosaccharide (Sprenger &
Duncan, 2012). It is undigestable, so remained at the intestinal microvilli of
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9
neonates and infants. This residual sialic acid would be energy source for C.
sakazakii to replicate, and make successful systemic infection. A cell-bound
zinc-containing metalloprotease, encoded by zpx, caused rounding of
Chinese hamster ovary (CHO) cells, which might be important in
dissemination of the pathogen into the systemic circulation (Kothary et
al., 2007).
I-2-3. Stress tolerance
PIF is not sterilized, but clean products must conform to specified
international microbiological criteria (Hunter & Bean, 2013). It is
manufactured in the process of high temperature heat treatment and
desiccation. Nevertheless, C. sakazakii has been isolated from milk powders
and desiccated products, substantiating that C. sakazakii has the privilege to
tolerate thermo-stress up to 60 °C for limited periods of time (Alvarez-
Ordonez et al., 2012; Arku et al., 2011). Moreover, C. sakazakii can survive
under desiccated environments for several weeks and gain resistance to
osmotic stresses, including powdered infant formula and the presence of
osmotic stress-causing agents; nisin and lactoferrin (Al-Nabulsi et al., 2009).
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I-3. Objectives of this study
Cronobacter sakazakii is an opportunistic pathogen leading to
bacteremia, meningitis and necrotizing enterocolitis, particularly in low-birth-
weight neonatal infants and/or immunocompromised patients. To control
C.sakazakii, the usage of antibiotics is prevalent. Originally, it is susceptible
to a wide range of antibiotics. However, unfortunately, a number of
Cronobacter isolates have exhibited the resistance to antibiotics (Kilonzo-
Nthenge et al., 2012). Additionally, several biocide compounds, including
essential oils and polyphenols, have turned out to be effective in mitigation of
C. sakazakii growth at laboratory level (Condell et al., 2012). However, these
are toxic to human beings. The addition of ultrasound or ultraviolet radiation
was used to sterilize PIF, but it led to the loss of nutrients and/or safety
(Adekunte et al., 2010; Arroyo et al., 2012). In this respect, a new paradigm
of alternatives to control C. sakazakii infection should be required. As neither
the mechanisms of pathogenicity nor identification of virulence factors remain
unveiled, it is urgently necessary to investigate them. To elucidate the
understanding of C. sakazakii pathogenesis, the genomic insights of complete
genome sequences of C. sakazakii ATCC 29544 and molecular studies of
virulence factors were performed.
The whole genomic analysis of C. sakazakii
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11
By sequencing of whole genomic DNA of C. sakazakii ATCC 29544,
the complete genome sequences were obtained and analyzed to reveal
virulence-associated factors and understand of its pathogenesis at genomic
level.
Characterization of the role of hfq in C.sakazakii
The impact of hfq on virulence of C. sakazakii ATCC 29544 was
investigated.
Discovery of a new target gene and investigation of its mechanism for
C.sakazakii virulence
A new target, highly conserved only in Cronobacter genus, was
discovered and its mechanism on C.sakazakii virulence was investigated.
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Chapter II.
Complete genome sequence analysis of
Cronobacter sakazakii ATCC 29544
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II-1. Introduction
Enterobacter sakazakii has been reclassified into seven species in the
genus Cronobacter according to biochemical and genetic evaluations
(Baldwin et al., 2009; Iversen et al., 2007a; Joseph et al., 2012a). Among
them, Cronobacter sakazakii is a well-known opportunistic food-borne
pathogen causing bacteremia, meningitis and necrotizing enterocolitis,
particularly in low-birth-weight neonatal infants. This species is a Gram-
negative, rod-shaped, peritrichous and yellow-pigmented facultative anaerobe
belonging to the Enterobacteriaceae family (Farmer et al., 1980b). Although
C. sakazakii food-borne outbreaks are quite low, the fatality to infants
generally ranges from 40 to 80% (Healy et al., 2010). Interestingly, C
sakazakii was reported to produce capsular or biofilm materials for its own
protection from extremely dry conditions, as in formula milk powder,
substantiating the high survival ability of C. sakazakii in the milk powder
(Hurrell et al., 2009). After human infection, C. sakazakii can invade the
intestinal epithelial cells and even the brain microvascular endothelial cells
(BMECs), demonstrating its potentials to cause meningitis (Giri et al., 2012).
Therefore, the biocontrol and regulation of C. sakazakii are urgently required.
However, C. sakazakii is resistant to some antibiotics, indicating a problem
with antibiotic therapies against C. sakazakii (Kilonzo-Nthenge et al., 2012;
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Xu et al., 2015), and its pathogenicity mechanism remains unknown. Recently,
to unveil the knowledge about the Cronobacter ecology, multilocus sequence
typing (MLST) using 7 housekeeping genes has been established to identify
the diversity of the Cronobacter genus from various sources, and its
application has facilitated understanding of the evolutionary relationships and
environmental fitness of Cronobacter species (Baldwin et al., 2009; Joseph
& Forsythe, 2012). To date, 45 genome sequences of Cronobacter sakazakii
are available in GenBank database (6 complete and 39 incomplete).
However, C. sakazakii CMCC 45402 is no longer C. sakazakii, but C.
malonaticus (Blazkova et al., 2015). Therefore, the exact number of total
genome sequences of C. sakazakii is 44 and that of complete ones is 5 at this
time. There have been two incomplete genome sequences of C. sakazakii
ATCC 29544, containing 100 and 280 contigs, respectively. To assemble and
complete these numerous contigs of C. sakazakii ATCC 29544T, I started to
sequence its genome for further understanding of this strain in genomic level.
In this study, to understand its infection and pathogenesis at the
molecular level, the genome of a representative C. sakazakii type strain,
ATCC 29544T, was completely sequenced and analyzed using bioinformatics.
This genome information would provide the researchers with genomic
insights into the virulence and pathogenicity mechanisms of this species for
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15
the further development of a rapid detection method and a novel biocontrol
strategy.
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II-2. Materials and Methods
II-2-1. Growth conditions and genomic DNA preparation
C. sakazakii ATCC 29544T was routinely cultivated using Luria-
Bertani (LB) or Tryptic Soy Broth (TSB) medium at 37˚C with shaking at 220
rpm. Bacterial cells were harvested in the mid-exponential growth phase using
centrifugation at 16,000 × g for 1 min and its genomic DNA was isolated
using G-spin™ Genomic DNA Extraction Kit for Bacteria (iNtRON
Biotechnology, Seongnam, South Korea). The concentration and purity of
extracted DNA were determined by NanoVue (GE healthcare, Little
Chalfont, United Kingdom).
II-2-2. Genome sequencing and assembly
The complete genome of C. sakazakii ATCC 29544T was generated
at Macrogen, Seoul, South Korea, using a hybrid of PacBio RS II (Pacific
Biosciences, Menlo Park, CA, USA) and Illumina HiSeq 2500 (San Diego,
CA, USA). An Illumina standard shotgun library, paired-end library and mate-
pair library were constructed and sequenced using the Illumina HiSeq 2500
platform. The filtered Illumina reads of 14,828,306 (paired end) and
12,583,144 (mate pair) were obtained and assembled using ALLPATHS-LG
(version r47449) (Gnerre et al., 2011). A PacBio SMRTbell library was
constructed using the P5 polymerase with C3 chemistry (P5-C3) and
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sequenced on the PacBio RS II platform. The total quality filtered reads were
93,528 and assembled using HGAP (version 2.0) and iCORN2 software (Otto
et al., 2010) with HiSeq 2500 paired-end sequence data. The total size of the
complete genome sequence is 4.6 Mb (a chromosome and three plasmids),
and the final genome coverages were on average 1,321 × Illumina and 73 ×
PacBio, respectively.
II-2-3. Genome annotation
The ORFs were predicted using Glimmer3 (Delcher et al., 1999),
FGENESB (Li, 2011), and GeneMark.hmm (Lukashin & Borodovsky, 1998).
The gene prediction results were confirmed by manual curation. The genes of
rRNA and tRNA were predicted using RNAmmer 1.2 (Lagesen et al., 2007)
and tRNAscan-SE (Lowe & Eddy, 1997), respectively. The genome
annotation was conducted using NCBI BLASTP (Altschul et al., 1990) and a
predicted protein analysis using InterProScan (Hunter et al., 2012) for the
prediction of protein functions.
II-2-4. Bioinformatics
Clusters of orthologous groups (COG) analysis was performed using
WebMGA (Wu et al., 2011). A circular genome map was generated by
GenVision (DNASTAR, Madison, WI, USA) based on the information
provided by the genome annotation. In addition, for comparative genomic
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18
analysis, average nucleotide identity (ANI) was performed by JSpecies based
on 12 complete genome sequences of Cronobacter. To calculate the ANI
among the complete genomes, each genome sequence was cut randomly into
1,020 base pairs, and each fragment was pairwise compared using BLASTN
algorithm. For multilocus sequence typing (MLST) analysis, seven house-
keeping genes were concatenated and ANI calculation was performed by
JSpecies.
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II-3. Results and Discussion
II-3-1. Genome properties of Cronobacter sakazakii ATCC 29544T.
The complete genome of C. sakazakii ATCC 29544T is composed of
a circular chromosome and three plasmids (Fig. II-1). The chromosome is
4,511,265 bps in DNA length with a GC content of 56.71%, 4,380 ORFs, 22
rRNA genes, and 83 tRNA genes, as listed in Table II-1. The plasmids,
designated pCSK29544_p1, pCSK29544_p2 and pCSK29544_p3, are 93,905,
4,938 and 53,457 bp in DNA length with GC contents of 57.02, 54.88 and
50.07%, respectively. The plasmids have predicted ORFs of 72, 6, and 57,
respectively, and no RNA gene in them. The genome annotation information
including a chromosome and three plasmids has been deposited under the
GenBank accession number, CP011047, CP011048, CP011049, and
CP011050, respectively, listed in Table II-2. The classification of ORFs into
functional categories based on COG (Clusters of Orthologous Groups)
(Tatusov et al., 1997) is summarized in Table II-3. In COG functional
categorization, 4,048 ORFs (89.64% of totally predicted ORFs) belonged to
COG functional categories.
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Fig. II-1. Genome map of a circular chromosome (A) and three plasmids (B).
The outer circle indicates the gene-coding regions by strand, and the genes are colored by COG categories. The blue and orange arrows
indicate rRNA and tRNA genes in the chromosome, respectively. The inner circle with a red line indicates the G+C content. The scale unit is a
base pair.
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Table II-1. Genome statistics
Attribute Value % of Totala
Genome size (bp) 4,663,565 100
DNA coding (bp) 4,116,350 88.27
DNA G+C (bp) 2,641,511 56.64
Total genes 4,620 100
Protein coding genes 4,515 97.72
RNA genes 105 2.27
rRNA operons 22 0.48
tRNA genes 83 1.8
Genes with function prediction 3,212 69.52
Genes assigned to COGs 4,048 87.21
Genes with Pfam domains 2,118 45.84
Genes with signal peptides 414 8.96
Genes with transmembrane helices 1,035 22.4
CRISPR repeats 2 0.04
a, The total is based on either the size of the genome (a chromosome and 3
plasmids) in base pairs or the total number of total genes in the annotated
genome.
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Table II-2. Summary of genome: one chromosome and 3 plasmids
Label Size (Mb) Topology Acession No.
Chromosome 4.51 Circular CP011047
Plasmid 1 0.093 Circular CP011048
Plasmid 2 0.005 Circular CP011049
Plasmid 3 0.053 Circular CP011050
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Table II-3. Number of genes associated with general COG functional categories
Code Value % of totala Description
J 187 4.14 Translation, ribosomal structure and biogenesis
A 1 0.02 RNA processing and modification
K 316 7.00 Transcription
L 248 5.49 Replication, recombination and repair
B 0 0 Chromatin structure and dynamics
D 41 0.91 Cell cycle control, Cell division, chromosome partitioning
V 46 1.02 Defense mechanisms
T 205 4.54 Signal transduction mechanisms
M 231 5.11 Cell wall/membrane biogenesis
N 119 2.63 Cell motility
U 107 2.37 Intracellular trafficking and secretion
O 157 3.48 Posttranslational modification, protein turnover, chaperones
C 225 4.98 Energy production and conversion
G 376 8.33 Carbohydrate transport and metabolism
E 338 7.49 Amino acid transport and metabolism
F 89 1.97 Nucleotide transport and metabolism
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H 161 3.56 Coenzyme transport and metabolism
I 96 2.13 Lipid transport and metabolism
P 216 4.78 Inorganic ion transport and metabolism
Q 66 1.46 Secondary metabolites biosynthesis, transport and catabolism
R 419 9.28 General function prediction only
S 404 8.95 Function unknown
- 467 10.34 Not in COGs
a, The total is based on the total number of ORFs in the genome.
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II-3-2. Pathogenesis and virulence factors
C. sakazakii infects human neonates and infants via mostly
contaminated reconstituted infant formula milk (Himelright et al., 2002),
causing serious human diseases, including bacteremia, necrotizing
enterocolitis, and even meningitis (Healy et al., 2010). The high survival rate
of C. sakazakii, even under extremely dried conditions, as in milk formula
powder, has not been investigated at the molecular level. To understand this
extraordinary property of C. sakazakii, molecular studies have been recently
performed. Interestingly, two gene clusters associated with biosynthesis of
capsular polysaccharides (CSK29544_00281-00284) and cellulose
(CSK29544_01124-01127) were detected. Recently, the biofilm formation and
cellulose (as a component of biofilm) production of C. sakazakii were
experimentally confirmed (Choi et al., 2015; Hu et al., 2015), suggesting that
these gene clusters may be involved in the biofilm formation. This biofilm
formation of C. sakazakii may contribute to the survival in the infant formula
conditions (Hu et al., 2015). However, exopolysaccharide (EPS) production
was not observed in C. sakazakii ATCC 29544 (Lehner et al., 2005),
suggesting that capsular protein gene cluster may be associated with biofilm
formation, not with EPS production. In particular, C. sakazakii is the only
Cronobacter species that has the nanKTAR gene cluster to utilize sialic acid
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(Joseph et al., 2013b). Interestingly, C. sakazakii ATCC 29544 has this gene
cluster (CSK29544_00587-590), indicating its ability to use sialic acid. This
unique ability may be involved in its adaptation to the milk conditions
because sialic acid is one of the components of milk (Wang et al., 2001).
Upon human infection, C. sakazakii invades brain microvascular
endothelial cells (BMECs), which are barriers to protect the brain from
infection of meningitic pathogens, including Escherichia coli K1 and
Neisseria meningitides, which cause meningitis in neonates and infants (Nair
et al., 2009; Nassif et al., 2002). Outer membrane protein A (OmpA) of C.
sakazakii is a critical determinant, contributing the in vitro invasion of human
BMECs by enhancing cell adhesion (Nair et al., 2009). In addition, a few
genes (ibeA, ibeB, and yijP) in meningitic E. coli were also suggested to be
associated with the invasion of human BMECs (Huang et al., 1999; Huang et
al., 2001; Wang et al., 1999). Interestingly, two genes, ompA
(CSK29544_03699) for BMEC adhesion and the ibeB-homolog cusC
(pCSK29544_3p0028) for the penetration of BMECs, were detected in the
genome of C. sakazakii ATCC 29544T, suggesting that this strain may invade
human BMECs. However, ibeA and yijP were not detected in the genome. It
is noteworthy that this ibeB-homolog cusC is located in the gene cluster of the
complete copper- and silver-resistance cation efflux system as encoded by
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cusABCF (CSK29544_3p0026-CSK29544_3p0031) in the plasmid
pCSK29544_3p, suggesting that this BMEC invasion ability may be
transferrable to other C. sakazakii or that the strain ATCC 29544T may have
been acquired from other C. sakazakii for human BMEC invasion. The
presence of a conjugational transfer protein encoded by traX
(CSK29544_3p0007) supports this hypothesis.
In addition, C. sakazakii ATCC 29544T harbors three plasmids,
pCSK29544_1p (1p), pCSK29544_2p (2p), and pCSK29544_3p (3p) (Fig. 4).
Interestingly, two large plasmids (1p and 3p) encode virulence-associated
genes that are related to iron uptake and BMEC invasion. Therefore, these two
plasmids may contribute to host dominance and pathogenesis, respectively.
Iron is an essential element for dominant survival and colonization via
bacterial competition for iron uptake because it plays an important role in the
electron transport system for energy production (Mietzner & Morse, 1994;
Neilands, 1995). To accomplish this dominant survival and colonization
against other bacteria, C. sakazakii has an iron acquisition system, including
siderophore production (iucABCD/iutA) and an ABC-type transport system
(eitCBAD) in the plasmid pESA3 (Franco et al., 2011a; Jaradat et al., 2014).
The strain ATCC 29544 also has this privileged iron acquisition system,
including siderophore biosynthesis (iucABCD/iutA; CSK29544_1p0024-
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CSK29544_1p 0028) and an ABC-type iron transport system (eitCBAD;
CSK29544_1p0056- CSK29544_1p 0059). However, this iron acquisition
system is present in the plasmid 1p but not in the chromosome, similar to
pESA3 of C. sakazakii BAA-894 (Franco et al., 2011a). Interestingly, C.
sakazakii BAA-894 has multiple copies of pESA3 plasmids, which is highly
homologous to the plasmid 1p, suggesting that the plasmid 1p may be multi
copied, too (Kucerova et al., 2010). These results indicate that C. sakazakii
ATCC 29544T may take advantage of better iron uptake ability in the given
environments, probably due to the presence of multiple copies of this iron
acquisition system encoded in the plasmid 1p.
Recent studies have revealed that milk formula contains an at least
six-times-higher arsenic concentration than that of breast milk, suggesting that
bacterial survival in milk formula may require arsenic resistance (Carignan et
al., 2015; Dakeishi M, 2006; Jackson et al., 2012). In addition to the
previously mentioned BMEC invasion ability of C. sakazakii ATCC 29544 via
the ibeB-homolog cusC gene in the cation efflux system, the plasmid 3p has
an arsenic resistance system as encoded by arsCBADR, suggesting that the
strain ATCC 29544 may have arsenic resistance activity for survival in
formula milk powder and that this ability may have been acquired via plasmid
conjugational transfer.
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II-3-3. Comparative genome analysis
To determine the unique features of C. sakazakii ATCC 29544, the
genome of this strain was compared using average nucleotide identity (ANI)
analysis with previously reported complete genome sequences of four C.
sakazakii strains and seven other Cronobacter strains (Kucerova et al., 2010;
Power et al., 2013; Shin et al., 2012a; Stephan et al., 2011; Zhao et al., 2014).
The ANI analysis of C. sakazakii genomes showed >98% ANI (average
nucleotide identity), suggesting that these sequences are highly homologous
(Fig. II-2). However, their sequence types (ST) are different, such as SP291
(ST4), NCTC 8155 (ST4), ATCC 29544 (ST8), ATCC BAA-894 (ST1), and
ES15 (ST125). ANI tree showed that SP291 is the closest to NCTC 8155
because they are ST4. In addition, ATCC 29544 (ST8) is very closely related
to ST4 strains, SP291 and NCTC 8155, but evolutionary distinct. Other
strains of ST1 and ST125 are a little bit far from them. They are in the same
group suggesting that all of them belong to C. sakazakii. However, C.
sakazakii CMCC 45402 was corrected to C. malonaticus CMCC 45402
(Blazkova et al., 2015). Indeed, the ANI tree revealed that the strain CMCC
45402 is much closer to C. malonaticus LMG 23826 rather than C. sakazakii
strains, substantiating that the strain CMCC 45402 belongs to C. malonaticus,
not to C. sakazakii.
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Interestingly, the strain ATCC 29544T contains three strain-specific prophages
(2,190,745-2,236,903, 2,248,908-2,269,664, and 2,360,824-2,368,684) with
no homology with other genomes. While these prophages are specific to only
ATCC 29544T, most of the encoded proteins are hypothetical proteins.
Therefore, we could not identify ATCC 29544-specific functions in the unique
prophages at this time, probably due to insufficient genome annotation
information of C. sakazakii or its prophages in the sequence databases.
