chapter 4: co-cultivation of a. polyphaga and l. monocytogenes

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Chapter 4: Co-cultivation of A. polyphaga and

L. monocytogenes

4.1 Introduction

Processes for intra-cellular invasion, dissemination and survival of L. monocytogenes in

mammalian cells have been well characterized using in vitro studies (Cossart, 2002;

Cossart and Sansonetti, 2004; de Chastellier and Berche, 1994). However, there is little

information about ecology of this intra-cellular pathogen in natural habitats. For example

there is no information to confirm whether this bacterium lives as a free organism or in

complex with other micro-organism including single cell protozoa. Given the ubiquity and

survival of L. monocytogenes over a wide range of natural habitats, it is hard to explain

how this bacterium has maintained its genetic capability for intra-cellular invasion and

maintenance of an intra-cellular lifestyle in mammalian cells.

The role of free-living amoebae in survival of intra-cellular bacterial pathogens and in

the environmental and their potential role as environmental reservoirs have been

investigated (Harb et al., 2000; Molmeret et al., 2005). Studies of the interaction of intra-

cellular pathogenic bacteria with free-living amoebae such as Acanthamoeba spp., has

gained a significant value since in vitro studies revealed amoebae could act as

environmental reservoirs (Greub and Raoult, 2004; Matz and Kjelleberg, 2005; Winiecka-

Krusnell and Linder, 1999). A number of well-known intra-cellular bacterial pathogens

including Salmonella enterica, Mycobacterium avium and Legionella pneumophilia are

able to survive and multiply within Acanthamoeba spp. (Bozue and Johnson, 1996; Gaze et

al., 2003; Steinert et al., 1998). Although the interaction L. pneumophilia with amoebae

has been well studied, much remains to be uncovered about the role of amoebae in

transmission and survival of other bacterial pathogens.

The interaction between L. monocytogenes and amoebae is less clear. Ly and Müller

(1990) carried out the preliminary study on the interaction of L. monocytogenes with single

cell protozoa. These authors argued that in co-culture with protozoans, L. monocytogenes

can survive or may multiply within these protozoa. Their work provided the first reports of

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the potential interaction of L. monocytogenes with single cell protozoa. However, detailed

study of the interaction between L. monocytogenes and Acanthamoeba spp. and the

potential role of the amoebae as an environmental reservoir for L. monocytogenes has yet

to be elucidated. For example, it is not clear the bacteria can multiply within amoebae, or

grow saprophytically on materials released from amoebae. Further, there is no

unequivocal information that describes the fate of intra-amoebic L. monocytogenes. It is

not known for example, whether phagocytosed L. monocytogenes cells are confined within

vacuoles or whether they can gain access to the cytoplasm of predator amoebae, as is the

case for mammalian cells. Furthermore, it is not known whether these bacteria can kill

amoebae or whether the reverse is true.

As described in Chapter 3 of this thesis, environmental isolates of L. monocytogenes

are apparently more likely to carry large plasmids than clinical isolates. Moreover, the

plasmid sequence analysis showed that the large plasmid found in L. monocytogenes

DRDC8 encoded for a number of important genes, including two cation transporters, ctpA

and cadA, which may play important roles in survival of bacteria in environmental niches.

Therefore, potential of free-living amoebae as environmental reservoir for

L. monocytogenes and the impact of plasmid-associated genes on interaction of bacteria

and amoebae needs to be investigated.

This chapter describes studies of the interaction between L. monocytogenes with

A. polyphaga at the cellular and sub cellular levels. The work provides the first evidence

of the impact of plasmid-associated determinants on interaction of L monocytogenes with

A. polyphaga.

4.2 Experimental Approach

The following approaches were used to study the interaction of A. polyphaga with

L. monocytogenes:

1. Growth of amoeba trophozoites on different bacterial lawns spread on NNA plates.

2. Co-cultivation assays for A. polyphaga and L. monocytogenes in 24-well trays and

in 75 cm2 flasks.

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3. The impact of plasmid associated determinants on interaction of L. monocytogenes

with A. polyphaga was assessed.

4. Fluorescence and TEM analysis of amoeba monolayers infected with bacteria was

carried out to identify the fate of intra-amoebic bacterial cells.

5. Promoter::GFP transcriptional fusions were constructed and used to assess whether

key genes are expressed by L. monocytogenes cells located within amoeba hosts.