II-3-4. Comparative phylogenetic tree analysis
To dissect evolutionary relationships of Cronobacter species, three
comparative phylogenetic tree analysis methods were used at the strain level,
including ANI, multilocus sequence typing (MLST), and 16S rRNA gene
sequencing. For the analysis, 11 complete genome sequences of Cronobacter
that are available in the NCBI Genbank database, including the strain ATCC
29544, were obtained and ANI values were calculated. A phylogenetic tree
was generated based on ANI values (Fig. II-2). The tree identified
Cronobacter sakazakii group. Within this group, the genome of Cronobacter
sakazakii ATCC 29544 is closely related with Cronobacter sakazakii NCTC
8155 and SP291, but evolutionarily distinct. The second phylogenetic tree was
generated by MLST using 7 house-keeping genes (Fig. 3B): atpD (ATP
synthase β chain), fusA (elongation factor), glnS (glutaminyl-tRNA synthase),
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gltB (glutamate synthase), gyrB (DNA gyrase B), infB (translation initiation
factor IF-2), and pps (phosphoenol-pyruvate synthase) (Baldwin et al., 2009).
In consistent with the previous report, MLST is a successful typing method to
discriminate Cronobacter genus between isolates (Joseph & Forsythe, 2012;
Joseph et al., 2012b; Joseph et al., 2013a). Contrary to the phylogenetic tree
by 16S rRNA gene, ANI and MLST showed higher resolution of
discrimination between Cronobacter genus (Fig. II-2)
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A
B
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35
C
Fig. II-2. Phylogenetic tree analysis using different methods.
(A) Phylogenetic tree analysis of C. sakazakii ATCC 29544T and other
Cronobacter strains. For the construction of the tree, MEGA6 with complete
16S rRNA sequences was used using the Neighbor-joining method (Tamura et
al., 2013). The percentage of trees in which the associated taxa clustered in
the bootstrap test (1,000 replicates) is shown nearby the branches (Sanderson
& Wojciechowski, 2000). The type strain is labeled with superscript T.
(B) ANI analysis of C. sakazakii ATCC 29544T and other Cronobacter strains
using average nucleotide identity (ANI), which was generated by JSpecies.
The type strain is labeled with superscript T.
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Chapter III.
The hfq, a RNA-binding protein, plays important
roles in virulence and stress adaptation in
Cronobacter sakazakii ATCC 29544
A part of this chapter was published in Infect. Immun. (2015) 83(5):2089-98.
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III-1. Introduction
Cronobacter genus (formerly Enterobacter sakazakii) is a Gram-
negative, rod-shaped, peritrichous and yellow-pigmented facultative anaerobe
belonging to the Enterobacteriaceae family. This family encompasses the
well-known enteric pathogens, Salmonella and pathogenic E. coli, but
Cronobacter are most closely related to the Enterobacter sakazakii and
Citrobacter genera. According to Farmer’s original 16 biogroups,
Enterobacter sakazakii has been reclassified into several Cronobacter spp.
based on geno- and phenotypic evaluations (Farmer et al., 1980a; Iversen et
al., 2007a; Iversen et al., 2007b; Iversen et al., 2008). Cronobacter spp. are
present in a wide range of environments, including water, soil, processed
foods, and facilities (Kandhai et al., 2004), and are composed of seven species:
Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter muytjensii,
Cronobacter turicensis, Cronobacter dublinensis, Cronobacter universalis,
and Cronobacter condimenti (Iversen et al., 2007a; Iversen et al., 2008;
Joseph et al., 2012a)
C. sakazakii is a foodborne opportunistic pathogen that causes
bacteremia, meningitis, and necrotizing enterocolitis, particularly in premature
infants (Bar-Oz et al., 2001; Drudy et al., 2006; Joker et al., 1965). Although
the incidence rate is quite low, mortality is high, ranging from 40 to 80%
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(Drudy et al., 2006; Mullane et al., 2007; Willis & Robinson, 1988). Despite
the serious perils associated with C. sakazakii, little is known at the molecular
level about the mechanism of its pathogenicity and about its virulence factors.
A cell-bound zinc-containing metalloprotease, encoded by zpx, caused
rounding of Chinese hamster ovary (CHO) cells, which might be important in
dissemination of the pathogen into the systemic circulation (Kothary et al.,
2007). The outer membrane proteins A (OmpA) and OmpX are reported to be
essential for adhesion to/invasion of Caco-2 and INT-407 cells (Kim et al.,
2010; Kim & Loessner, 2008). Studies have shown that C. sakazakii can
invade and translocate efficiently across cultured human intestinal epithelial
cells, as well as endothelial cells, experimentally mimicking the potential path
of meningitis (Giri et al., 2012; Kim & Loessner, 2008). A plasmid-borne
outer membrane protease (Cpa) is reported to be involved in C. sakazakii
protection against complement-dependent serum killing and efficient invasion
by activating plasminogen into plasmin (Franco et al., 2011b). Furthermore,
plasmid-borne iron acquisition systems, such as an aerobactin-like
siderophore (cronobactin) and an ABC ferric-iron transporter (eitABCE),
might enable Cronobacter spp. to obtain iron in a highly iron-restricted
environment (Grim et al., 2012). Flagella from Cronobacter spp. can induce
an inflammatory response dependent on Toll-like receptor 5 (TLR5)
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recognition, so they could contribute to the pathogenesis of the bacteria (Cruz-
Cordova et al., 2012). As a global regulator, LysR-type transcriptional
regulator (LTTR) in C. sakazakii impacts pathogenesis, invasion, biofilm
formation, and in vivo challenge (Choi et al., 2012).
The Hfq protein, which was first discovered in E. coli nearly half a
century ago as a host bacterial factor required for the RNA synthesis of
bacteriophage Qβ (Franze de Fernandez et al., 1968), is widely conserved as
an RNA chaperone in many bacterial species (Kajitani & Ishihama, 1991). It
oligomerizes into a hexameric ring structure (Sauter et al., 2003), enhances
the formation of small RNA (sRNA)–mRNA duplexes, and contributes to
RNA regulation by interacting with RNA turnover enzymes, RNase E,
polynucleotide phosphorylase, and poly(A) polymerase (Vogel & Luisi, 2011).
Thus, Hfq is regarded as a posttranscriptional global regulator involved in the
biogenesis of outer membrane proteins (OMPs) (Guillier et al., 2006), quorum
sensing (Lenz et al., 2004), and various stress responses (Repoila et al., 2003).
In addition, recent studies have demonstrated the importance of Hfq in the
pathogenesis of various bacteria, including Listeria monocytogenes, Yersinia
pseudotuberculosis, Francisella tularensis, Salmonella enterica serovar
Typhimurium, and E. coli (Christiansen et al., 2004; Kulesus et al., 2008;
Meibom et al., 2009; Schiano et al., 2010; Sittka et al., 2007). For example, a
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lack of Hfq in S. Typhimurium caused attenuated virulence in vivo, reduced
host cell adhesion and invasion in vitro, defectiveness in secretion of effector
proteins, chronic envelope stress (accumulation of periplasmic and outer
membrane proteins), loss of motility, and reduced survival within cultured
macrophages (Sittka et al., 2007). Such pleiotropic functions of the hfq gene
product were also observed in other bacterial species (Chiang et al., 2011;
Schiano et al., 2010), but the effects of hfq deletion could be different from
one species to another; the deletion of hfq in S. Typhimurium and Brucella
abortus reduced bacterial growth and survival within the host cells, but not in
L. monocytogenes and F. tularensis (Christiansen et al., 2004; Meibom et al.,
2009; Robertson & Roop, 1999)
The aim of this study was to elucidate the function of the hfq
homolog of C. sakazakii in virulence or stress responses, such as motility,
invasion of host epithelial cells, intracellular survival in macrophages, and
resistance to hydrogen peroxide. Here, I demonstrate that Hfq is essential for
virulence and stress adaptation in C. sakazakii ATCC 29544.
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III-2. Materials and Methods
III-2-1. Bacterial strains, plasmids, and culture conditions.
The bacterial strains used in this study are listed in Table 1. C.
sakazakii and E. coli were routinely grown in Luria-Bertani (LB) medium at
37°C with constant shaking unless otherwise indicated. C. sakazakii strains
ATCC 29544 (SK001), Δhfq (SK005), and Δhfq harboring pHFQ (SK007)
were cultured overnight in LB medium at 37°C and subcultured with a 1%
overnight culture in 50 ml LB medium. The cultures were incubated with
shaking at 220 rpm in 250-ml Scott flasks at 37°C. Ampicilin,
chloramphenicol, and arabinose were used at 50 μg/ml, 25 μg/ml, 50 μg/ml,
and 1.33 mM, respectively.
III-2-2. Construction of a hfq deletion mutant using the Lambda-Red
recombination method.
The whole genome sequence of C. sakazakii BAA-894 was obtained
from GenBank (Grim et al., 2012) and adopted to manipulate the C. sakazakii
ATCC 29544 genome. Site-specific mutation of C. sakazakii ATCC 29544
was generated by the Lambda-Red recombination method, as described by
Kim et al. and Datsenko and Wanner (Datsenko & Wanner, 2000; Kim et al.,
2010). Briefly, the kanamycin resistance cassette from plasmid pKD13 was
amplified using the following primers (Table 2): for hfq deletion mutant
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construction, hfq-lamb-F (5’-ATG GCT AAG GGG CAA TCT TTG CAA
GAT CCG TTC CTC AAC GCG CTG CGT CGT GTA GGC TGG AGC TGC
TTC G-3’) and hfq-lamb-R (5’-TTA CTC GGC GTC TTC GCT ATC CTG
AGA GGC AGC GGA AGA TGG CTG TGC GTA TTC CGG GGA TCC
GTC GAC C-3’). (The nucleotide sequences originating from pKD13 are
underlined, and those from the C. sakazakii gene of interest are shown in
italics.) The PCR products were transformed into the wild-type (WT) strain, C.
sakazakii ATCC 29544, harboring the pKD46 plasmid, by electroporation, and
cells were selected for kanamycin-resistant transformants, hfq::kan. Finally,
the kanamycin resistance cassette was removed using the pCP20 plasmid, as
described previously (Choi et al., 2012)
IIII-2-3. Complementation study.
Two hfq complementation plasmids (a pACYC184 derivative, pHFQ,
with its own hfq promoter and a pBAD18 derivative, pHFQ-A, under an
arabinose-inducible promoter) were constructed. To make pHFQ, the entire
309-bp hfq coding sequence (accession number KC866358) and 782 bps of
upstream sequence comprising miaA (accession number KC866359), which
includes promoter regions of hfq, were amplified from C. sakazakii ATCC
29544 chromosomal DNA (Chiang et al., 2011) with primer sets hfq-
pACYC184-SphI-F (5’-AAA GCA TGC AGC GAT GGC GGA GAT TGT
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43
CG-3’) and hfq-pACYC184-SalI-R (5’-AAA GTC GAC GTA AAA TAG
ATG TGT ACC AG-3’) (the artificial restriction enzyme digestion site is
underlined) and cloned into pACYC184 (Hanahan, 1983). For pHFQ-A, hfq-
pBAD18-EcoRI-F (5’-AAA GAA TTC GGT TCA AGA GTA TAA ACA
AC-3’) and hfq-pACYC184-SalI-R (5’-AAA GTC GAC GTA AAA TAG
ATG TGT ACC AG-3’) were used to amplify the hfq coding region into the
pBAD18 vector (the underlined nucleotide sequences are artificially added
restriction recognition sites) (Guzman et al., 1995). The plasmids were used to
transform the mutant to generate hfq harboring pHFQ and hfq harboring
pHFQ-A.
III-2-4. Gentamicin protection (invasion) assay.
To determine bacterial invasion of mammalian Caco-2 cells, a
gentamicin protection assay was performed as described previously (Kim et
al., 2010). Briefly, bacteria were prepared by transferring a 1% inoculum from
overnight cultures into fresh LB medium, followed by incubation for 2.5 h at
37°C with constant shaking. C. sakazakii cells were collected by
centrifugation at 20,000 x g, washed with phosphate-buffered saline (PBS)
(pH 7.4), and resuspended in 1 ml of pre-warmed fresh Eagle’s minimum
essential medium (EMEM) (ATCC). Mammalian cells were seeded in EMEM
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44
supplemented with 20% fetal bovine serum (FBS) (Gibco, Invitrogen) in 24-
well tissue culture plates with a cell density of 2 x 105 per well. The cell
monolayers were incubated for 1 day, infected with bacteria at a multiplicity
of infection (MOI) of 100, and incubated for 1.5 h in the presence of 5% CO2.
After the cells were washed once with PBS, fresh medium containing
gentamicin (100 μg/ml; Sigma) was added, and the plates were further
incubated for 1.5 h, followed by three washes with PBS. Then, 500 μl of 1%
Triton X-100 was added, and the plates were incubated for a further 15 min
before the bacteria were collected and plated on tryptic soy agar (TSA).
III-2-5. Motility assay.
The C. sakazakii ATCC 29544 (SK002) and hfq (SK006) strains and
the complemented strain (SK008) were cultured overnight in LB medium at
37°C, which was used to subculture (1% inoculum) fresh LB medium.
Bacteria were grown to mid-exponential phase and then diluted to adjust the
OD600 to 1.5. Aliquots (2 μl) of each strain were injected onto soft-agar
motility plates (LB medium containing 0.3% agar) and incubated at 37°C for
7 h. A GelDoc EZ imager (Bio-Rad) was used to take a photograph.
III-2-6. Bacterial survival assay in animal cells.
A long-term survival assay was performed as described previously
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45
(Townsend et al., 2007b). Briefly, RAW264.7 murine macrophage-like cells
were seeded in Dulbecco’s modified Eagle medium (DMEM) (Gibco,
Invitrogen) supplemented with 10% FBS in 24-well plates at a density of 5 x
105 cells per well and incubated for 1 day at 37°C with 5% CO2. C. sakazakii
strains were prepared as described for the invasion assay, and cell monolayers
were infected with bacteria at an MOI of 100 for 45 min at 37°C. The medium
was replaced with DMEM (plus 10% FBS) supplemented with 100 μg/ml of
gentamicin, and the cells were incubated for an additional 45 min. The plates
were washed twice with PBS, and the cells were lysed with 1% Triton X-100,
followed by serial dilution and plating onto TSA to determine the numbers of
intracellular bacteria at various time points. For further study of bacterial
persistence (long-term survival), the cells were replenished daily with fresh
medium containing 10 μg/ml of gentamicin, and the numbers of intracellular
bacteria were determined at 3, 8, 24, 48, 72, and 96 h.
III-2-7. Bacterial resistance against hydrogen peroxide.
A hydrogen peroxide assay was performed as described previously
with minor modifications (Schiano et al., 2010). A C. sakazakii culture was
prepared as for the motility assay. Hydrogen peroxide (Sigma) was added to
the culture to a final concentration of 100 mM, and the culture was incubated
at 37°C for 10 min, followed by counting of viable cells on TSA. For
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46
quantitative real-time (qRT)-PCR analysis (see below), each sample was
exposed to 100 mM H2O2 for 5 min at 37°C.
III-2-8. qRT (quantitative)-PCR.
A C. sakazakii culture was prepared as for the motility assay. Five
hundred microliters of the culture was mixed with 1 ml of RNA Protect
Bacteria Reagent (Qiagen), and total RNA was isolated using an RNeasy mini
kit (Qiagen). The RNA sample was then treated with RNase-free DNase
(Ambion) to remove residual DNA. cDNA was synthesized using
Omnitranscript Reverse Transcription reagents (Qiagen) and random
hexamers (Invitrogen), and quantification of the cDNA was performed using
2x iQ SYBR green Supermix (Bio-Rad). The real-time amplification of the
PCR products was performed using the CFX Connect Real-Time PCR
Detection system (Bio-Rad). The mRNA level of each gene was divided by
the mRNA level of the 16S rRNA. The mRNA expression values in strains
SK005 and SK007 were further normalized to the transcription levels in the
wild-type strain. For mRNA stability, the samples were taken after the
addition of rifampicin (working conc. 250 μg). The following steps were the
same as stated above.
III-2-9. Animal study in vivo.
Three- to 4-day-old Sprague-Dawley (SD) rat pups were used to
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47
assess the virulence of the WT (SK001) and the hfq mutant (SK004) strains.
Mid exponential-phase bacteria were collected, washed, and resuspended in
PBS. For competitive assays, a mixed inoculum of 1 x 109 CFU/ml of the
fully virulent WT (SK001) and the hfq mutant (SK004) harboring a kan
cassette was orally administered to rat pups. To analyze bacterial colonization
in organs, all the rat pups were killed with CO2 at 20 h post-infection. The
spleens and livers were aseptically removed, homogenized, and serially
diluted. Bacterial loads were determined for the WT and mutant by plating
onto TSA in the presence or absence of kanamycin. The results are presented
as competitive-index (CI) values, which were calculated as follows:
(hfqoutput/hfqinput)/(WToutput/WTinput).
III-2-10. TEM (transmission electron microscopy) analysis.
Flagellar morphology was visualized by negative staining, followed
by transmission electron microscopy (TEM). After being negatively stained
with 2% uranyl acetate for 1 min on carbon-Formvar copper grids, flagella
were examined with an energy-filtering transmission microscope (EF-TEM;
Libra 120, Germany) at a voltage of 120 kV.
III-2-11. Western blot analysis.
C. sakazakii ATCC 29544 was cultured overnight in LB medium at
37°C, which was used to subculture (1% inoculum) fresh LB medium. The
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bacteria were grown to mid-exponential phase, collected, and disrupted in B-
PER (Thermo Scientific) according to the manufacturer’s instructions. The
protein samples were loaded on a 12% SDS-polyacrylamide gel. The
separated proteins on the gel were transferred to a polyvinylidene difluoride
(PVDF) membrane and blocked with 5% nonfat dry milk–1x Tris-buffered
saline–Tween 20 (TBST) buffer. The membrane was probed with anti-
OmpA/OmpX polyclonal antibody and anti-DnaK antibody (Enzo Life
Science) as primary antibodies and then treated with anti-mouse IgG
conjugated with peroxidase (Santa Cruz Biotechnology) as the secondary
antibody in all of the immunoblot experiments. The chemiluminescent signals
were developed with a West-Zol plus Western blot detection system (Intron
Biotechnology, South Korea).
III-2-12. Primer extension analysis.
Primer extension assays were performed as described previously with minor
modifications (Kim et al., 2011b). Briefly, C. sakazakii was grown to the mid-
exponential phase for about 3 h in LB broth with constant shaking and
harvested by centrifugation at 20,000 x g for 2 min at 4ºC. The total RNA was
purified with TRIzol (Invitrogen) according to the manufacturer’s instructions.
Purified RNA was resuspended in nuclease-free water and the RNA
concentration was determined by measuring the optical density of the solution
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49
at 260 and 280 nm using NanoVue (GE Healthcare). A portion (20 pmol) of
P1_R and P2_R primers was labeled with 32P at the 5’ end by 10U of T4
polynucleotide kinase (Invitrogen) and 40μCi of [γ-32P]ATP for 30min at 37ºC
in 40 μl, respectively. The labeling mixture was heated at 70ºC for 10 min and
purified with MicroSpin G-25 columns (GE healthcare). For annealing, the γ-
32P-end labeled primers (1 pmol each) were incubated with 30 μg of total
RNA in 20 μl of 250 mM KCl, 2 mM Tris (pH 7.9), and 0.2 mM EDTA. The
mixture was heated at 70ºC for 3 min and then was allowed to cool to room
temperature for 1 h. After annealing, 50 μl of extension reaction solution
containing 100 μg/ml of actinomycin D, 10 mM MgCl2, 5 mM dithiothreitol,
20 mM Tris-Cl (pH 7.6), 770 μm deoxynucleotide triphosphates and 100 U of
Superscript Ⅲ reverse transcriptase (Invitrogen) was added to the mixture.
The mixture was incubated at 42ºC for 70 min and treated with 100U of
RNase T1 (Roche) at 37ºC for 15min in the presence of 2 μl of 0.5M EDTA.