4.3 Results

4.3.1 Construction of promoter::gfp Transcriptional Fusions

Sequence data for putative promoter regions of three L. monocytogenes strain 4b

(GenBank Accession NC 002973) genes; positive regulatory factor A (prfA), antibiotic

resistance gene B (abrB) and atpI, were identified from Listeria specific genomic

sequences available in the GenBank Nucleotide Database. The prfA gene product

regulates expression of gene products required for establishment of an intra-cellular life

style within mammalian cells (Cossart, 2002; Vega et al., 1998). The abrB gene is similar

to that encoded by Bacillus subtilis and is apparently expressed during the stationary phase

of bacterial growth (O'Reilly and Devine, 1997). The atpI gene is a constitutively

expressed gene found in most bacteria and is involved in ATP biosynthesis and turnover.

Prokaryotic atp operons encode the cell membrane F-type ATPase (ATP synthase) that

mostly contains nine genes for the ATPase, atpIBEFHAGDC (Kasimoglu et al., 1996;

McCarn et al., 1988). This operon is carried by Gram-positive such as Bacillus

pseudofirmus and Gram- negative bacteria such as E. coli. atpI is the first gene in this

polycistronic operon, and is assumed to be co-expressed with the other downstream genes

in this operon (Hicks et al., 2003).

The sequence data was used to design oligonucleotide primer pairs that allowed PCR

amplification of the promoter regions for each of the three genes. Restriction endonuclease

recognition sites were encoded in the 5’ ends of each primer to facilitate directional

cloning of the amplified DNA into a vector carrying a promoterless gfp. The

promoter::gfp transcriptional fusions were finally subcloned into a shuttle vector (pAT18)

and these constructs used to transform E. coli and L. monocytogenes strain DRDC8.

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Figure 4.1 outlines the construction of promoter::gfp constructs from PCR amplified

DNA encoding atpI, prfA and abrB promoter regions. Briefly, the amplified DNA

encoding the promoters, were cloned upstream of the promoterless green fluorescence

protein gene (gfp) in similarly digested pREP-GFP vector. This resulted in the creation of

pREP-abr (carrying the PabrB::gfp transcriptional fusion), pREP-prf (carrying the PprfA::gfp

transcriptional fusion) and pREP-atp (carrying the PatpI::gfp transcriptional fusion)

plasmids (Figure 4.1). Each construct was used to electro-transform competent E. coli

DH5α cells. Transformants were plated on LB agar containing chloramphenicol

(50 µg.mL-1

). Plasmid DNA prepared from selected chloramphenicol resistant

transformants was digested with BamHI and SphI to confirm that the vector contained an

insert of the correct size (data not shown). The promoter plus gfp gene fusion of each of

the constructed plasmids was sequenced to confirm the appropriate orientation of DNA

encoding the promoter with respect to the gfp gene and to identify the transcriptional

fusion joint (Figure 4.2). Resultant plasmids that contained appropriate constructs were

then used to transform E. coli DH5 to propagate and purified the plasmids. Only

transformants carrying the PatpI::gfp fusion expressed GFP when plated on LB agar

containing chloramphenicol. The colonies first appeared as normal white colonies, but

after 48 to 72 h incubation, gradually changed to a green colour. Colonies of tranformants

expressing GFP were usually smaller and stickier than those of the wild type host strain.

As the pREP-GFP shuttle vector could not be replicated by L. monocytogenes cells, the

promoter::gfp gene transcriptional fusions were subcloned into the E. coli –

L. monocytogenes shuttle vector pAT18 (Fortinea et al., 2000). Figure 4.3 outlines the

construction of these subclones. Oligonucleotide primer pairs were designed to amplify

the entire promoter::gfp fusion from plasmids pREP-abr, pREP-prf and pREP-atp (Figure

4.3). Each primer was designed to encode either a KpnI or a PstI restriction endonuclease

recognition site to facilitate directional cloning of each construct into the multiple cloning

site of pAT18. PCR products of the appropriate size were determined by gel

electrophoresis, purified, digested with KpnI and PstI, and ligated into similarly digested

pAT18 shuttle vector to create plasmids pAT-abrB, pAT-prfA and pAT-atpI (Figure 4.3).

The DNA encoding the promoter::gfp transcriptional fusions were deliberately cloned into

the multiple cloning site within the lacZ gene of pAT18 so that transcription of the gfp

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gene could be placed under the control of either the lac promoter or the cloned promoter.

Plasmids pAT-abrB, pAT-prfA and pAT-atpI, were then used to electro-transform

competent E. coli DH5 . Transformants were plated on LB agar containing erythromycin

(150 µg.mL-1

), IPTG and X-gal. White colonies (LacZ-) were selected and used to prepare

plasmid DNA. Restriction endonuclease digestion analysis was used to confirm presence

of insert DNA of the correct size (data not shown). PCR was also used to confirm

presence of the gfp gene in the isolated plasmid DNA. Incubation of transformants on LB

agar containing erythromycin and IPTG resulted in green colonies that produced a green

fluorescence when exposed to UV light (data not shown). This indicated that each

construct resulted in expression of GFP in E. coli when the constructs were under the

control of the lac promoter of pAT18. GFP expression was greatest for transformants

carrying the PatpI::gfp transcriptional fusion.