The respective samples were ethanol precipitated after addition of 1.4 μl of
5M NaCl with 2.5 volumes of absolute ethanol and then washed with 75%
ethanol. Each sample was resuspended with 6 μl of stop/loading buffer
(Epicentre) and 4 μl of Tris-EDTA (pH 8.0) buffer and then denatured at 90ºC
for 3 min. The samples were resolved on 6% polyacrylamide-8 M urea gels
and signals were analyzed by BAS 2500 (Fuji Film). The PE_1R and P2_R
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primers were used for sequencing the hfq promoter region with a SequiTherm
EXCELⅡ DNA sequencing system (Epicentre).
III-2-13. Statistical analysis.
Statistical analysis was conducted using the GraphPad Prism
program (version 5.01). All results were analyzed by Student’s unpaired t test.
The data are presented as means and standard deviations. A P value of 0.05
was considered statistically significant.
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Table III-1. Bacterial strains and plasmids used in this study
Strain or plasmid Genotype and/or characteristics Reference or source
C. sakazakii ATCC 29544 Wild-type strain (Kim et al., 2010)
ES1001 29544 harboring pKD46 (Kim et al., 2010)
SK001 29544 harboring pACYC184 This study
SK002 29544 harboring pBAD18 This study
SK003 Δhfq This study
SK004 hfq::kan harboring pACYC184 This study
SK005 Δhfq harboring pACYC184 This study
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SK006 Δhfq harboring pBAD18 This study
SK007 Δhfq harboring pHFQ This study
SK008 Δhfq harboring pHFQ-A This study
E.coli
DH5α λ- Φ80dlacZΔ(lacZYA-argF)U169 recA1 endA1 hsdR17(rK-
mK-) supE44 thi-1 gyrA relA1
(Hanahan, 1983)
Plasmids
pKD13 oriR6K AmpR FRT KanR FRT (Datsenko & Wanner, 2000)
pKD46 oriR101 repA101(Ts) AmpR ara BADpgam-bet-exo (Datsenko & Wanner, 2000)
pCP20 oripSC101(TS) AmpRCmR cΙ857λ PRflp (Datsenko & Wanner, 2000)
pACYC184 TetR CmR p15A ori (Chang & Cohen, 1978)
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pBAD18 AmpR, araC, PBAD, pBR322 ori, expression vector (Guzman et al., 1995)
pHFQ pACYC184-hfq This study
pHFQ-A pBAD18-hfq This study
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Table III-2. Oligonucleotides used for the construction of strains and plasmids in Chapter III
Oligonucleotide name Oligonucleotide sequence (5’ to 3’) Purpose
Mutant construction
hfq-lamb-F ATG GCT AAG GGG CAA TCT TTG CAA GAT CCG TTC CTC AAC
GCG CTG CGT CGT GTA GGC TGG AGC TGC TTC G
SK003 construction
hfq-lamb-R TTA CTC GGC GTC TTC GCT ATC CTG AGA GGC AGC GGA AGA
TGG CTG TGC GTA TTC CGG GGA TCC GTC GAC C
SK003 construction
hfq-lamb-conF GTA CAG CGA TGT GTT ACA GGT SK003 confirmation
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hfq-lamb-conR CCA ACA AAA TAC TTT GGG TGC G SK003 confirmation
Cloning
hfq- pACYC184-R-SalΙ AAA GTC GAC GTA AAA TAG ATG TGT ACC AG pHFQ construction
hfq-pACYC184-F-SphΙ AAA GCA TGC AGC GAT GGC GGA GAT TGT CG pHFQ construction
pACYC184-seq-F GGC TCA TGA GCG CTT GTT TC pHFQ confirmation
pACYC184-seq-R TGT CCT ACG AGT TGC ATG ATA pHFQ confirmation
hfq-pBAD18-F-EcoRΙ AAA GAA TTC GGT TCA AGA GTA TAA ACA AC pHFQ-A construction
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pBAD18-seq-F GTC CAC ATT GAT TAT TTG CAC G pHFQ-A confirmation
pBAD18-seq-R CAG GCT GAA AAT CTT CTC TCA T pHFQ-A confirmation
a Restriction enzyme sites are underlined.
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Table III-3. Primers for qRT-PCR analysis in Chapter III
Name Target genes Sequences (5’ to 3’)
Control-RT-F control GGG CCT CAT GCC ATC AGA T
Control-RT-R control TCT CAG ACC AGC TAG GGA TCG T
flhC-RT-F flhC GCA ACT TAG CCG CGG TAG AC
flhC-RT-R flhC TGA ACC AGT CCG TGG AAA AGG
fliA-RT-F fliA GCA GGA ACT GGG ACG TAA CG
fliA-RT-R fliA GTG TCG AGC AAC ATC TGA CGA T
flgK-RT-F flgK CGC TAT GAG CAG ATG TCG AAA AT
flgK-RT-R flgK GTC TGC AGG CTT TTG AAG AAA TC
fliC-RT-F fliC CGT ATC GCT GGT GGT GCT AA
fliC-RT-R fliC CAG CGC CAA CCT GAA TTT TC
miaA-RT-F miaA GAG CAG CGT TTT CAC CAG AT
miaA-RT-R miaA AGG CAT GTC CGT ATG CAA AT
hflX-RT-F hflX TGT CGA AGC ATT ACA GGT GAT T
hflX-RT-F hflX AAT CTC AAC GGC TTT ACC TTC A
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III-3. Results
III-3-1. hfq mutant in C. sakazakii was constructed and defective in
growth.
As numerous studies have demonstrated that Hfq contributes to the
pathogenesis of many bacteria differently (Christiansen et al., 2004; Meibom
et al., 2009; Robertson & Roop, 1999), I studied the effects of an Hfq-like
protein on the pathogenesis of C. sakazakii ATCC 29544. To understand the
roles of hfq in C. sakazakii ATCC 29544 pathogenesis, I generated an
unmarked mutant lacking the entire hfq gene using the Lambda Red-
recombination technique. The loss of hfq was verified by sequencing, and I
confirmed no polar effect on miaA and hflX, located upstream and
downstream of hfq, respectively, by qRT-PCR (Fig.III-1A). When I compared
the growth of the strains in LB medium with constant aeration at 37°C, the
deletion mutant showed a slightly lower growth rate than the wild type
(Fig.III-1B). The plasmid carrying the hfq gene with its cognate promoter
region, which was identified by primer extension analysis (Fig.III-2),
complemented the reduced growth of the hfq strain (Fig.III-1B).
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A
B
Fig. III-1. hfq mutant showed defectiveness of growth, but had no polar
effects.
(A) The expression level of miaA and hflX was checked by qRT-PCR to
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validate non-polar effects on hfq deletion in C. sakazakii.
(B) Growth of C. sakazakii hfq in LB medium. C. sakazakii wild-type and hfq
strains and hfq harboring pHFQ were cultured in LB broth at 37°C. The
OD600 value of each strain harboring the pACYC184 control plasmid or the
complementation plasmid pHFQ was measured hourly. The error bars
represent means and standard errors of the mean (SEM) from three
independent biological replicates.
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A
B
P1
C T A G C T A G
P2
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Fig. III-2. The promoter regions of hfq were identified by primer extension analysis.
The transcription start site was determined by primer extension of RNA derived from C. sakazakii ATCC 29544 grown to mid-exponential
phase in LB broth. Lanes G, A, T and C represent the nucleotide sequencing ladders of predicted promoter regions in C. sakazakii ATCC
29544. The transcription start site for P1 and P2 are indicated by triangles, respectively. (D) Schematic diagram showing the position of
proposed promoters (denoted as P1 and P2) of hfq in C .sakazakii ATCC 29544. Transcription start sites are indicated by boldface in bigger
font. Possible promoters (-10 and -35) are shown underlined and the hfq coding region is denoted as hfq start codon with an arrow.
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III-3-2. The loss of hfq attenuates colonization by C. sakazakii in rat
pups.
Hfq is a global regulator known to modulate physiological fitness
and pathogenesis at multiple levels in various bacteria (Chiang et al., 2011;
Ding et al., 2004; Schiano et al., 2010; Sittka et al., 2007). A Klebsiella
pneumoniae mutant lacking hfq was unable to disseminate into extraintestinal
organs (spleen and liver) and was attenuated in induction of a systemic
infection in a mouse model (Chiang et al., 2011). Similarly, Vibrio cholerae
hfq mutants were highly attenuated in virulence in the suckling mouse model
of cholera, showing 1,000-fold less efficacy than wild-type bacteria at
colonizing the murine small intestine (Ding et al., 2004).
To examine whether the hfq gene in C. sakazakii contributes to
pathogenesis, an in vivo challenge assay using newborn rats was conducted as
performed in other studies (Hunter et al., 2008; Townsend et al., 2007b). To
minimize individual variations between infant rats, competitive assays were
carried out by orally inoculating the bacterial mixture containing equal
numbers of hfq and wild-type strains. At 20 h post-infection, the hfq deletion
mutant was outcompeted by the wild type, with CIs of 0.05 0.06 and 0.08 0.09
in the spleen and liver, respectively (Fig.III-3). These results suggest that hfq
is critical for the ability of C. sakazakii to disseminate into deeper organs. The
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attenuated virulence of the hfq mutant might be attributable to multiple
defects during the infection processes, including decreased invasion into host
cells and impaired resistance against environmental stresses.
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Fig. III-3. The virulence of C. sakazakii was diminished due to the
absence of hfq in vivo.
Two- to 3-day-old rat pups were orally administered a mixture of wild-type C.
sakazakii harboring pACYC184 (SK001) and an hfq::kan strain (SK004), with
approximately 5 x 108 CFU/ml of each. To determine the bacterial loads in the
spleen and liver, the organs were removed aseptically, homogenized at 20 h
post-infection, and plated onto selective agar. The CI was calculated as
follows: (hfqoutput/hfqinput)/(WToutput/WTinput). A CI of < 1 indicates attenuation
of virulence. Each circle or square represents the CI for a single animal in the
spleen and liver of each group, respectively. The geometric means of the CIs
for all the rat pups are shown as solid lines, and the asterisks indicate
significant differences (**, P < 0.05; *, P < 0.1). CI values of less than 1
indicate that hfq::kan (SK004) strains were outcompeted by the wild type
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(SK001).
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III-3-3. Hfq is involved in the invasion of C. sakazakii into human
epithelial cells.
The loss of hfq caused a reduction in invasion ability in S.
Typhimurium and E. coli (Kulesus et al., 2008; Simonsen et al., 2011). To
explore whether hfq in C. sakazakii affects its invasion ability, a gentamicin
protection assay was performed using Caco-2 cells. As expected, the hfq strain
showed approximately 3-fold reduced invasion into these cells compared with
the WT. Complementation with the plasmid harboring the hfq fully restored
the invasion ability of the hfq strain to wild-type levels (Fig.III-4A).
Additionally, I checked the invasiveness of the strains in HepG2 cells, which
are derived from liver epithelia, and the result was comparable to that with
Caco-2 cells (Fig.III-4A), suggesting that impaired invasiveness may be
common for the epithelium-derived cell lines.
As outermembrane protein A (OmpA) and B (OmpX) are known to
be invasins in C. sakazakii (Kim et al., 2010), it was necessary to compare the
expression of OmpA and OmpX between WT and hfq mutant strains via
western-blot analysis. However, substantial differences in OmpA and OmpX
expression were observed neither total lysate nor outer membrane fraction
(Fig.III-4B).
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A
B
Anti-OmpX
Anti-DnaK
WT Δhfq
Anti-OmpA
Anti-DnaK
WT Δhfq
HepG2 Caco-2
Anti-OmpA
WT
Anti-OmpX
WT Δhfq
Total lysate Outer membrane
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Fig. III-4. Loss of Hfq impairs the invasion of epithelial cells.
(A) Confluent monolayers of eukaryotic cells were infected with C. sakazakii grown to the exponential phase and incubated for 1.5 h,
followed by gentamicin treatment (100 μg/ml) for a further 1.5 h. The Caco-2 (left) and HepG2 (right) cells were lysed with 1% Triton X-100
to recover the intracellular bacteria. The data are representative of 3 independent experiments. The bars represent the means and SEM from
independent experiments performed in triplicate. The asterisks indicate significant differences (***, P < 0.001).
(B) Levels of OmpA and OmpX from total lysate (left) and outer membrane (right) were assessed by immunoblotting in exponentially grown
cells. The blots are representative of 3 independent experiments. DnaK was used as a loading control.
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III-3-4. Hfq is required for oxidative-stress resistance in C. sakazakii.
In aerobic environments, many bacteria generate or encounter
reactive oxygen species (ROS) (superoxide anion, hydrogen peroxide, and
hydroxyl radicals), which damage lipids, proteins, and nucleic acids (Imlay,
2003). It is well known that pathogenic bacteria have the ability to cope with
oxidative stresses, which is essential for their pathogenesis (Hassett & Cohen,
1989). Here, I tested the role of Hfq in bacterial resistance to oxidative stress
by exposing wild type and hfq strains of C. sakazakii to H2O2. The hfq mutant
showed significantly (100-fold) lower survival than the WT in the presence of
100 mM H2O2 after 10 min of exposure (Fig.III-5A). In agreement with this
result, the hfq strain exhibited a reduction in bubble formation, which is
indicative of decomposition of H2O2 into water and oxygen (Fig.III-5B). katG
is one of the H2O2-inducible genes and encodes hydroperoxidase I or catalase,
a primary detoxifier of H2O2 in many bacteria, including C. sakazakii
(Alvarez-Ordonez et al., 2012; Storz et al., 1990b). As katG is positively
regulated by OxyR, which is a H2O2-inducible regulator in E. coli and S.
Typhimurium (Storz et al., 1990a; Storz et al., 1990b), both oxyR and katG
expression levels were monitored by qRT-PCR. As a result, the addition of
H2O2 significantly increased katG expression in wild-type bacteria, but the
absence of Hfq decreased its expression approximately 3-fold (Fig.III-5C),
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implying that Hfq plays an important role in inducing katG expression for
protection of C. sakazakii against oxidative stress. Complementation with a
plasmid containing hfq in trans restored the impaired resistance of the hfq
strain to H2O2 (Fig.III-5C). Interestingly, the expression of oxyR was not
induced even in the presence of H2O2 (Fig.III-5C).
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A B
C
1 : WT
2: Δhfq
3: Δhfq+pHFQ
1 3 2
H2O
2-treated
H2O
2-non-treated
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Fig. III-5. Stress tolerance is attenuated in C. sakazakii in the absence of Hfq.
(A) Survival of C. sakazakii strains harboring the pACYC184 control plasmid or a complementation plasmid, pHFQ, upon treatment with 100
mM H2O2 for 10 min.
(B) Indirect catalase activities of strains treated with H2O2. 1, wild type (SK001); 2, hfq (SK005); 3, hfq harboring pHFQ (SK007). The
bubbles indicate oxygen gas, produced by decomposition of H2O2.
(C) qRT-PCR analysis of oxyR and katG expression levels. To obtain the expression level relative to the WT on the y axis, the mRNA level of
each gene was divided by the mRNA level of the 16S rRNA. The mRNA expression values in strains SK005 and SK007 were further
normalized to the transcription levels of the wild type. The bars represent means and SEM from independent experiments performed in
triplicate. The data are representative of 3 independent experiments. The asterisks indicate significant differences (**, P < 0.05).
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III-3-5. Loss of the hfq gene reduces intracellular survival of C. sakazakii
in macrophage-like cells.
A previous study showed that Cronobacter spp. were able to persist
or replicate within macrophages (Townsend et al., 2007b), and Salmonella hfq
was shown to be critical for bacterial survival in cultured host macrophages
(Sittka et al., 2007). Thus, I examined the impact of hfq deletion on the
survival of C. sakazakii within cultured macrophages. Although the initial
uptake of Δhfq bacteria by macrophages was 5-fold lower than that of the WT,
clearance of the mutant (2-log-unit and 5-log-unit decreases in survival after
24 and 72 h, respectively) was significantly faster than for the WT (0.5 and
1.5 log units at the respective time points) on average (Fig.III-6A).
Furthermore, the hfq deletion mutants were cleared completely after 72 h
post-infection, whereas WT strains (approximately 4-log CFU/ml) were still
able to persist within macrophages at 96 h (Fig. 6A). A plasmid containing
hfq complemented the ability to survive within macrophages (Fig.III-6B).
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A
B
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Fig. III-6. Lack of Hfq results in reduced intracellular survival of C.
sakazakii in macrophage-like cells.
(A) Intracellular bacteria of the wild type and hfq mutant (SK003) were
evaluated for the number of CFU at uptake (1.5 h) and at 8, 24, 48, 72, and 96
-h post-infection.
(B) Intracellular bacteria of C. sakazakii strains harboring the pACYC184
control plasmid or a complementation plasmid, pHFQ, were evaluated for the
number of CFU at uptake (1.5 h) and at 8 and 24 h post-infection. The bars
represent means and SEM from independent experiments performed in
triplicate. The data are representative of 3 independent experiments. The
asterisks indicate significant differences (***, P < 0.001; **, P < 0.05).
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III-3-6. A C. sakazakii Δhfq strain is hypermotile due to higher expression
of flagella.
As motility is an important virulence determinant (Duan et al., 2013),
I examined the effect of Hfq on the swimming motility of C. sakazakii using
0.3% soft-agar plates. The absence of the hfq gene resulted in increased
motility (Fig.III-7A), and the transmission electron microscope image showed
that the hfq mutant was more flagellated than the WT (Fig.III-7B). These
results suggested the possibility of negative regulation of flagellar synthesis
by Hfq. Therefore, the expression of flagellum-associated genes was studied
in the hfq mutant by qRT-PCR (Fig.III-7C). The transcription of fliA, which
encodes an alternative sigma factor specific for the late operons of flagellar
regulons, including flgK and fliC (Ohnishi et al., 1990), was increased 30-fold
in the hfq mutant compared with the wild type. In accordance with the
increased fliA transcription, genes under the control of FliA, including flgK
and fliC, encoding a flagellar-hook-associated protein and a flagellin protein,
respectively, showed increases in transcription in the absence of Hfq. On the
other hand, the transcriptional level of flhDC, encoding a master regulator of
flagellar regulons, was not affected by the lack of Hfq. To see whether the
transcriptional expression of those genes resulted from different mRNA
stability between WT and Δhfq, mRNA stability of flagellar genes were
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measured by qRT-PCR after the addition of rifampicin (working conc. 250 μg).
No difference of mRNA stability between WT and Δhfq were detected, which
substantiated hfq may indirectly regulate flagellar genes (Fig. III-7D).
Interestingly, introducing a plasmid harboring hfq into the hfq mutant could
partially complement the transcription levels of fliA, flgK, and fliC (Fig.III-7C)
but could not recover the hypermotility (data not shown). However, when the
expression of hfq is tightly regulated by arabinose pBAD promoter (pHFQ-A),
the hypermotility phenotype of hfq mutant was reduced compared with wild
type (Fig.III-7A)
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WT
Δhfq
Δhfq+pHFQ-A
0.5 µm
WT
0.5 µm
Δhfq
A B
C
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D
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Fig. III-7. Loss of Hfq causes enhanced motility.
(A) Motility of the C. sakazakii strains harboring the pBAD18 control plasmid or the complementation plasmid pHFQ-A on semisolid agar
plates. The strains were cultured on semisolid agar plates containing 1.33 mM arabinose at 37°C, and motility was monitored. The image is
representative of several experiments.
(B) Morphology of wild-type and Δhfq (SK003) strains imaged by transmission electron microscopy.
(C) qRT-PCR of fliA, flgK, fliC, and flhC transcripts from WT (SK001) and Δhfq (SK005) strains and Δhfq harboring pHFQ (SK007). To
obtain the expression level relative to that of the WT on the y axis, the mRNA level of each gene was divided by the mRNA level of the 16S
rRNA. The mRNA expression values in strains SK005 and SK007 were further normalized to the transcription levels of the wild type. The
bars represent means and SEM from independent experiments performed in triplicate. The data are representative of 3 independent
experiments.
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(D) mRNA stability of flagellar genes (flhDC, fliA, fliC, and flgK) was estimated by qRT-PCR. Samples were taken after rifampicin (working
conc. 250 μg) at indicated time points. The transcripts were normalized by an endogenous control (16s rRNA).
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III-4. Discussion
The gene (KC866358) homologous to the hfq gene is located in a
clockwise orientation in the genome of C. sakazakii ATCC 29544, as shown in
Fig. III-8A and is also found in C. sakazakii ATCC BAA 894
(YP_001436319.1) and C. turicensis z3032 (YP_003212051.1) (Kucerova et
al., 2010; Stephan et al., 2011). The gene (KC866358) showed high protein
sequence similarity to the hfq genes of E. coli K-12 (89%), S. Typhimurium
(91%), and Y. pseudotuberculosis (84%) (Fig. III-8B).
In most bacteria, the N-terminal domain of Hfq is highly conserved
and is responsible for RNA binding and protein-protein interactions among
Sm (the core of small nuclear ribonucleoprotein particles [snRNPs]) and Sm-
like proteins (Kambach et al., 1999; Moller et al., 2002), whereas the C-
terminal fragments are variable in length and amino acid composition
(Arluison et al., 2004). The C-terminal domain of Hfq is reported to play roles
in gene regulation and protein stability (Arluison et al., 2004; Vecerek et al.,
2008), suggesting that the variable regions at the C termini may be
responsible for the differential gene regulation by Hfq observed in diverse
bacterial species.
To explore whether the hfq could contribute to C. sakazakii virulence,
I conducted invasion assay using human–derived epithelial cells (Caco-2 and
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HepG2 cells). In the assay, hfq affect invasion ability of C. sakazakii (Fig. III-
4). In Salmonella, Hfq controls Salmonella pathogenicity island I (SPI1),
which encodes a type III secretion system (T3SS) and several effector
proteins that are involved in the invasion of Salmonella into host cells (Sittka
et al., 2007). A secretion system equivalent to the T3SS has not been
identified in Cronobacter; rather, it invades host cells via a receptor-mediated
mechanism (Kim & Loessner, 2008). Previously, outer membrane proteins
(OmpA and OmpX) were reported to be involved in C. sakazakii invasion of
human intestinal Caco-2 cells as invasins (Kim et al., 2010). In this study, the
effect of Hfq on the expression of OmpA and OmpX was studied with
Western blot analysis using anti-OmpA and anti-OmpX polyclonal antibodies.
However, I did not observe any differences in OmpA and OmpX expression
between wild and the mutant strains (Fig. 5B), suggesting that Hfq might not
be associated with outer membrane protein expression. However, the
possibility that Hfq may regulate other, yet-unidentified invasins in C.
sakazakii ATCC 29544 cannot be ruled out.
In survival assay, the loss of hfq attenuated survival of C.sakazakii
within the murine-derived macrophages. These data suggest that hfq is
necessary for intracellular survival of C. sakazakii in host macrophages. F.
tularensis lacking katG caused increases in hydrogen peroxide production and
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tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) expression
within macrophages, leading to attenuation of bacterial survival inside host
cells (Melillo et al., 2010). In this regard, the accelerated clearance of the
Δhfq strain within host cells might be attributable, at least in part, to decreased
katG expression (Fig. III-5C), which causes H2O2 accumulation and
stimulates inflammatory cytokine production. Another probable explanation
for the attenuated Δhfq persistence inside the macrophage-like cells is that
flagellar synthesis is altered by the removal of hfq, as described below.
In Fig. III-5C, although the expression levels of katG were increased
under H2O2 treatment, those of oxyR were not induced, suggesting that OxyR-
independent katG induction may be possible, as has been reported in
Neisseria gonorrhea and Bradyrhizobium japonicum (Panek & O'Brian, 2004;
Tseng et al., 2003).
It is interesting that the C. sakazakii hfq mutant showed an increased
motility phenotype, unlike other bacteria, i.e., Salmonella and uropathogenic
E. coli (Kulesus et al., 2008; Sittka et al., 2007). One explanation for this
phenomenon might be the variable region at the C-terminus of Hfq, as shown
in Fig. III-8B. According to a previous report, an Hfq variant lacking its C
terminus was defective in autoregulation and riboregulation (Vecerek et al.,
2008). Therefore, C-terminal variation might enable differential gene
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regulation from species to species. Alternatively, the hypermotility of the hfq
strain may be associated with Hfq mediated RpoS regulation in flagellar
biosynthesis. It has been reported that Hfq is required for efficient translation
of the rpoS sigma factor (Brown & Elliott, 1996; Muffler et al., 1996), and
RpoS negatively regulates operons involved in flagellar biosynthesis and
functions in E. coli by decreasing the amount of RpoF, which competes with
RpoS in binding to the core RNA polymerase (Makinoshima et al., 2003;
Patten et al., 2004). In the absence of RpoS, the expression of RpoF (FliA)
and FliC was increased, and the surface of the bacteria was found to be more
flagellated than that of the WT strain. In this regard, it is plausible that the hfq
null mutation caused poor translation of rpoS in C. sakazakii and that the
lowered level of RpoS resulted in higher flagellar biosynthesis and formation.
Further study would be needed to shed light on the mechanisms of flagellar
regulation mediated by hfq.
An Acinetobacter baylyi Δhfq strain showing a retarded growth and
abnormal morphology was fully complemented with the introduction of hfq
and its cognate promoter region into the chromosome, while a low-copy-
number plasmid harboring hfq under a leaky inducible promoter could not
complement it, indicating that fine-tuning of hfq expression is important for
its tight regulation (Schilling & Gerischer, 2009). The failure of the pHFQ
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plasmid to complement the hypermotility of the hfq strain might be caused by
the imbalance of Hfq production from the low-copy-number plasmid
pACYC184 (Chang & Cohen, 1978). To rule out that possibility, the pHFQ-A
plasmid, in which hfq expression is under an arabinose-inducible promoter
(Guzman et al., 1995), was used to optimize Hfq expression and
accomplished partial restoration of motility with 1.33 mM arabinose (Fig. III-
7A). Taken together, these data suggest that hfq in C. sakazakii is involved in
the negative regulation of flagellar biosynthesis.
The ability of pathogenic microbes to survive within cultured
phagocytic cells is associated with the ability to escape or remodel an innate
immune response that employs antimicrobial defense mechanisms, such as
oxidative burst and acidic compartmentalization (Townsend et al., 2007b). To
establish a good niche within the host, bacteria should have the ability to
modulate immune system activation by harnessing cytokine production
(Cummings et al., 2005; Finlay & McFadden, 2006). It has been reported that
C. sakazakii flagella and flagellin proteins stimulate the production of
inflammatory cytokines (IL-8, TNF-α and IL-10) in a dose-dependent manner
(Cruz-Cordova et al., 2012). To dampen cytokine stimulation, pathogens, such
as Pseudomonas and Salmonella, reduce flagellar synthesis after entering host
cells (Bardoel et al., 2011; Cummings et al., 2005; Eriksson et al., 2003).
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Bacterial flagella activate the signaling pathway by binding to TLR5 during
infection (Hayashi et al., 2001). Indeed, secreted flagellin from Pseudomonas
triggered the expression of NF-κB and secretion of IL-8. To avoid the
activation of TLR5, the enzyme AprA, which cleaves flagellin, is produced
(Bardoel et al., 2011). Likewise, flagellin from Salmonella also stimulates the
activation of immune cells, leading to proinflammatory cytokine expression.
However, intracellular Salmonella represses flagellar expression to modulate
the immune response, whereas extracellular bacteria express flagella. Overall,
it is important for pathogenic bacteria to harness flagellar expression for
successful infection (Bardoel et al., 2011; Cummings et al., 2005). In this
regard, hyperflagellation of the hfq mutant (Fig. III-7B) might result in higher
production of inflammatory cytokines. Accordingly, the increase in cytokine
production might elicit an immune response, leading to accelerated clearance
of intracellular bacteria or causing local inflammation and necrosis (Slifka &
Whitton, 2000).
In the present study, I demonstrated that the loss of Hfq remarkably
attenuated C. sakazakii virulence in a rat pup model of infection due to defects
in growth, invasion ability, resistance to oxidative stress, and intracellular
survival. In addition, to our surprise, the Hfq-deficient strain showed
hypermotility, which is a distinctive phenotype among bacteria.
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Fig. III-8. hfq homolog in C. sakazakii ATCC 29544.
(A) Genomic organizations of hfq (KC866358), miaA (KC866359), and hflX (KJ577586) in C. sakazakii ATCC 29544. The promoter regions
of hfq are indicated as P1 and P2.
(B) Multiple-sequence alignment of hfq sequences in Gram-negative pathogens with a functionally identified hfq gene by Clustal Omega
(version 1.2.1) and Genedoc. The uppercase letters in the consensus sequence indicate conserved amino acids appearing in all the aligned
sequences, and the lowercase indicates conserved amino acids appearing in at least two sequences. Identical and conserved residues are in
black and shaded gray (dark gray, 70% conserved; light gray, 40% conserved), respectively. Every 10 amino acids are indicated by asterisks.
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Chapter IV.
CSK29544_02616, a newly identified protein,
influences C. sakazakii virulence by modulating
LPS production
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IV-1. Introduction
Lipopolysaccharides (LPS) are the major constituents of the outer
membrane of almost all Gram-negative bacteria and extremely heat-stable
amphiphilic substances (Holst et al., 1996). It has been reported that LPS
plays crucial roles in maintaining outer membrane integrity, eliciting immune
responses in humans and other animals, and protecting bacteria from
membrane disruptive agents (Klein et al., 2009; Miller et al., 2005)
The outer membrane of Gram negative bacteria is composed of an
asymmetric bilayer, containing phospholipids in the inner leaflet and lipid A
in the outer leaflet (Kamio & Nikaido, 1976). Phospholipids consist of a
glycerol molecule, a phosphate group, and two fatty acid moieties (except for
cardiolipin), whereas LPS is structurally composed of 3 distinct regions: lipid
A, a hydrophobic moiety anchored in outer membrane, core oligosaccharide
(inner- and outer-core), and O-antigen (Raetz, 1990). LPS roughly exists 106
copies in a single cell of E. coli, covering about 75% of the cell surface area
(Galloway & Raetz, 1990; Raetz, 1990). Lipid A, known as an endotoxin,
includes six hydrophobic acyl chains located in the outer leaflet of outer
membrane. They are connected to a glucosamine and phosphate head group.
The core oligosaccharides start with the addition of a pair of Kdo sugar
residues (3-deoxy-D-manno-oct-2-ulosonic acid) to lipid A moiety. This core
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then connects to the O-antigen, a polysaccharide and hyper-variable region
between bacterial species and different stains within the same species (Raetz
& Whitfield, 2002; Raetz et al., 2009). Of these three components, the lipid A
moiety is of interest because it is the only residue that is essential for bacterial
cell viability and is highly conserved (Raetz & Whitfield, 2002).
The lipid A biosynthesis pathway has been recently unveiled through
several decades of careful experimentations (Emiola et al., 2014). Its
biosynthesis initiates delivery of R-3-hydroxyacyl chain from R-3-
hydroxyacyl-acyl carrier protein (ACP) to the glucosamine 3-OH of UDP-N-
acetylglucosamine (GlcNAc), resulting in UDP-3-(R-3-hydroxyacyl)-GlcNAc
(Anderson et al., 1985; Anderson & Raetz, 1987; Anderson et al., 1993). This
reaction is mediated by lpxA encoding UDP-GlcNAc acyltransferase, which is
the very first step in the biosynthesis of lipid A (Raetz, 1990). It has been
reported that lipid A biosynthesis is regulated by several enzymes, including
lpxC, kdtA, lpxD, and lpxK. LpxC, UDP-3-(R-3-hydroxyacyl)-GlcNAc
deacetylase, and KdtA, a Kdo transferase, are regulated by the protease FtsH
degrading both enzyme substrates (Fuhrer et al., 2007; Katz & Ron, 2008). It
has been known that the catalytic activity of LpxK, encoding a
tetraacyldisaccharide 4’-kinase, is dependent on the concentration of
phospholids (Ray & Raetz, 1987). In particular, the activity of LpxD is
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modulated by NPr (encoded by ptsO) through a direct interaction (Kim et al.,
2011a)
Interestingly, the research of crosstalk between LPS and
phospholipid biosynthesis has been investigated for several decades. There are
a number of studies that indicate a strong relationship between both
biosynthetic pathways as they share the same precursor, β-OH-myristoyl-ACP
(Galloway & Raetz, 1990; Ogura et al., 1999; Zeng et al., 2013). As these
substances are the major components of outer membrane, the proper
equilibrium between them is important for bacterial cells to maintain outer
membrane integrity. The ratio of lipid A to glycerophospholips has been
reported to be 0.12 in E. coli (Galloway & Raetz, 1990). If the level of lipid A
biosynthesis is reduced by mutation of lpxC, the amount of phospholipid
production is increased, which results in unbalance between lipid A and
phospholipid. This altered ratio influences outer membrane protein assembly
in E. coli (Kloser et al., 1998). Furthermore, various studies have verified that
LPS is required for the correct assembly of OMPs (de Cock & Tommassen,
1996; de Cock et al., 1999; Koplow & Goldfine, 1974; Ried et al., 1990; Sen
& Nikaido, 1991).
Infectious diseases caused by Gram negative pathogenic bacteria
have been one of serious health concerns. Antibiotics, as a panacea, have been
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widely used to treat bacterial infections. However, unfortunately, many
pathogens, including strains of Pseudomanas aerusinosa in cystic fibrosis
patients, have emerged as being antibiotic-resistant to commercially available
antibiotics (Conway et al., 2003; Garau & Gomez, 2003; Sherrard et al.,
2014). In this regard, the attempts to develop new antibacterial agents have
been spotlighted for several decades. As a part of efforts, Lipid A biosynthetic
enzymes have been attractive targets for developing a new paradigm of
antibacterial agents because they are conserved enzymes within almost all
Gram negative bacteria (Jackman et al., 2000). Recently, a pentadecapeptide,
known as peptide 920, has been discovered and shown to have high affinity
for LpxA. The functionality of this peptide was proved by inhibiting E. coli
growth (Benson et al., 2003). It may be a promising agent to control
infections from gram negative pathogens as the structure of lipid A-kDO
moiety is highly conserved within them (Raetz & Whitfield, 2002).
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IV-2. Materials and Methods
IV-2-1. Strains, plasmids, and culture conditions.
Strains and plasmids used in the present study are listed in Table IV-
1. Bacteria cells were culture at 37°C with constant shaking (220 rpm), unless
otherwise stated.
IV-2-2. Construction of random transposon mutant libraries and
screening.
Random mutagenesis was performed using the EZ-Tn5TMpMOD-2<
MCS> transposon system (Epicentre, Madison, WI) as described previously.
Briefly, the transposon construct was released by restriction digestion with
PvuII and then electroporated (1.8 kV) (MicroPulser; Bio-Rad, Hercules, CA)
into competent C. sakazakii ATCC 29544. The transformants were selected on
tryptic soy agar (TSA; Difco) plates containing kanamycin (50 µg/ml). The
resulting colonies were individually cultured and stored at 80°C in TSB
containing 15% (vol/vol) glycerol. This work was performed by the aid of
colleague (Y.Choi).
IV-2-3. Determination of the transposon insertion sites.
To locate the transposon insertion site, genomic DNA was isolated
from candidate clones that were defective in invasion (see below for invasion
assay). After the self-ligation of restriction enzyme-digested DNA according
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to the manufacturer’s protocol (Epicentre), the ligation mixture was PCR
amplified and sequenced with Tn5-specific primers provided by the
manufacturer (pMOD<MCS> forward sequencing primer and pMOD <MCS>
reverse sequencing primer).
IV-2-4. Generation of ΔCSK29544_02616 deletion mutant by using site-
specific mutagenesis.
To design all PCR primers used in this study, partial sequences of C.
sakazakii ATCC 29544 were used. Site-specific mutagenesis of C. sakazakii
ATCC 29544 was performed by Dasenko and Wanner (Datsenko & Wanner,
2000). In short, the kanamycin resistance cassette from plasmid pKD13 was
amplified using the following primers: CSK29544_02616-lamb-F (CGC GTG
GTG GAT GCT ATC CGC GCG CAG GCC GCG CTC GGC CTT GCG
GAG AAA GTG GCA TGA TGT AGG CTG GAG CTG CTT CG) and
CSK29544_02616-lamb-R (CGT CAT TGA CTA CTG CCG CTC CCA TGC
CAA CGA TAG CGT CGT TAC CGA TGT GGA TTT GCT ATT CCG GGG
ATC CGT CGA CC). (The nucleotide sequences originating from pKD13 are
underlined, and those from C. sakazakii gene of interest are shown in italics.)
The PCR products were transformed into C. sakazakii ATCC 29544 harboring
pKD46 plasmid by electroporation and cells were selected for Km-resistant
transformants. Finally, the kanamycin resistance-cassette was removed using
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the pCP20 plasmid, as described by Datsenko and Wanner (Datsenko &
Wanner, 2000).
IV-2-5. Construction of plasmids.
The plasmids used in the study are listed in Table IV-I. To generate
the recombinant plasmid, primers pACYC184-CSK29544_02616-F-SphΙ and
pACYC184-CSK29544_02616-R-BamHΙ amplified the entire
CSK29544_02616 coding region of 639 bps and 616 bps of upstream
sequences, which might contain cognate promoter of CSK29544_02616. The
purified product was digested with restriction enzymes, SphΙ and BamHΙ and
ligated into pACYC184 vector cut with the same enzymes. The recombinant
vector was transformed into DH5α at 42ºC and selected on LB agar
containing chloramphenicol resistance. The selective clone was sequenced.
IV-2-6. Gentamicin protection (invasion) assay.
To determine bacterial invasion of mammalian Caco-2 cells, a
gentamicin protection assay was performed as described previously (Kim et
al., 2010). Briefly, bacteria were prepared by transferring a 1% inoculum from
overnight cultures into fresh LB medium, followed by incubation for 2.5 h at
37°C with constant shaking. C. sakazakii cells were collected by
centrifugation at 20,000 x g, washed with phosphate-buffered saline (PBS)
(pH 7.4), and resuspended in 1 ml of pre-warmed fresh Eagle’s minimum
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essential medium (EMEM) (ATCC). Mammalian cells were seeded in EMEM
supplemented with 20% fetal bovine serum (FBS) (Gibco, Invitrogen) in 24-
well tissue culture plates with a cell density of 2 x 105 per well. The cell
monolayers were incubated for 1 day, infected with bacteria at a multiplicity
of infection (MOI) of 100, and incubated for 1.5 h in the presence of 5% CO2.
After the cells were washed once with PBS, fresh medium containing
gentamicin (100 μg/ml; Sigma) was added, and the plates were further
incubated for 1.5 h, followed by three washes with PBS. Then, a five-hundred
μl of 1% Triton X-100 was added, and the plates were incubated for a further
15 min before the bacteria were collected and plated on tryptic soy agar
(TSA).
IV-2-7. Survival assay.
A long-term survival assay was performed as described previously
(Townsend et al., 2007b). Briefly, RAW264.7 murine macrophage-like cells
were seeded in Dulbecco’s modified Eagle medium (DMEM) (Gibco,
Invitrogen) supplemented with 10% FBS in 24-well plates at a density of 5 x
105 cells per well and incubated for 1 day at 37°C with 5% CO2. C. sakazakii
strains were prepared as described for the invasion assay, and cell monolayers
were infected with bacteria at an MOI of 100 for 45 min at 37°C. The medium
was replaced with DMEM (plus 10% FBS) supplemented with 100 μg/ml of
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gentamicin, and the cells were incubated for an additional 45 min. The plates
were washed twice with PBS, and the cells were lysed with 1% Triton X-100,
followed by serial dilution and plating onto TSA to determine the numbers of
intracellular bacteria at various time points. For further study of bacterial
persistence (long-term survival), the cells were replenished daily with fresh
medium containing 10 μg/ml of gentamicin, and the numbers of intracellular
bacteria were determined at 3, 8, 24, 48, 72, and 96 h.
IV-2-8. In vivo animal study.
Three- to four-day-old Sprague-Dawley (SD) rat pups were used to
assess the virulence of the WT and the at mutant (SK010) strains. Mid
exponential-phase bacteria were collected, washed, and resuspended in PBS.
For competitive assays, a mixed inoculum of 1 x 109 CFU/ml of the fully
virulent WT and the CSK29544_02616 mutant (SK010) harboring a kan
cassette was orally administered to rat pups. To analyze bacterial colonization
in organs, all the rat pups were killed with CO2 at 20 h post-infection. The
spleens and livers were aseptically removed, homogenized, and serially
diluted. Bacterial loads were determined for the WT and mutant by plating
onto LB agar in the presence or absence of kanamycin. The results are
presented as competitive-index (CI) values, which were calculated as follows:
(CSK29544_02616output/ CSK29544_02616input)/(WToutput/WTinput).
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IV-2-9. Autoaggregation (suspension-clearing) assay.
Assays were performed as described by previously with
modifications. Briefly, bacteria were prepared by transferring 1% inoculum
from overnight cultures into fresh LB medium, followed by incubation until
mid-exponential phase at 37ºC with constant shaking. Exponentially grown
cells were diluted to adjust the OD600 of 1.5 to 5 ml LB broth in 14 ml round-
bottom tube (BD) and left to sit at room temperature. The rate of suspension
clearance was measured over 7 h by carefully removing 100 µl from the top
of the suspension and measuring the OD600.
IV-2-10. Overexpression and purification of recombinant proteins.
Each open reading frame (ORF) of the genes encoding
CSK29544_02616 and lpxA was amplified by PCR using a pair of
oligonucleotide primers as listed in Table IV-2. The amplified PCR products
were cloned into a pBAD24, Hisx6 -tag expression vector, pETDuet 1, and
GST tag expression vector, pGST parallel 1 to result in pHis-
CSK29544_02616, pHis-lpxA, pGST-CSK29544_02616, pLpxA, and pACP
as described in Table IV-1. For the induction, 13.3 mM of arabinose and 0.5
mM of IPTG were added to the culture at OD600 of 0.5 at 37°C, otherwise
indicated. After additional 4 h induction at 37°C, cells were harvested. For
nickel chelated nitrilotriacetic acid (Ni-NTA) affinity chromatography
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(Qiagen, USA), the cells were resuspended in lysis buffer (20 mM Tris-Cl,
300 mM NaCl and 5 mM imidazole, adjusted to pH 8.0) and lysed by
sonication on ice, centrifuged at 21,130 x g and 4 °C for 1 h to remove cell
debris, and the supernatant was subjected to lysis buffer-equilibrated nickel
chelated nitrilotriacetic acid (Ni-NTA) affinity chromatography (Qiagen,
USA). After incubation with gentle end-over-end rotation at 17 rpm at 4°C for
1 h, protein-bound Ni-NTA resin was washed 10 times with 1 ml of washing
buffer (lysis buffer containing 20 mM imidazole) and twice with 1 ml of
washing buffer (lysis buffer containing 50 mM imidazole). The Hisx6-tagged
target proteins were eluted with elution buffer (20 mM Tris-Cl, 300 mM NaCl
and 250 mM imidazole, adjusted to pH 8.0) and concentrated with AmiconR
Ultra-4 (10,000 MWCO cellulose; Milipore, USA) according to the
manufacturer’s instructions. The buffer was changed to the storage buffer (20
mM Tris-Cl, 300 mM NaCl and 50% glycerol, adjusted to pH 8.0) using PD
midiTrapTM G-25 (GE healthcare, UK) according to the manufacturer’s
instructions, and then aliquots of protein were stored at - 20°C, until further
usages.
IV-2-11. Ligand fishing to search for proteins interacting with Hisx6-
tagged CSK29544_02616.
SK015, which expressing CSK29544_02616 with N-terminal six
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histidines was used for ligand fishing. One mg of N-terminally Hisx6-tagged
CSK29544_02616 was incubated cell lysate of WT and that of
ΔCSK29544_02616, respectively, in the Poly-Prep chromatography column
(Bio-Rad, Hercules, CA) that had been pre-equilibrated with Ni-NTA resin
(Qiagen, USA), then incubated for 1 h at 4°C with gentle end-over-end
rotation at 17 rpm. After incubation, each column was washed with 1 ml of
washing buffer 5 times (lysis buffer containing 10 mM imidazole), and the
proteins bound to the column were eluted with the elution buffer. Aliquots of
the eluted protein sample (15 μl each) were analyzed by SDS-PAGE and then
were stained with Coomassie brilliant blue R. The protein band specifically
bound to His-CSK29544_02616rom the gel, and in-gel digestion and peptide
mapping of tryptic digests were carried out by Yonsei Proteome Research
Center (Korea).
IV-2-12. LC-MS/MS.
Nano LC–MS/MS analysis was performed with a nano HPLC system
(Agilent, Wilmington, DE). The nano chip column (Agilent, Wilmington, DE,
150 mm × 0.075 mm) was used for peptide separation. The mobile phase A
for LC separation was 0.1% formic acid in deionized water and the mobile
phase B was 0.1% formic acid in acetonitrile. The chromatography gradient
was designed for a linear increase from 3% B to 50% B in 25 min, 90% B in 5
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min, and 3% B in 15 min. The flow rate was maintained at 300 nL/min.
Product ion spectra were collected in the information-dependent acquisition
(IDA) mode and were analyzed by Agilent 6530 Accurate-Mass Q-TOF using
continuous cycles of one full scan TOF MS from 350-1200 m/z (1.0 s) plus
two product ion scans from 100-1700 m/z (1 s each). Precursor m/z values
were selected starting with the most intense ion, using a selection isolation
width of ~4 Da. The rolling collision energy feature was used, which
determines collision energy based on the precursor value and charge state. The
dynamic exclusion time for precursor ion m/z values was 20 s.
IV-2-13. Database search.
The mascot algorithm (Matrixscience, USA) was used to identify
peptide sequences present in a protein sequence database. Database search
criteria were, taxonomy; Cronobacter sakazakii (NCBInr database
downloaded on Nov 8 2013) + BAA 894(Accession No.:
NC_009778.1)+ATCC 29544 (Accession No.: CP011047.1) fixed
modification ; carboxyamidomethylated at cysteine residues ; variable
modification ; oxidized at methionine residues, maximum allowed missed
cleavage; 2, MS tolerance; 100 ppm, MS/MS tolerance ; 0.1 Da. Only
peptides resulting from trypsin digests were considered.
IV-2-14. BACTH (bacterial adenylate cyclase based two-hybrid system)
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assay.
The protein-protein interaction was verified by the reconstitution of
adenylate cyclase (CyaA) in E. coli through heterodimerization of fusion
proteins (Battesti & Bouveret, 2012). The gene CSK29544_02616 was
PCR-amplified from the genomic DNA of C. sakazakii ATCC 29544 using a
pair of primers pKT25-CSK29544_02616-F-BamHI and pKT25-
CSK29544_02616-R-EcoRI. The resulting PCR product was digested with
BamHI and EcoRI, and ligated into pKT25 digested with the same restriction
enzymes to fuse CSK29544_02616 at the C-terminal end of T25 fragment,
generating pKT25-CSK29544_02616. The construction of pUT18C-lpxA was
conducted as stated above, except for a different pair of primers and a
backbone plasmid (pUT18C). E. coli BTH101 reporter strain (cyaA-) was co-
transformed with the recombinant plasmids (pKT25-CSK29544_02616 and
pUT18C-lpxA), and appropriate transformants were selected on LB agar
containing Kan, Amp, and X-gal. The BTH101 strain harboring pKT25-
zip/pUT18C-zip and pKT25/pUT18C was used as positive or negative control,
respectively. The positive transformants were streaked on LB agar
supplemented with Kan, Amp, and X-gal to determine the CSK29544_02616-
LpxA interaction by monitoring colony color. To quantify the interaction
between CSK29544_02616-LpxA, β-galactosidase assays were performed as
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previously described, and the results were expressed in Miller units (Miller,
1972)
IV-2-15. β-galactosidase assay.
β-galactosidase assays were performed in duplicates as described
previously (Miller, 1972). Briefly, 1 ml of culture was centrifuged at 16,000 x
g for 1 min and re-suspended in Z-buffer (60 mM Na2HPO4 40 mM
NaH2PO4, 10 mM KCl, 2 mM MgSO4 and 40 mM β-mercaptoethanol,
adjusted to pH 7.0), and OD600 was measured. Aliquots (0.1 ml) of the cell
suspensions were added to Pyrex tubes containing 20 μl of 0.1% SDS, 40μl of
chloroform and 0.9 ml of Z-buffer, followed by vortexing for 10 s. The
samples were incubated for 10 min, vortexed every 3 min at RT, and then 0.2
ml of 2-nitrophenyl β-D-galactopyranoside (ONPG; 4 mg/ml) to start the
reaction. As appropriate yellow color appeared, 0.5 ml of 1 M Na2HCO3 was
added. The samples were centrifuged at 16,000 x g for 1 min to sediment all
cell debris, then the optical density of the supernatants was measured at 420
nm or 550 nm. The β-galactosidase activity (arbitrary units) was calculated as
previously described and expressed in Miller units (Miller, 1972)
IV-2-16. In vivo GST pull-down assay.
Constructs comprising LpxA and CSK29544_02616 were cloned
into pETDuet 1 and pGST parallel-1 vectors in frame with an N-terminal 6x-
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histidine tag and an N-terminal glutathione S-transferase (GST) tag,
respectively. LpxA and CSK29544_02616 were expressed separately in E.
coli ER2566 and BL21(DE3) cells and induced with 0.5 mM isopropyl-β-D-
1-thiogalactopyranoside (IPTG). Cells were harvested by centrifugation at
16,000 x g and cell pellets were mixed in a 1:1 ratio. For cell lysis, the mixed
pellets were resuspended in IP150 buffer (50 mM Tris-HCl pH 7.4, 150 mM
NaCl, 2 mM MgCl2, and 0.1% NP-40) containing protease inhibitor cocktail
(P8849, Sigma, Korea). Cells were ultra-sonicated and centrifuged at 21,130 x
g and 4 °C for 1 h to remove cell debris. Concentration of the supernatant was
measured by Brad-ford assay and a sample at a concentration of 1 mg was
incubated with 20 μl of glutathione-agarose beads for 3 h at 4 °C using gentle
end-over-end rotation at 17 rpm. After incubation, the beads were washed
three times with IP150 buffer and the bound proteins were eluted by boiling in
Laemmli’s SDS-Sample buffer (GeneDEPOT, USA) for 10 min. The samples
were resolved by SDS-PAGE and proteins were detected by immunoblotting
with antibodies against GST and His epitope tag.
IV-2-17. Western blot analysis.
GST or His epitope-tagged proteins from the GST pull-down assay
were loaded into 12% SDS-polyacrylamide slab gel. After electrophoresis, the
separated proteins were electro-transferred to PVDF membrane. The
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membrane was blocked with TBST buffer (10 mM Tris-Cl, 150 mM NaCl, 0.1%
Tween 20, adjusted to pH 8.0) supplemented with 0.45% skim milk using a
SNAP i.d.TM Protein Detection System (Milipore), and then it was incubated
for 30 min with a TBST buffer supplemented with primary anti-GST or anti-
His antibodies. After 3 times washing with 20 ml TBST buffer, secondary
antibody solution [TBST buffer supplemented with 0.45% skim milk and the
goat anti-mouse IgG-HRP (Santa Cruz Biotechnology)] was applied onto the
membrane, and incubated for 20 min and another wash with TBST buffer was
performed. The chemiluminescence signals were developed with WEST-
ZOL○R plus Western Blot Detection System (iNtRON Biotechnology, South
Korea) according to the manufacturer’s instructions, and then X-ray film was
exposed to the light to detect the signals.
IV-2-18. LpxA enzyme assay
IV-2-18-1. Cell cultures
Strains of interests were used to inoculate LB broth containing the
proper antibiotics and incubated at 37 °C, 220 rpm until OD600 of 0.5 to 1 was
reached. The cultures were then induced with IPTG of 0.5 mM and incubated
at 37 °C for 4 h post-induction unless otherwise stated. Cells were harvested
by centrifugation at 10,000 x g, suspended in proper buffer with appropriate
amount of protease inhibitor cocktail (P8849, Sigma), and ultra-sonicated.
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Cellular debris were removed by centrifugation at 21,130 xg for 1 h at 4 °C,
and the resultant crude cytosol was used for further protein purifications
unless otherwise indicated.
IV-2-18-2. Purification of LpxA
Free tagged-LpxA was purified as described previously (Jenkins &
Dotson, 2012) with minor modifications. In brief, cell extracts in 20 mM
potassium phosphate (KPhos) buffer and 20% glycerol (buffer A) were
filtered, and applied to Green-SeparoporE○R4B-CL (20181099-2, bioWORLD,
USA) that had been pre-equilibrated in the same buffer. The column was
incubated at 4 °C for 18 h by end over and rotation (F1 mode, 18 rpm). The
column was washed 3 times with 0.5 ml of buffer A, and eluted with 0.5 ml of
buffer A containing 200, 300, and 1M NaCl, respectively. Eluted LpxA
fractions (1M NaCl for high purity) were used for further study.
IV-2-18-3. Purification of Holo-Acyl Carrier Protein
Holo-ACP was produced in cells expressing both apo-ACP, encoded
by acpP, and holo-ACP synthetase, encoded by acpS, according to a previous
study (Broadwater & Fox, 1999). Holo-ACP was purified as described
previously with slight modifications (Jenkins & Dotson, 2012). Protein was
expressed as described in III-2-19-1. Cell cultures section, except that cultures
were induced at 18 °C and incubated for 12 h. To the cellular lysate suspended
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in 10 ml of ACP buffer containing 20 mM HEPES and 1 mM TCEP (Tris(2-
carboxyethyl)phosphine hydrochloride) (pH 8.0), 10 ml of 2-propanol (Final
conc. 50%) was added slowly, and gently mixed by end-over-end rotation (F1
mode, 12 rmp) at 4°C for 1 h in 50 ml falcon tube. The resulting suspension
was centrifuged at 13,000 xg at 4°C for 30 min, and the supernatant was taken
and diluted with 20 ml of ACP bfr (to lower isopropanol conc.). This solution
was loaded onto a Source 15Q column (6 ml). A gradient of ACP buffer
containing 0-500 mM NaCl was carried out, and holo-ACP was eluted at 300
mM NaCl as judged by SDS-PAGE analysis. The eluted protein was desalted
on a PD midiTrapTM G-25 (GE healthcare, UK) in ACP buffer and stored at
4 °C. Protein concentrations were measured by Bio-Rad protein assay.
IV-2-18-4. Purification of V. harveyi AasS-Hisx6
Expression and purification of AasS-Hisx6 were performed as
described above (III-2-19-1. Cell cultures section). After washing and elution
steps from the Ni-NTA resin, the tagged-AasS was subsequently desalted on a
PD midiTrapTM G-25 (GE healthcare, UK) in 20 mM Tris-HCl (pH 7.5), 10%
glycerol, 1mM ethylenediaminetetraacetic acid (EDTA), 0.1 mM TCEP, and
0.002% Triton X-100 as reported earlier for optimal storage (Jiang et al.,
2006). The resulting protein was aliquoted into microcentrifuge tubes and
stored at deep-freezer. Protein concentrations were measured by Bio-Rad
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protein assay.
IV-2-18-5. Acylation of holo-ACP
Acylation of holo-ACP was performed as described previously with
slight modifications (Jiang et al., 2006). A forty μM of holo-ACP was first
reduced for 1 h at 25°C in the presence of two equivalents of TCEP (80 μM)
prior to addition of fatty acid. Acylation reaction containing 5 mM ATP, 5 mM
MgCl2, 100 µM TCEP, 0.01% Triton X-100, 100 µg of AasS, and 150 µM R-
3-hydroxymyristic acid in 100 mM Tris (pH 7.5) in total volume of 10 mL
was incubated at 30°C for 50 min, and another 50 µg of AasS was added.
After 20 min of additional incubation at 30°C, the reaction was loaded onto a
Source 15Q column (6 ml) equilibrated with 20 mM HEPES (pH 8.0). Acyl-
ACP was eluted at approximately 300 mM NaCl and subsequently desalted by
a PD midiTrapTM G-25 and stored at 4 °C.
IV-2-18-6. Fluorescent enzyme assay for LpxA and CSK29544_02616
Assays were performed as previously reported with a few
modifications (Jenkins & Dotson, 2012). All substrates used in the assay were
dissolved in 20 mM HEPES (pH 8.0) with a final volume of 100 μl. A
spectraMax i3 plate reader (Molecular Devices, California, USA) was used to
monitor fluorescence, with photomultiplier tube (PMT) sensitivity set to low
to prevent saturation and number of readings set to 40. Initially, ten μl of 100
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μM Thioglo○R3 (T-003, COVALENT associates Inc, USA) was added in 96
well optical bottom plates (165305, Thermo Fisher Scientific, USA), followed
by the addition of 3-hydroxymyristoyl-ACP at a final conc. of 8 μM and 4
mM of UDP-GlcNAc. This mixture was incubated in the dark at 30 °C for 40
min to allow any unacylated ACP to react with the ThioGlo solution. After
incubation, either 10 nM of LpxA solution or 300 nM of Hisx6-
CSK29544_02616 was added directly to the well and mixed gently before
measurement, and the plate was read continuously at λex =378 nm and λem
=446 nm for 2 h at 15s intervals. Negative control was set to include all
substrates without any enzymes (either LpxA or Hisx6-CSK29544_02616),
and positive control was set to all substrates, excluding Hisx6-
CSK29544_02616.
IV-2-19. LPS extraction
LPS extraction was performed as described previously with
modifications. In brief, LPS was extracted from overnight bacterial cultures
using hot phenol-water micro-extraction methods (Kim & Ryu, 2012; Wang et
al., 2010). One millilitre of bacterial cultures (ca. 2 x 109 cfu ml-1) was
harvested, washed once with 1 ml DPBS (Dulbecco’s PBS containing 0.15
mM CaCl2 and 0.5 mM MgCl2), and re-suspended in 100 µl of DPBS. After
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sonication, proteinase K was treated at a concentration of 100 µg ml-1 and
incubated for 1 hour at 37°C followed by addition of 200 µl of ddH2O. An
equal volume of preheated (68°C) phenol solution was added prior to
incubation at 68°C with vigorous vortex mixing every 5 min. The samples
were chilled on ice for 5 min, and the aqueous phases were separated by
centrifugation at 10 000 g, 4°C for 5 min. The LPS was extracted again from
the phenol phase with another 300 ml of ddH2O. After the addition of sodium
acetate to the pooled aqueous phases at a final concentration of 0.5 M, 10
volumes of 95% ethanol was added, and the mixture was incubated overnight
at -20°C. The crude LPS were sedimented by centrifugation at 16 000 g, 4°C
for 5 min, re-suspended in 100 ml ddH2O, and precipitated again with 95%
ethanol. Finally, the precipitated LPS was re-dissolved in 50 ml ddH2O and
stored at -20°C. The extracted LPS were analyzed by DOC-PAGE on a 15%
acrylamide gels (Reuhs et al., 1998). Briefly, the resolving gel (15%) was
prepared with 5 ml of monomer stock solution [30% (w/v) acrylamide, 0.8%
(w/v) N,N′-methylenebisacrylamide], 2 ml of resolving gel buffer solution
(1.875 M Tris base, adjust to pH 8.8), 3 ml of ddH2O, 17.5 ml of 10%
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ammonium persulphate and 8.75 ml of N,N,N′,N′-
tertramethylethylenediamine (TEMED). The stacking gel (4%) consisting of
0.33 ml of monomer stock solution, 0.5 ml of stacking gel buffer solution
(0.635 M Tris base, adjust to pH 6.8), 1.67 ml of ddH2O, 12.5 ml of 10%
ammonium persulphate and 6.25 ml of TEMED was laid onto the solidified
resolving gel. The gels were pre-run with a running buffer (290 mM glycine,
37 mM Tris base and 6 mM sodium deoxycholate) for 10 min at 15 mA using
Bio Rad Mini-PROTEAN® Tetra Cell. Four microlitres of extracted LPS was
mixed with equal volumes of sample buffer (containing 2 ml of stacking gel
buffer solution, 1 ml of glycerol and 2.5 mg of bromophenol blue, brought up
to 10 ml with ddH2O), and the mixtures were loaded onto the gels prior to
running each gel for ~ 60 min at 15 mA. The gels were fluorescently stained
using the Pro-Q® Emerald 300 Lipopolysaccharide Gel Stain Kit (Molecular
Probes, Cat. No. P20495; Eugene, OR, USA) according to the manufacturer’s
instructions. The samples were visualized under the 300 nm UV by Gel
DocTM EZ imager (BIO-RAD)
IV-2-20. Outer membrane fraction
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Strains of interest were cultured overnight at 37°C, which was used
to subculture (1% inoculum) fresh LB medium. Bacteria were grown to mid-
exponential phase and then collected by centrifugation at 10,000 xg for 5min,
followed by resuspended in 10 ml of HEPES buffer (10 mM, pH 7.4). the
resuspension was ultra-sonicated, centrifuged, and filterated using 0.22 μm
pore-size. The membranes were collected by ultracentrifugation at 100,000 xg
for 1h at 4°C. The pellet was resuspended in 2 ml of 10 mM HEPES, pH7.4,
washed in a total volume of 10 ml of 10 mM HEPES, pH7.4, and spun again
in the ultracentrifugation (100,000 xg for 1h at 4°C). The pellet was
resuspended in 10 ml of 10 mM HEPES, pH 7.4 containing 2 % N-
lauroylsarcosine (wt/vol) (Sarkosyl) (Sigma) and incubated at 37°C for 30
min with shaking. The Sarkosyl-treated membranes were centrifuged at
100,000 xg for 1h at 4°C and the pellet was resuspended in 50 mM Tris-cl
(pH 8.0) containing 1% (wt/vol) Zwittergent 3-14 (Calbiochem) and 10 mM
EDTA.
IV-2-21. Phospholipid quantitation
Phospholipids were quantified by methods described previously with
some modifications (Kloser et al., 1998; Mrsny et al., 1986). In brief,
overnight grown cells (500 mg wet weight) were centrifuged, resuspended in
1 ml of deionized water, and mixed with 2.5 ml of Methanol and 1.25 ml of
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chloroform according to a single phase Bligh and Dyer solvent mixture
(methanol-chloroform-water 2:1:0.8, v/v). This suspension was incubated at
25°C for 2 h with periodic vortexing (every 30 min). After centrifugation at
2800 xg for 15 min at 4°C, 3 ml of the extract from the top was taken, and
mixed with 1.25 ml of each chloroform and water. After thorough vortexing,
the mixture was centrifuged as above. A 1 ml of lower chloroform phase was
removed and the solvents were removed with N2. One ml of 70 % perchloric
acid was added to the sample-containing tubes as well as tube with no sample
as a negative control. The tubes were covered with Teflon tape and placed in
as multi-block heater preheated to 150°C for 2 h 30 min to completely digest
phospholipids. All tubes were heated for the same period until the sample that
required for the longest time to be digested. The tubes were cooled and
aliquots (100 μl) were taken to 600 μl of water, 200 μl of 2.5% ammonium
molybdate, and 200 μl of freshly prepared 10% ascorbic acid. The mixtures
were vortexed and phosphomolybdate color was developed for 1.5 h in a 37°C
water bath. Following reactions, samples were measured using
spectrophotometer at absorbance of 820 nm.
IV-2-22. Hydrophobicity assay
The surface hydrophobicity was measured as described previously
(Wang et al., 2012). In brief, bacterial cells grown overnight were harvested,
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washed twice with PBS, pH7.4, and resuspended in 2 ml of the same buffer to
OD600 around 1. The exact OD600 was measured and recorded as H0. A eight-
hundred μl of xylene was added to the bacterial suspension, mixed for 2 min
thoroughly, and incubated at room temperature for 1 h. The OD600 of the
aqueous phase was measured and recorded as H. The formula representing
bacterial hydrophobicity is as follows: [(H0-H)/H0]x 100, H0 : OD600of before
xylene extraction and H : OD600of after xylene extraction.
IV-2-23. Biofilm assay
The experiment was performed as previously described with slight
modifications (Choi et al., 2015). Briefly, bacteria were inoculated into 3 ml
of LB and incubated at 37°C for 3 h. The culture was diluted to 1.5 of OD600,
an aliquot of 500 μl was loaded in triplicate into a 24-well polystyrene plate,
and incubated at 37°C for 48 h in static condition. To fix the biofilm, 100 μl of
99% methanol was added for 15 min, the supernatant were removed, and the
plates were air-dried. Subsequently, a 500 μl of 0.1% crystal violet (CV)
solution was added. After 20 min, the excess CV was removed, washed with
PBS three times. Finally, the bound CV was released by addition of 250 μl of
95% ethanol. The absorbance was measured at 570 nm using SUNRISE-
BASIC TECAN micro plate reader (Tecan, Austria).
IV-2-24. RNA isolation and sequencing
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Bacteria cells (WT and ΔCSK29544_02616) were culture at 37°C
with constant shaking (220 rpm) until mid-exponential phase. Total RNA was
extracted by RNeasy mini kit (Qiagen, Germany) according to the
manufacturer’s instructions and treated with Ambion Turbo DNA-freeTM
(AM1907, USA) to remove residual DNA. RNA sequencing and alignment
processes were performed by Chunlab, Inc (Seoul, Korea). The RNA wa
subjected to a substractive hybridization/bead capture using Ribo-Zero kit
(Epicentre, USA) to get rid of rRNA. The purified RNA was used to construct
mRNA-seq library using Illumina TruSeq RNA Sample Preparation kit v2
(Illumina, USA). RNA sequencing was performed by Hiseq to generate
single-end-reads. Sequencing reads were mapped to C. sakazakii ATCC
29544 genome sequences using CLC Genomics Workbench (CLC bio,
Denmark). The results were visualized by CLRNAseq program (Chunlab)
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Table IV-1. Bacterial strains and plasmids used in Chapter IV
Strain or plasmid Genotype and/or characteristics Reference or source
C. sakazakii ATCC 29544 Wild-type strain (Kim et al., 2010)
ES1001 29544 harboring pKD46 (Kim et al., 2010)
SK001 29544 harboring pACYC184 (Kim et al., 2015)
SK009 29544 harboring pBAD24 This study
SK010 CSK29544_02616::kan This study
SK011 Δ CSK29544_02616 This study
SK012 Δ CSK29544_02616 harboring pACYC184 This study
SK013 Δ CSK29544_02616 harboring p CSK29544_02616 This study
SK014 Δ CSK29544_02616 harboring pBAD24 This study
SK015 Δ CSK29544_02616 harboring pHis- CSK29544_02616 This study
SK016 BTH101 harboring pKT25 and pUT18C-lpxA This study
SK017 BTH101 harboring pKT25-CSK29544_02616 and
pUT18C-lpxA
This study
SK018 BL21(DE3) harboring pGST- CSK29544_02616 This study
SK019 ER2566 harboring pHis-lpxA This study
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SK020 ER2566 harboring pLpxA This study
SK021 ER2566 harboring pACP This study
SK022 lpxA::KanR harboring pBAD33-CSK29544_02616 This study
E. coli
DH5α λ- Φ80dlacZΔ(lacZYA-argF)U169 recA1 endA1 hsdR17(rK-
mK-) supE44 thi-1 gyrA relA1
(Hanahan, 1983)
BTH101 F- cya-99 araD139 galE15 galK16 rpsL1(StrR) hsdR2
mcrA1 mcrB1; reporter strain in bacterial two-hybrid assay
(Karimova et al., 1998)
BL21 (DE3) F- ompT hsdS (rB- mB
-) gal (DE3); protein overexpression Laboratory collection
ER2566 F¯ λ¯ fhuA2 [lon] ompT lacZ::t7 gene1 gal sulA11 ∆(mcrC-
mrr)114::IS10 R(mcr-73::miniTn10-TetS )2 R(zgb-
210::Tn10--TetS ) endA1 [dcm]
(Lee et al., 2007)
Plasmids
pKD13 oriR6K AmpR FRT KanR FRT (Datsenko & Wanner, 2000)
pKD46 oriR101 repA101(Ts) AmpR ara BADpgam-bet-exo (Datsenko & Wanner, 2000)
pCP20 oripSC101(TS) AmpRCmR cΙ857λ PRflp (Datsenko & Wanner, 2000)
pACYC184 TetR CmR p15A ori (Chang & Cohen, 1978)
pBAD24 AmpR, araC, PBAD, pBR322 ori, expression vector (Guzman et al., 1995)
pKT25 ori p15A, Plac::cyaA 1–224, KanR (Karimova et al., 2001)
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pUT18C ColEI-ori, Plac::cyaA 225–399, MCS, AmpR (Karimova et al., 2001)
pKT25-zip ori p15A, Plac::cyaA 1–224ΦGCN4-zip, KanR (Karimova et al., 2001)
pUT18C-zip ColEI-ori, Plac::cyaA 225–399ΦGCN4-zip, AmpR (Karimova et al., 2001)
pKT25-CSK29544_02616 pKT25-based expression vector for CSK29544_02616 with
N-terminal T25 fragment of adenylate cyclase
This study
pUT18C-lpxA pUT18c-based expression vector for lpxA with N-terminal
T18 fragment of adenylate cyclase
This study
pETDuet 1 LacI, pBR322ori, pT7, two MCS, AmpR, expression vector Novagen
pGST parallel 1 LacI, tac promoter, AmpR,, expression vector (Sheffield et al., 1999)
pCSK29544_02616 pACYC184 with CSK29544_02616 This study
pHis- CSK29544_02616 pBAD24-based expression vector for CSK29544_02616
with N-terminal six histidines
This study
pHis-lpxA pETDuet 1-based expression vector for LpxA with N-
terminal six histidines, AmpR
This study
pGST- CSK29544_02616 pGST parallel 1-based expression vector for
CSK29544_02616 with N-terminal GST , AmpR
This study
pacpP pETDuet 1-based expression vector for AcpP This study
pacpS pETDuet 1-based expression vector for AcpS This study
pACP pETDuet 1-based expression vector for AcpP and AcpS This study
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pLpxA pETDuet 1-based expression vector for free tagged-LpxA This study
pAasS pET28a expression vector for AasS with C-terminal six
histidines
This study
plpxA pUHE21-lacIq-based expression vector for lpxA This study
pBAD33-CSK29544_02616 pBAD33-based expression vector for CSK29544_02616 This study
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Table IV-2. Oligonucleotides used for the construction of strains and plasmids in Chapter IV
Oligonucleotide name Oligonucleotide sequences (5’ to 3’) Purpose
CSK29544_02616-lamb-F CGC GTG GTG GAT GCT ATC CGC GCG CAG GCC
GCG CTC GGC CTT GCG GAG AAA GTG GCA TGA
TGT AGG CTG GAG CTG CTT CG
SK011 construction
CSK29544_02616-lamb-R CGT CAT TGA CTA CTG CCG CTC CCA TGC CAA
CGA TAG CGT CGT TAC CGA TGT GGA TTT GCT
ATT CCG GGG ATC CGT CGA CC
SK011 construction
motA-lamb-F GGC AAC GTT TTC TGC TCA CCT GAA CAT CCT
CGC CAT AGC CAA CAG CGG AAG GAT GAT GTC
TGT AGG CTG GAG CTG CTT CG
Mutant construction
motA-lamb-R GCG ACA CGA AGC CGT AAG CCA GCA GGA TGC
CGA GGA ATG TCC CCA CCA TCG CGT GAG CGA
ATT CCG GGG ATC CGT CGA CC
Mutant construction
flhD-lamb-F GGC AAC GTT TTC TGC TCA CCT GAA CAT CCT
CGC CAT AGC CAA CAG CGG AAG GAT GAT GTC
TGT AGG CTG GAG CTG CTT CG
Mutant construction
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flhD-lamb-R GCG ACA CGA AGC CGT AAG CCA GCA GGA TGC
CGA GGA ATG TCC CCA CCA TCG CGT GAG CGA
ATT CCG GGG ATC CGT CGA CC
Mutant construction
motA-con-F GAA TGC TTG AAT TAT CGC GCT G Mutant construction
motA-con-R GTC TTA CGA CCA AAC TCT ACG Mutant construction
flhD-con-F CAC TGT TCA CCG ACA AGT C
flhD-con-R CAC TTT CCA GCA TCT GTA AAC
pBAD24- Hisx6- CSK29544_02616-F AAA GAA TTC ATG CAT CAT CAT CAT CAT CAC GGC
AGC GGC AGC GGC AGC GGC AGC ATG AAG CTT
GGC ATT TAC GGC G
pHis- CSK29544_02616
construction
pBAD24- Hisx6- CSK29544_02616-R AAA GTC GAC CAT CTT CAA TAA CAG ACA CCG
CA
pHis- CSK29544_02616
construction
pBAD24-seq-F GGA TCC TAC CTG ACG CTT TT Cloning confirmation
pBAD24-seq-R TTA TCA GAC CGC TTC TGC GT Cloning confirmation
pACYC184-CSK29544_02616-F-
SphI
AAA GCA TGC GGG AAA CGA CCG TTG TGG C pCSK29544_02616
construction
pACYC184-CSK29544_02616-R-
BamHI
AAA GGA TCC CCG GCA CTT CAA AGG TTT CC pCSK29544_02616
construction
pACYC184-seq-F CTA CTT GGA GCC ACT ATC GAC T Cloning confirmation
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pACYC184-seq-R TGT CCT ACG AGT TGC ATG ATA Cloning confirmation
pKT25-CSK29544_02616-F-BamHI AAA GGA TCC TGG CAT GAA GCT TGG CAT TT pKT25-CSK29544_02616
and pGST-
CSK29544_02616
construction
pKT25-CSK29544_02616-R-EcoRI ATA GAA TTC CCA TCT TCA ATA ACA GAC ACC pKT25-CSK29544_02616
and pGST-CSK29544_02616
construction
pKT25-con-F GCC ATT ATG CCG CAT CTG Cloning confirmation
pKT25-con-R CTT CGC TAT TAC GCC AGC Cloning confirmation
pUT18C- lpxA-F-SalI AAA GTC GAC CGT GAT TGA TAA GAC CGC CT pUT18C- lpxA construction
pUT18C- lpxA-R-BamHI AAA GGA TCC CGA CAA AAC GCG CGT TCG pUT18C- lpxA construction
pUT18C-con-F CGT TCG AAG TTC TCG CCG pUT18C- lpxA confirmation
pUT18C-con-R CTG GCT TAA CTA TGC GGC AT pUT18C- lpxA confirmation
pGST parallel 1-con-F CCA GCA AGT ATA TAG CAT GG Cloning confirmation
pGST parallel 1-con-R CAG GCT CTA GAT TCG AAA G Cloning confirmation
pETDuet1-lpxA-F-BamHI GAG GCC GGA TCC AGT GAT TGA pLpxA construction
pETDuet1-lpxA-R-HindIII CGT TCG GCA AGC TTG CTT T pLpxA construction
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126
pETDuet1-acpS-F-NcoI AAA CCA TGG CGA TTC TGG GGC TCG GTA CC pacpS construction
pETDuet1-acpS-R-EcoRI GAT CCC CAA CAC GAA TTC TAA GGA pacpS construction
pETDuet1-acpP-F-NdeI AAA CAT ATG AGC ACT ATC GAA GAA pacpP construction
pETDuet1-acpP-R-XhoI AAA CTC GAG GTA GAT ACT TGT GGG ACT AAA A pacpP construction
pETDuet1-LpxA-F-NdeI AAA CAT ATG ATT GAT AAG ACC GCC TTT pLpxA construction
pETDuet1-LpxA-R-XhoI AAA CTC GAG GAC CGG CAC CAA GAA TAT C pLpxA construction
pETDuet1-con-F TCT CGA TCC CGC GAA ATT AA Cloning confirmation
pETDuet1-con-R GGC CGT GTA CAA TAC GAT TA Cloning confirmation
pET28a-AasS-F-NcoI TTT CCA TGG ATA TGA ACC AGT ATG TAA ATG A pAasS construction
pET28a-AasS-R-XhoI TTT CTC GAG CAG ATG AAG TTT ACG CAG TTC pAasS construction
pUHE21-lpxA-RBS-F-EcoRI AAA GAA TTC AGG AGG ATA CGT GAT TGA TAA
GAC C
plpxA construction
pBAD33-CSK29544_02616-F-EcoRI TTT GAA TTC AGG AGG GGC ATG AAG CTT GGC
ATT T
pBAD33-CSK29544_02616
construction
lpxA-lamb-F TTA ACG AAT CAG ACC ACG CGT GGA ACG GGC
GAA GAA ATC GTA GAA CGG CTG CAC TTC AGG
TGT AGG CTG GAG CTG CTT CG
SK022 construcution
lpxA-lamb-R GTG ATT GAT AAG ACC GCC TTT ATT CAT CCC
ACC GCC ATT GTG GAA GAG GGT GCC ATT ATC
SK022 construcution
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ATT CCG GGG ATC CGT CGA CC
pUHE21-con-F AGA TTC AAT TGT GAG CGG ATA AC Cloning confirmation
pUHE21-con-R GGT CAT TAC TGG ATC TAT CAA CA Cloning confirmation
a Restriction enzyme sites and Hisx6-tag coding sequences are underlined and itaclicized, respectively.
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IV-3. Results
IV-3-1. A gene discovered in an invasion-attenuated mutant.
To identify genes related to the virulence of C. sakazakii, a
transposon-mediated random mutant library in C. sakazakii was screened to
find out invasion-defective clones, which showed more than 5 folds compared
to wild type. Three hundred clones were screened with an invasion assay, and
four clones showing defective invasion ability were found (Tn10, 24, 28, and
54). However, among them, a clone showing the most defective invasiveness
(Tn 54) was selected and analyzed further. After the boundary region between
the transposon and the C. sakazakii genome cut with EcoRV restriction
enzyme was PCR-amplified (inverse PCR, Fig. IV-1A) and sequenced
according to the manufacturer’s instructions, I found that the clone most
defective in invasion had the transposon insertion in a CSK29544_02616
region of DNA through nucleotide blast search of the transposon-flanking
region.
To confirm the transposon insertion site in the clone, primers were
designed using the boundary sequence, and the size of PCR-amplified region
was compared to that of wild type: the clone showed bigger size (Fig. IV-1B)
However, the function of CSK29544_02616 gene product has not been
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129
identified on the database.
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130
A B
Fig. IV-1. PCR confirmation of several mutants from invasion-screening.
(A) PCR products of inverse PCR from each denoted mutants.
(B) Lane M, the nucleotide size marker; Lane 1, WT band from Tn10 primer sets; Lane 2, a mutant band from Tn10 primer sets; Lane 3, WT
band from Tn24 primer sets; Lane 4, a mutant band from Tn24 primer sets; Lane 5, WT band from Tn54 primer sets; Lane 6, a mutant band
from Tn54 primer sets.
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IV-3-2. The CSK29544_02616 gene is required for C .sakazakii invasion.
To understand the roles of CSK29544_02616 in C. sakazakii ATCC
29544 invasiveness, I generated an unmarked mutant lacking the entire
CSK29544_02616 gene using the Lambda Red recombination technique. The
loss of CSK29544_02616 was verified by sequencing. When I compared the
growth of the strains in LB medium with constant aeration at 37°C, the
deletion mutant showed the same growth rate as the wild type (data not
shown). The plasmid carrying the CSK29544_02616 gene with its probable
cognate promoter region was also constructed. Before studying on
CSK29544_02616, the confirmation of its effect on the reduced invasion
ability was carried out. The CSK29544_02616 mutant showed 1,000
folds attenuated invasion ability into human–derived epithelial Caco-2
cells compared with wild type strain (Fig. IV-2), in consistent with the
screening data. Complementation with the plasmid harboring the
CSK29544_02616 gene fully restored the invasion ability of the mutant strain
to wild-type levels, substantiating that the possibility of polar effects and
secondary site mutations are excluded (Fig. IV-2).
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132
Fig. IV-2. CSK29544_02616 is required for C. sakazakii invasion into
intestinal epithelial cells.
The absence of CSK29544_02616 gene disrupted invasion ability of C.
sakazakii. Confluent monolayers of eukaryotic cells were infected with C.
sakazakii grown to the exponential phase and incubated for 1.5 h, followed by
gentamicin treatment (100 μg/ml) for a further 1.5 h. The Caco-2 cells were
lysed with 1% Triton X-100 to recover the intracellular bacteria. The data are
representative of 3 independent experiments. The bars represent the means
and SEM from independent experiments performed in triplicate. The asterisks
indicate significant differences (***, P < 0.001).
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IV-3-3. The CSK29544_02616 gene contributes to C .sakazakii virulence.
To explore whether CSK29544_02616 gene could contribute to C.
sakazakii virulence, I initially performed an in vivo challenge assay using
newborn rats as performed in other studies (Hunter et al., 2008;
Townsend et al., 2007b). To minimize individual variations between infant
rats and compare the fitness of the mutant to compete with wild type for
survival in the host environment, competitive assays were carried out by
orally inoculating the bacterial mixture containing equal numbers of
CSK29544_02616 mutant and wild-type strains. At 20 h post-infection, the
CSK29544_02616 deletion mutant was outcompeted by the wild type, with
CIs of 0.002 and 0.001 in the spleen and liver, respectively (Fig. IV-3A).
These results suggest that CSK29544_02616 gene is responsible for the
ability of C. sakazakii to disseminate into deeper organs. The attenuated
virulence of the CSK29544_02616 mutant might be attributable to multiple
defects during the infection processes. To make successful systemic infection
for pathogenic bacteria into hosts, they should actively either invade or be
phagocytosed into/by immune cells, and then survive within them (Sansonetti,
2001). To see the impact of CSK29544_02616 on both phagocytosis and
survivals by/within macrophages, survival assay was performed using
RAW264.7 (murine-derived macrophages). At first, I could see that
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134
phagocytosis of the mutant was about 100 folds significantly reduced than
that of wild type (Fig. IV-3B, uptake). Moreover, the CSK29544_02616
deletion mutants were cleared completely after 72 h post-infection,
whereas WT strains (approximately 3-log CFU/ml) were still able to
survive within macrophages at 96 h. A plasmid containing
CSK29544_02616 complemented the ability to survive within
macrophages (Fig. IV-3B).
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135
A
B
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136
Fig IV-3. CSK29544_02616 is required for the pathogenesis of C.
sakazakii.
(A) in vivo animal study reveal that the virulence of C. sakazakii was
diminished in the absence of CSK29544_02616. Two to three days old rat
pups were administered orally by a mixture of wild type and
CSK29544_02616::kanR strains with about 4 x 109 and 3 x 109 CFU/ml
respectively. To determine bacterial loads in spleen and liver, respect organs
were removed aseptically, homogenized at 20 h post-infection and plated onto
selective agar plates. Competitive index (CI) was calculated as follows:
ΔCSK29544_02616output / wtoutput÷ΔCSK29544_02616input / wtinput.
(B) The survival assay using RAW264.7 demonstrate the lack of
CSK29544_02616 is important for both phagocytosis and survival with the
macrophage cells. The procedure is described in Materials and Methods.
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IV-3-4. CSK29544_02616 directly interacts with LpxA.
I have seen that CSK29544_02616 is involved in the pathogenesis of
C. sakazakii, however, the function of CSK29544_02616 remained unknown
due to lack of information. To search for its function, I chose to find out its
binding partner and used ligand fishing. N-terminally Hisx6–tagged form of
CSK29544_02616 was well purified and the functionality of Hisx6–tagged
CSK29544_02616 was verified by swimming motility in the
CSK29544_02616 mutant (Fig. IV-4). A crude cell-free extract from C.
sakazakii ATCC 29544 was mixed with N-terminally Hisx6–tagged form of
CSK29544_02616 or no addition of the protein or the tagged-
CSK29544_02616 without a crude cell extract, and subjected to pull-down
assays by using a metal-affinity chromatograpy. I found a protein band
(approximate molecular weight of 28 kDa) specifically eluting in a fraction
containing His-CSK29544_02616 (Fig. IV-5). Peptide mapping of the protein
band indicated that it corresponds to the UDP-N-acetyl glucosamine
acyltransferase, encoded by lpxA (Wyckoff & Raetz, 1999).
To verify whether CSK29544_02616 interacts specifically with
LpxA in vivo, I used bacterial adenylate cyclase based two-hybrid system
based on the complementation of β-galactosidase activity of E. coli cyaA
mutant strain BTH101 (Karimova et al., 1998). CSK29544_02616 and LpxA
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138
were fused with T25 and T18 fragments of the adenylate cyclase catalytic
domain from Bordetella pertussis, respectively as shown in Fig. IV-6A. The
reporter strain E. coli BTH 101, which expressed the combination of the
hybrid protein: T25- CSK29544_02616/T18-LpxA, produced blue color in
LB broth containing X-gal (Fig. IV-6B), indicating an interaction between
CSK29544_02616 and LpxA.
This functional restoration of adenylate cyclase activity was further
quantitatively measured through a β-galactosidase assay. As shown in Fig. IV-
6C, the β-galactosidase activity of BTH 101 strain expressing the combination
proteins (T25-CSK29544_02616/T18-LpxA) was 50-60 folds much higher
than that of negative control strain, which harbors the plain T-25 and T-18
fragments only. Furthermore, basal level of the β-galactosidase activity was
detected in the strain containing one fusion protein only, supporting that the
interaction between CSK29544_02616 and LpxA specifically complemented
the β-galactosidase activity.
To corroborate the direct interaction between CSK29544_02616 and
LpxA, in vitro GST pull-down assay was performed. For the assay, I set two
groups: C (control), which mixed a cell lysate from the strain overexpressing
N-Hisx6–LpxA with that of the strain expressing GST, and E (experimental
group), which mixed a cell lysate from the strain overexpressing N-GST-
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139
CSK29544_02616 with that of the strain overexpressing N-Hisx6–LpxA (Fig.
IV-7A). These proteins were well overexpressed and solubilized in the buffer
(data not shown). As shown in Fig. IV-7B, group E only showed the band
when immunoblotted with anti-His antibody after GST pull-down, whereas
group C was not detected as immunoblotted with anti-His antibody. Taken
together, CSK29544_02616 and LpxA directly bind with each other.
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A B
Fig. IV-4. N-terminally Hisx6 –tagged CSK29544_02616 was successfully purified with its functionality.
(A) A gel image of N-Hisx6-CSK29544_02616 from SDS-PAGE.
(B) The confirmation that N-Hisx6-CSK29544_02616 has it functionality by recovering motility as that of wild type.
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Fig. IV-5. CSK29544_02616 was shown to be a binding partner of LpxA
from ligand fishing assay.
Ligand fishing assay was performed to find out protein binding partner(s).
The procedure is described in Materials and Methods.
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142
A
B
C
T25 02616 pKT25::CSK29544_02616
T18 LpxA pUT18C::lpxA
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Fig. IV-6. CSK29544_02616 specifically binds with LpxA in vivo.
(A) The construction of tagging plasmids for bacterial two hybrid based on
adenylate cyclase reconstitution. CSK29544_02616 was N-terminally tagged
with T25 fragment and LpxA was N-terminally tagged with T18 fragment.
(B) A bacteria two hybrid assay reveal the direct binding of CSK29544_02616
with LpxA by color change, when the indicated strains were in the presence of
X-gal.
(C) A bacterial two hybrid assay reveals the direct interaction of
CSK29544_02616 with LpxA. E.coli BTH101 reporter strains harboring the
indicated plasmid pair were subjected to β-galactosidase assay to
quantitatively measure β-galactosidase activity, calculated as Miller units.
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A
B
IB : immunoblotting
GST
pull-down
IB : anti-GST
Lysate
C E C E
IB : anti-His
GST : glutathione-S-transferase
pETDuet::lpxA
PT7-1
lpxA
pGST parallel 1
GST
PTac
+
pGST parallel 1::at
02616 GST
PTac
+ pETDuet::lpxA
His
PT7-1
lpxA
C (GST+His-LpxA)
E (GST- CSK29544_02616+His-LpxA)
Hisx6
Hisx6
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Fig. IV-7. CSK29544_02616 specifically binds to LpxA in vitro.
(A) Construction of strains used in in vitro GST pull-down assay; C, a mixed lysate of pETDuet::N-Hisx6-lpxA plus pGST parallel 1,
respectively. E, a mixed lysate of pETDuet::N-Hisx6-lpxA plus pGST parallel 1::CSK29544_02616, respectively.
(B) The cell lysate from each group C and E were incubated with glutathione-agarose beads for 3 h at 4 °C. After GST pull-down experiment,
the samples were analyzed by immunoblotting (IB) with the indicated antibodies.
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IV-3-5. CSK29544_02616 increases LpxA enzyme activity.
It was verified that CSK29544_02616 specifically binds with LpxA.
Because LpxA is a first enzyme of LPS biosynthesis that transfers β-
hydroxymyristoyl group to UDP-N-acetylglucosamine to make UDP-3-(R-3-
hydroxyacyl)-GlcNAc, at first, I checked the presence or absence of LPS in
CSK29544_02616 mutant. As shown in Fig. IV-11A, the lack of
CSK29544_02616 could still produce LPS as the same as that of WT.
Likewise, CSK29544_02616 had nothing to do with expression of lpxA in
transcriptional level, revealed by RNA-seq data (data not shown).
CSK29544_02616 mutation did not affect either LPS structure or expression
of lpxA, so I speculated that CSK29544_02616 may bind with LpxA to
modulate the enzymatic activity of LpxA either positively or negatively. To
figure it out, enzyme assay for LpxA was established by using fluorescent dye
(Thioglo-3) as described previously (Jenkins & Dotson, 2012). Before
performing the enzyme assay, I prepared all the substrates required for the
assay (Fig. IV-8).
I set the standard reaction condition for LpxA activity, including the
concentration of substrates. After adjustment of the standard reaction, I
checked the concentration of CSK29544_02616, which did not show any
enzymatic activity because CSK29544_02616 has bacterial transferase
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hexapeptide repeat domain, which most of acyl transferases have. As shown
in Fig. IV-9-B, CSK29544_02616 helped LpxA enzyme activity by increasing
reaction velocity in vitro. To verify if the increment of LpxA enzymatic
activity was caused by specific interaction between CSK29544_02616 and
LpxA, BSA (bovine serum albumin) was used for negative control. As a result,
the enzymatic activity of LpxA with BSA did not increase LpxA activity as
CSK29544_02616 did (data not shown). To further check whether
CSK29544_02616 increase LpxA activity or not in vivo, I tried to construct
lpxA mutant, which has been only reported in Neisseria meninhitidis and E.
coli in a conditional temperature, nonpermissive temperature (Galloway &
Raetz, 1990; Steeghs et al., 1998). Fortunately, I got the mutant having
lpxA/lpxA::kan, in turn, the mutant still has wild type copy of lpxA (Fig. IV-
10), as previously reported in N. meningitides (Zarantonelli et al., 2003). In
consistent with the previous data (Galloway & Raetz, 1990), mutation of lpxA
in E. coli showed the retarded growth compared with wild type. Likewise,
lpxA mutation in C. sakazakii also caused severe growth defect, however, the
introduction of lpxA into the mutant fully recovered its growth as the same as
wild type (Fig. IV-10B), suggesting the success of lpxA mutant construction in
C. sakazakii. With this mutant, I transformed the pBAD33::CSK29544_02616
to check its effect on the reduced lpxA activity. As expected, the defective in
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growth was comparable with that of wild type when the plasmid was induced
by 13.3 mM of arabinose, substantiating that CSK29544_02616 is involved in
increasing LpxA activity via direct interaction each other (Fig. IV-9C). Taken
together, CSK29544_02616 helps LpxA enzyme activity, verified by in vitro
enzyme assay and in vivo.
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A B
28 kDa →
35 kDa →
W1-3 200 mM 300 mM S F 1M NaCl M : marker
1 : Holo-ACP (1 st FPLC, Q column-anion exchange chromatography)
2 : Acyl-ACP (2 nd FPLC, Q column-anion exchange chromatography)
Ø Acyl -ACP
M 1 2
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C D
Fig. IV-8. Each substrate used for LpxA enzymatic assay was well purified.
(A) AcpP and AcpS, encoding apo-ACP and holo-ACP, respectively, were coexpressed in E.coli ER2566 without any tags. The holo-ACP was
purified using anion-exchange chromatography, followed by acylated by AaaS, acyl-ACP synthetase. The acyl-ACP was purifited by anion-
exchange chromatography.
20 mM 50 mM 250 mM S F 20 mM 50 mM 250 mM S F
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(B) Free tagged-LpxA was purified by Green-SeparoporE○R4B-CL.
(C) AasS, N-terminally Hisx6-tagged, was purified.
(D) CSK29544_02616, N-terminally Hisx6-tagged, was purified.
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A
B C
+
SH
ACP
SH
+ Thioglo-3 Fluorescence
λex
=378 nm
λem
=446 nm
ACP
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Fig. IV-9. CSK29544_02616 increases the enzymatic activity of LpxA.
(A) A schematic representation of LpxA enzymatic assay. A generated Thioglo-3-ACP containing free thiol-group (-SH) conjugated was
excited at 378 nm and measured at 446 nm, respectively.
(B) in vitro LpxA enzymatic activity depending on the presence of CSK29544_02616 or absence of CSK29544_02616. The activities were
presented in RFU value.
(C) in vivo LpxA activity depending on the various concentration of CSK29544_02616, ranging from 0 to 13.3 mM.
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① ② ③
M 1 2 1 2 1 2
A B
Fig. IV-10. lpxA mutant having lpxA/lpxA::kan was constructed.
(A) PCR-confirmation of lpxA::kan by using various primer sets; ①: forward and reverse primer from external region of lpxA, ②: K1 primer,
annealing to interal region of kan cassette, and the reverse primer from external region of lpxA, ③: the forward primer from external region of
lpxA and K2, annealing to inter region of kan cassette.
1 2 3 4 5 6 7 8 9 100
2
4
6WT
lpxA::kan
plpxA/lpxA::kan (0 mM)
plpxA/lpxA::kan (50 mM)
plpxA/lpxA::kan (100 mM)
plpxA/lpxA::kan (500 mM)
hour
OD
60
0
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(B) The complementation of lpxA::kan mutant by introduction of lpxA. The mutant strain harboring pUHE21-lacIq::lpxA was induced by IPTG
ranging from 0 to 500 μM.
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IV-3-6. CSK29544_02616 is involved in modulating the ratio of lipid A
and phospholipid.
Outer membrane is an asymmetric structure, composed of two
different substances, including phospholipids in the inner leaflet and lipid A in
the outer leaflet, which serve as a physical barrier for gram negative bacteria
(Kamio & Nikaido, 1976). In order to maintain cell integrity, the ratio of
between phospholipids and lipid A should in balance. In E.coli, the ratio
between them is reported to be 0.12 (Galloway & Raetz, 1990). The disrupted
balance affect outer membrane biogenesis by altering the composition of outer
membrane proteins (de Cock & Tommassen, 1996; de Cock et al., 1999;
Koplow & Goldfine, 1974; Ried et al., 1990; Sen & Nikaido, 1991).
I have checked that CSK29544_02616 specifically binds with LpxA
to synergistically help its enzymatic activity (Fig. IV-9B and C). In this regard,
I speculated that the lack of CSK29544_02616 might cause the decreased
production of LPS. To verify the hypothesis, I extracted LPS using hot
phenol-water extraction method. As expected, the resultant in Fig. IV-11A
showed that CSK29544_02616 mutant had the reduced lipid A-core region,
approximately 50% as quantified by imageJ, compared with wild type. It has
been well-known that the biosynthetic pathways, including lipid A and
phospholipids, utilize the same precursors, β-OH-myristoyl-ACP (Galloway
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157
& Raetz, 1990; Ogura et al., 1999; Zeng et al., 2013). Thus, I hypothesized
that the low level of LPS production in CSK29544_02616 mutation may
elevate the production of phospholipids, as previously reported in E. coli
(Kloser et al., 1998), which finally results in the unbalanced ratio between
phospholipids and lipid A followed by changed outer membrane proteins. To
check the hypothesis, I extracted total lipids and quantitated the amount of
phospholipids by measuring lipid phosphorous. As my expectations, the level
of phospholipids in CSK29544_02616 mutant showed approximately 20%
increment on average compared with wild type as shown in Table IV-3.
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IV-3-7. The altered balance between Lipid A and phospholipids in
CSK29544_02616 mutation influenced outer membrane assembly.
To check whether the changed ratio between Lipid A and
phospholipids cause outer membrane assembly, outer membrane proteins
were purified and resolved on SDS-PAGE to compare any differences
between wild type and the mutant. Indeed, the result in Fig. showed that some
bands are missing in the mutant, and some bands are more abundant in the
mutant (Fig. IV-11B). For identification of protein bands, liquid
chromatography orbitrap mass spectrometry was performed. Interestingly, the
black triangle, an abundant band in wild type, identified proteins involved in
flagellar biosynthesis, including fliC, flgG, and flgF encoding flagellin,
flagellar basal protein, and flagellar basal body rod protein, respectively. In
the black triangle, there are more proteins, but I excluded proteins, not
matched with the size (about 28 kDa). In contrast, the red triangle, an
abundant band in CSK29544_02616 mutant, identified fecA and fhuA,
encoding for iron(III) dicitrate transport protein and ferric hydroxamate outer
membrane receptor, respectively, in consistent with that the transcriptional
expression of fecA and fhuA was overexpressed about 6 and 8 folds,
respectively in RNA sequencing data (Table IV-4). Furthermore, the
expression level of other outer membrane proteins was shown to be changed
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159
(Table IV-4). In conclusion, the decreased level of LPS production caused by
CSK29544_02616 disrupts the proper ratio between Lipid A and
phospholipids, leading to altered outer membrane profiles by changing their
expression level and/or assembly.
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1 2 3
LipidA-core
A B
Fig. IV-11. CSK29544_02616 modulate the production of lipid A,
resulting in alteration of outermembrane assembly.
(A) Lipopolysaccharide (LPS) analysis. LPS was extracted by hot phenol-
water method, separated on deoxycholate (DOC) PAGE and visualized by
Pro-Q Emerald 300 Lipopolysaccharide Gel Stain Kit (Molecular Probes).
(B) The profile of outer membrane proteins of wild type (lane 1) and
ΔCSK29544_02616 (lane 2), respectively; M: marker. Ten μg of outer
membrane protein was loaded.
M 1 2
70
56 43
35
28
17
kDa
►
►
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Table IV-3. LPS and phospholipid contents of WT and
ΔCSK29544_02616
Strain LPSa (%) Phospholipidsa (%)
WT 100±6.1 100±6
ΔCSK29544_02616 54.7±6.1 123±8
aLPS and phosphate analyses for phospholipid determination were carried out
in duplicate and from equal wet weights of cells. LPS was quantified using
ImageJ from the gel image. Values for the parental strains were adjusted to
100% and mutant values were normalized accordingly.
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Table IV-4. The expression level of membrane protein from RNA
sequencing data
a The number of reads, which was normalized by RLE (relative log expression)
method. The red letters indicate overexpression compared with wild type.
Filtration criteria are as follows: p value was less than 0.05
Gene WTa ΔCSK29544_02616a Fold change (log2)
Outer memebrane proteins
fhuA 792.2 4534.64 2.52
lamB 1916.2 429.16 2.16
fecA 1436.12 12714.74 3.15
ompW 2342.55 135488.42 5.85
Inner membrane proteins
fepD 13.48 85.83 2.67
ycjO 29.97 118.21 2.08
mglC 40369.12 3313.95 3.61
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IV-3-8. Reduction of LPS production in ΔCSK29544_02616 increased cell
surface hydrophobicity and biofilm formation.
It has been previously demonstrated that the loss of LPS in G(-)
bacteria may be replaced with phospholipids, which facilitates hydrophobicity
of cell surface (Nikaido & Nakae, 1979). Recently, the relationship between
the degree of LPS presence and hydrophobicity of cell surface has been
verified that the lost LPS resulted in increased hydrophobicity of cell surfaces
in Pseudomonas aeruginosa (Al-Tahhan et al., 2000). In this regard, I
questioned whether CSK29544_02616 mutation affected cell surface
hydrophobicity or not. To confirm it, I performed hydrophobicity assay using
xylene. As expected, the mutant showed about 3 folds rise in hydrophobicity
as compared with that of wild type (Fig. IV-12A). The microscopic image of
WT and ΔCSK29544_02616 demonstrated that the mutant showed the rapid
cell autoaggregation than WT, suggesting increased hydrophobicity, in part,
caused the rapid cell autoaggregation as shown in Fig. IV-12B. Interestingly,
C. sakazakii in the absence of CSK29544_02616 elevated the performance to
form biofilm by approximately 4 folds higher than WT on abiotic surfaces in
static conditions (Fig. IV-12C). The higher ability to form biofilms in the
mutant is attributable to the increased hydrophobicity of cell surface and
autoaggregation.
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A B
C
30 min
WT
60 min
ΔCSK29544_02616
30 min 60 min
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Fig. IV-12. Increased lipid amount in outer membrane elevated hydrophobicity.
(A) Hydrophobicity was measured using xylene extraction. The formula to calculate the hydrophobicity is as follows: [(H0-H)/H0]x 100.
(B) The image of wild type (upper panel and ΔCSK29544_02616 (lower panel), respectively, taken from optical microscope. The adjusted
OD600 of each cell was incubated in 1.5 ml e-tube for the indicated time, respectively. Then, the photos were taken at the indicated time point
from the samples.
(C) Biofilm formation. The assay using crystal violet was performed in both technical and biological triplicate. The biofilm formation is
quantitated by the absorbance at 570 nm from the WT, ΔCSK29544_02616, and ΔCSK29544_02616 harboring pCSK29544_02616 plasmid.
Each value was normalized to that of WT.
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IV-3-9. The loss of CSK29544_02616 abrogated flagella-mediated motility.
Previous reports have demonstrated that flagella formation and
motility/chemotaxis are dependent on composition of membrane
phospholipids, including cardiolipin (CL) and phosphoethanolamine (PE) in E.
coli (Inoue et al., 1997; Kitamura et al., 1994; Nishino et al., 1993; Shi et al.,
1993). In this regard, I speculated that the altered composition of lipid A and
phospholipids in outer membrane might cause flagellar formation and/or
motility. At first, I tested flagella-mediated motility using soft agar plate. An
aliquot of the strains (WT, ΔCSK29544_02616 ΔCSK29544_02616+p
CSK29544_02616) were injected onto 0.3% soft agar plate containing
chloramphenicol (25 μg/ml). The absence of CSK29544_02616 was shown to
be nonmotile, but the motility was fully restored upon complementation with
ΔCSK29544_02616+pCSK29544_02616 (Fig. IV-13A). To check whether the
loss of motility in the mutant was ascribed to a defect in flagellar assembly,
the photographs of the strains were taken by an energy-filtering transmission
microscope (EF-TEM; Libra 120, Germany) at a voltage of 120 kV. I found
out that the mutant had also flagella on its body as that of WT (Fig. IV-13B).
Interestingly, however, the flagella of the mutant seemed to be far thicker than
that of wild type. These abnormal flagella caused by CSK295444_02616
mutation may elicit paralyzed flagella, leading to nonmotility even the
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presence of flagella on its body surface. To further demonstrate the effect on
motility in the mutant, I performed suspension-clearing assays. As shown in
Fig. IV-13C, the wild type showed good stability in the suspension throughout
the time, whereas the mutant settled rapidly within 2 hour. However, the
complementing strain expressing CSK29544_02616 exhibited a better
stability in suspension than the mutant, but not as the same as wild type.
These data demonstrate that disruption of CSK29544_02616 resulted in the
loss of active movement to maintain bacteria in suspension. Taken all together,
CSK29544_02616 is involved in flagellar functionality.
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A B
1: WT
2: ΔCSK29544_02616
3: ΔCSK29544_02616+p02616
WT
1 μm 1 μm
ΔCSK29544_02616
1 μm
ΔCSK29544_02616 1
2 3
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C
Fig. IV-13. The loss of CSK29544_02616 caused paralyzed flagella.
(A) Motility on 0.3% agar plate.
(B) Confirmation of flagellar presence by transmission electron microscopy
(TEM).
(C) Suspension clearing assay. The rate of suspension clearance was measured
over 7 h by carefully removing 100 µl from the top of the suspension and
measuring the OD600.
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IV-4. Discussion
In this work, I sought to identify virulence factors associated with
invasion ability of C. sakazakii ATCC 29544. Among several targets from
screenings, One of the mutants showing the most reduced invasion ability was
found to have transposon insertion in the CSK29544_02661 encoding a
putative NeuD family acyltransferase. Due to the lack of information, I
decided to find out whom CSK29544_02616 plays with. Interestingly, LpxA,
encoding a very first enzyme involved in biosynthesis of LPS, was shown to
interact with CSK29544_02616. CSK29544_02616 did not affect expression
of lpxA, but modulate its enzymatic activity in a synergistic manner. In the
absence of CSK29544_02616, LpxA still has its activity at a slower velocity
than the presence of CSK29544_02616, which resulted in the reduction of
LPS production compared with wild type. When it comes to that LPS plays a
key role in disrupting tight junctions of intestinal epithelial cells, which
function as physical barriers from bacterial invasion (Kim & Loessner, 2008),
and C. sakazakii invades into host cells through either apical side or
basolateral side (Kim et al., 2010), the highly attenuated invasiveness in
CSK29544_02616 mutant may be, in part, attributable to the reduced LPS
production.
The loss of LPS was compensated by a concomitant increase in the
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171
amount of phospholipids as shown in Table IV-3. In N. meningitis, lpxA
mutant changed the composition of phospholipids, especially PE
(phosphoethanolamine), the most abundant species of phospholipids (ca.
75%). As the fatty acyl chains of lipid A are generally saturated and shorter,
the shorter and saturated fatty acyl chains of phospholipids were increased to
compensate for the membrane fluidity caused by LPS deficiency (Steeghs et
al., 2001). In this respect, the reduction in LPS production may alter the
composition of phospholipid species. Overall, the ratio between LPS and
phospholipids was shown to be about 1:2.5 as compared with that of wild type
(1:1), which made cell surface more hydrophobic. This elevated
hydrophobicity caused by CSK29544_02616 deletion facilitated cell
autoaggregation shown in Fig. IV-12B. It has been reported that macrophages,
required for both innate and humoral immune defense against pathogenic
bacteria, recognize their size and shape. In the case of rod-shaped bacteria, the
attachment of bacteria-mimic particles is decreased at 3 μm in diameter,
suggesting that the degree of phagocytosis from macrophages is dependent on
the bacteria size (Doshi & Mitragotri, 2010). The optical microscopic image
showed the occurrence of cell aggregation after 30 min (Fig. IV-12B). In
survival assay (Fig. IV3-B), CSK29544_02616 deletion mutant showed 100
folds impaired phagocytosis than wild type. The time for measuring
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172
phagocytosis was 45 min, indicating cell autoaggregation occurred. In this
respect, the decreased phagocytosis may be ascribed to increment in cell size
caused by cell autoaggregation. In addition, the mutant was shown to be more
rapidly cleared out compared with wild type. This may be, in part, resulted
from the impaired phagocytosis in CSK29544_02616 mutant. Alternately, it
has been reported that C. sakazakii can persist for 96 h within human-derived
macrophage cells by harnessing the expression of inflammatory cytokines,
including interlukin-10 (IL-10, an anti-inflammatory cytokine) and interlukin-
12 (IL-12, a pro-inflammatory cytokine). C. sakazakii induces type II immune
responses from macrophages, the state that macrophages decrease
inflammation, but encourage anti-inflammation presented by their metabolism
(Townsend et al., 2007b). Thus, ΔCSK29544_02616 may possibly fail to
harness the ratio between IL-10 and IL-12, which resulted in turning them
into type I state. Those attenuations in invasiveness, phagocytosis, and
survival ability contributed to the colonization and dissemination into the
deeper organs, including the spleen and the liver.
The knock-out mutant has thicker flagella on it body than wild type,
in turn, a paralyzed flagella, which may cause nonmotility. I hypothesized that
nonmotility of CSK29544_02616 mutation might be driven by torque
generation. To validate this hypothesis, I used CR3 bacteriophage, targeting
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173
flagella as a receptor for the infection (Shin et al., 2012b) and motA deletion
mutant. In E.coli, motA encoding an integral membrane protein is necessary
for flagella torque generation. It is not needed for flagellar assembly, but
required for motility by rotating flagella. (Braun et al., 1999). CR3 could not
infect flgK deletion mutant, lacking flagellin, and motA deletion mutant,
suggesting phage CR3 requires not only flagellin, but flagellar torque;
interestingly, it could infect CSK29544_02616 mutant, which suggests that
CSK29544_02616 does not affect flagellar assembly and torque generation
(data not shown). It is of interest to note that the whole gene clusters involved
in flagellar structure biosynthesis (hook-basal body) were shown to be
overexpressed compared with WT, which may be driven, in part, by
unbalanced membrane phospholipid compositions. In other words, the altered
balance between lipid A and phospholipids may possibly cause the changes of
phospholipid species, particularly cardiolipin, supported by overexpression of
gens involved in cardiolipin biosynthesis (data not shown). As the previous
data demonstrated the deficiency of acidic phospholipids repress flagellar
biosynthesis (Inoue et al., 1997; Kitamura et al., 1994), the increased
cardiolipin may affect flagellar formation.
Taken together, the lack of CSK29544_02616 had C. sakazakii
nonmotile even having intact flagellar structure and torque generation.
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174
Therefore, I reasoned that this phenomenon may possibly be caused by
biased-rotation, but further study needed to be performed.
LpxA catalyzes the transfer of R-3-OH-myristoyl group of R-3-OH-
myristoyl-ACP to UDP-GlcNAc to produce UDP-3-O-(R-3-
hydroxymyristoyl)-GlcNAc. In Fig. IV-9, CSK29544_02616 elevated the
enzymatic reaction of LpxA by directly binding to it. It has been demonstrated
that the enzymatic reaction of LpxA is thermodynamically unfavorable, in
turn, it is a reversible reaction. Although thioesters are energy-rich compounds
that S→O acyl transfer is normally thermodynamically favorable, LpxA
reaction is in contrary to this notion (Anderson et al., 1993). I hypothesized
that CSK29544_02616 helps LpxA activity to increase because the interaction
may elicit conformational change of LpxA structure, which may either gain
access to UDP-GlcNAC, one of substrates, with ease or prevent the forward
reactions from the reverse reactions. This mechanism further needs to be
elucidated to answer the question how CSK29544_02616 specifically binds to
LpxA and help its activity.
In Fig. IV-9, LpxA itself without CSK29544_02616 still has its
enzymatic activity. I questioned that why Cronobacter genus has highly
conserved CSK29544_02616 homologs, unidentified in other genus. As the
major transmission vehicle of Cronobacter genus is reconstituted PIF, derived
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175
from either intrinsic or extrinsic contaminant including under poor good
manufacturing (GMP) or reconstitution of PIF (Singh et al., 2015), I reasoned
that the LPS has something to do with the tolerance to osmotic stresses, such
as dried infant formula. A previous report investigated that the role of outer
membrane lipopolysaccharide, especially O-antigen, in the protection of
S.Typhimurium from desiccation stress (Garmiri et al., 2008). Given that
polysaccharides has the ability to retain water and/or to slow down both the
rate of cellular water loss and uptake, O-antigen may function as a osmotic
protectant (Dudman, 1977). For this reason, I postulated that to resist under
dried infant formula would be the plausible speculation. In addition,
endotoxin, LPS, was shown to be prevalent in powdered infant formula at
elevated levels, which enhanced the ability of C. sakazakii to invade into host
cells (Townsend et al., 2007a), substantiating that 10 folds higher incidence
rate of necrotizing enterocolitis (NEC) was observed in infants fed with PIF
than breast fed infants (Iversen & Forsythe, 2003)
In summary, these data indicate that CSK29544_02616 plays a
critical role in pathogenesis of C. sakazakii by modulating the enzymatic
activity of LpxA through the direct interaction.
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176
fliR fliQ
fliP
fliO
fliN fliM
fliL fliK
fliJ fliI
fliH fliG fliF
fliE flhB flhA
flhE flgJ
flgI flgH flgG
flgF
flgE flgD
flgC
flgB
flgA
A B
OM
IM
T3SS
Motor/switch C ring
MS ring
Proximal rod
L ring P ring
Distal rod Proximal rod
Basal
body
RLE
500 1000 5000
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Fig. IV-14. C. sakazakii in the absence of CSK29544_02616 overexpressed
gene clusters encoding hook-basal body structures.
(A) Heat-map analysis. Filtered genes were plotted by sequencing reads,
normalized by RLE, between WT and ΔCSK29544_02616. Genes encoding
flagellar hook-basal body structure were listed in the left with italics.
(B) A schematic illustration showing flagellar structures (Chevance & Hughes,
2008). The purple-colored stars indicate overexpressed genes in
ΔCSK29544_02616.
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178
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국
Cronobacter sakazakii (크 노 사카자키)균 면역체계가 약한
신생 또는 면역 이 약한 · 를 주 하여 사 장염,
증, 뇌 막염 같 질병 일 키는 회 감염 병원 균 높
사 나타낸다. 이러한 명 병원 에도 불구하고 병원
커니즘과 병원 인자들에 한 연구는 미 한 실 이다. 라 ,
본 연구에 는 Cronobacter sakazakii 주인 C. sakazakii ATCC 29544
체 연구 병원 과 새 운 인자를 규명하고 그 조
커니즘에 한 연구를 행하고자 하 다.
체 게놈 시퀀싱 통해 C. sakazakii ATCC 29544는 4.6 M 규모
크 모좀 (chromosome) 하나 3 개 플라스미드 (plasmid)를
체 가지며 4,620 개 개 해독틀 (open reading frame, ORF)
가진다. 본 연구에 는 C. sakazakii 균 감염 매개 경 가 개
원우 (reconstituted milk)인 감 하여, C. sakazakii 균이 건조한
분 경 조건하에 생존에 여하는 자들 주 침입시
병원 일 키는데 요한 독 인자들에 해 분 하 다. 체
체 분 결과, C. sakazakii 는 삼 충격에 할 있는
막 다당 (capsular polysaccharide) 합 자, 생 막 (biofilm)
하는 구 요소인 cellulose 합 자들 가지며, 분 에
존재하는 시 산 에 지원 이용할 있는 nanKTAR 페
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(operon) 가진다. 또한, 분 에 존재하며 균에 독한 질인
산염 원시 산염 만드는 산염 항 페
(arsCBADR) 가진다. 이러한 특징 인 자들 C. sakazakii가
주 침입시 매개체 쓰이는 분 원우 (건조 경) 내
에 생존 능 에 여할 것 사료 다. 또한, C. sakazakii 는
외막 단 질인 ompA (장상피 포 침 ) ibeB-homolog cusC
(brain microvascular endothelial cell, BMEC 침 ) 자를 가지며, 이
는 공 인 병원 첫 단계인 주 포 침입에 여할 것
사료 다. 주 포 침입 후 주 내 경 C. sakazakii 생존
에 명 인데, 특히 생 학 지닌 철 (iron) 가용 생
장에 필 인 요소이다. 이러한 철 가용 높이 해 C.
sakazakii는 siderophore (iucABCD/iutA) ABC-type iron 송 시스
(eitCBAD) 가진다. 본 연구에 는 C. sakazakii ATCC 29544 주
체 게놈 시퀀스 분 통해 병원 인자 커니즘에 한 이
해를 높이는데 여했다.
다양한 병원 균주에 병원 자 진 hfq (RNA-
binding protein)를 타 하여 C. sakazakii 내 역할에 해
규명하 다. 이 자는 사 에 결합하여 사후 과 에
자 사 조 한다. hfq 자가 결여 균주
는 장 상피 포 침 능 식 포 내에 생존 능 이
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매우 감소하며 에 도 그 병원 이 히 감소 는 것 인
할 있었다. 또한 식 포가 생산하는 산소종 (ROS) 하
나이며 균에 산 스트 스를 가하는 하이드 퍼 사이드 (H2O2)
존재하에 생존 능 이 감소하는 것 인 하 다. 미롭게도,
다른 병원 hfq 결여 균주 는 다르게, C. sakazakii hfq 결여 균주는
편모 생합 자들 사 량 편모 생
(flagellation)이 히 증가 었고, 이는 과도한 운동 (hypermotility)
래하 다. 결 , hfq 자는 C. sakazakii 병원 에
요한 역할 하는 것 인 하 다.
주 포 침 에 여하는 새 운 인자 (invasion)를 찾
해, C. sakazakii random mutant libarary를 구축하 다. 스크리닝 통해
침 능이 가장 많이 감소한 클 (clone) 별하여 자 결여
를 인 결과, CSK29544_02616 자에 트랜스포존 (transposon)
삽입 인 하 다. 이 자는 연구 지 미지 단 질
이며 Cronobacter 속에 높 단 질 열 상동 보존 어 있
인 하 다. CSK29544_02616 자가 C. sakazakii 병원 에
미 는 향 인 하고자 CSK29544_02616 자 결여 균주를
구축하고 병원 스트 연구를 행하 다. CSK29544_02616
자 결여 균주는 장 상피 포 침 능, 식 포에 한 식균작
용 식 포 내 생존능, 편모 매개 운동 이 모 감소 었
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며, 에 도 그 병원 이 히 감소 었다. CSK29544_02616
자 분자 능 증명하 해 단 질 구 도 인 보를
토 능 추를 시도하 지만, 보 부재 인해 실 하 다.
라 , 본 연구에 는 CSK29544_02616 자 능 추하
해 ligand fishing 시도한 결과, LpxA (LPS lipid A를 생산 하는
데 여하는 효소)가 CSK29544_02616 단 질과 결합함 인하
다. CSK29544_02616 단 질 LpxA 결합하여 효소 높이는
것 있었다. CSK29544_02616 자 결여 균주에 는 LpxA
효소 이 감소 함에 라 LPS 생 량이 감소했고 이는 인지질
(phospholipid) 생 량 증가시 다. LPS 인지질 (phospholipid)이
외막 구 하는 요소임 감 하면, LPS 생 감소는 인지질 생
증가에 해 보 있었다. LPS는 다당체를 함 하고
있는 과 소 동시에 지닌 질이다. 외막에 감
소하고 인지질 증가 인해 C. sakazakii cell surface 소
(hydrophobicity)이 증가 어 autoaggregation이 일어났다. 이 게 증가
cell autoaggregation 생 막 (biofilm) 증가시킴 인 하
다. 또한, LPS 인지질 간 진 balance는 외막 단 질 구
에 향 미쳤다. 미롭게도, CSK29544_02616 자 결여 균주
는 편모를 가짐에도 불구하고 운동 이 없었다. 라 ,
CSK29544_02616 자는 LpxA 효소 조 함 써 C.
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sakazakii 생장 해 없이 병원 에 요한 역할 하는 병원
인자 써 항 균 써 가능 인하 다.
결 , 본 연구에 는 게놈 분 과 분자 연구를 통해 C.
sakazakii 병원 에 한 이해를 높이고 병원 자 특
과 커니즘 규명하여 C. sakazakii 감염 구축 한
다지는 연구를 행하 다.
주요어 : 크 노 사카자키 (Cronobacter sakazakii), 지놈 시
퀀싱 (genome sequencing), hfq (RNA-binding protein),
CSK29544_02616, 병원 , LpxA,
학 번 : 2010-23444