Plasmids pAT-abrB, pAT-prfA and pAT-atpI were purified from selected E. coli DH5

transformants and used to transform L. monocytogenes DRDC8 with selection on BHI

media containing erythromycin (10 µg.mL-1

). Transformants carrying the pAT-atpI

plasmid construct could not be isolated. L. monocytogenes DRDC8 cells transformed with

pAT-prfA (strain ADGP) and pAT-abrB (strain ADGA) were confirmed by restriction

endonuclease digestion analysis of plasmid DNA isolated from these transformants, and

using PCR amplification of gfp gene from plasmid DNA (Figure 4.4). These transformants

produced sticky colonies which were smaller than colonies produced by the DRDC8

parental strain. Furthermore, colonies of these transformed bacteria changed from white to

a green colour with extended storage at 4ºC and emitted green fluorescence characteristic

of GFP expression when exposed to UV light. These observations indicated the abrB and

prfA promoter::gfp transcriptional fusions were capable of driving GFP expression in

L. monocytogenes (data not shown).

4.3.2 Co-culture of A. polyphaga AC012 and L. monocytogenes

The interaction of A. polyphaga and bacteria has been extensively studied for a number

pathogenic bacteria and this amoeba has been shown to be capable of harbouring several

types of pathogenic bacteria (Lamothe et al., 2004). To test this observation for

L. monocytogenes, and to obtain detailed information about the cell biology of the

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interaction, co-culture experiments involving A. polyphaga strain AC012 with various

L. monocytogenes strains were carried out.

4.3.2.1 Co-culture of A. polyphaga AC012 on Lawns of Bacterial Cells

The ability of axenic A. polyphaga trophozoites to survive in co-culture with

environmental and clinical isolates of L. monocytogenes was assessed by placing an

inoculum of trophozoites on lawns of bacteria spread on NNA plates. Table 4.1 shows the

characteristics of the bacterial strains used for this experiment. Identically prepared plates

were incubated at 22, 30 and 37°C to assess the impact of temperature on the outcome of

the interaction of amoeba trophozoites with bacterial cells. Incubation temperatures of

22ºC and 30ºC were selected because these are representative of environmental conditions

normally experienced by A. polyphaga. An incubation temperature of 37ºC was chosen

because at this temperature, L. monocytogenes is known to optimally express its virulence

genes required for establishment of an intra-cellular lifestyle within mammalian cells.

Furthermore, L. monocytogenes cells express flagella at temperatures below 30ºC, whereas

no flagella are produced at temperatures in excess of 30ºC. All co-culture plates were

examined under an inverted microscope daily over a period of 3 d. The size of the amoeba

growth zones was measured and counts of amoeba trophozoites and cysts were estimated

qualitatively.

Figure 4.5a, b and c presents a summary of the results obtained for the co-culture

experiments. When co-culture plates were incubated at 22 and 30°C, the size of the

trophozoite growth zone and numbers of amoebae increased rapidly over a period of 3 d,

irrespective of whether the amoebae were cultured on lawns of L. monocytogenes or E.

coli. After 3 d, about 50% of amoeba cells had encysted (Figure 4.5a, b, c). Amoeba cells

had grown and multiplied at the initial site of inoculation on the centre of plates and

gradually migrated to marginal zones. After 3 d of incubation, amoebae completely

covered the whole NNA plate surface. No evidence was obtained to indicate killing of

amoebae by bacteria on plates or growth of bacteria. No bacterial colonies grew on the

non-nutrient medium at any stage of co-culture.

Growth of the amoeba resulted in a zone at the centre of plates of non nutrient media

that was apparently cleared of bacteria. Marginally different growth rates for A. polyphaga

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cells were observed when these cells were grown on lawns of different types of bacteria.

For example, A. polyphaga grew more rapidly when cultured on lawns of live E. coli than

when grown on lawns of heat-killed E. coli as previously reported (Wang and Ahearn,

1997). However, there was no apparent difference in estimates of growth rates when

cultured on lawns of different strains of L. monocytogenes.

Examination of the co-culture plates under a dissecting microscope showed no obvious

morphological differences were observed for trophozoite cells at any stage of incubation

on bacterial lawns. No enlargement of amoeba cytoplasmic vacuoles was observed, nor

was any amoeba cell debris found on the co-culture plates while the A. polyphaga cells

were growing in the presence of different types of bacteria (data not shown).

When A. polyphaga was co-cultured on lawns of bacteria with incubation at 37°C, few

trophozoites were observed, irrespective of the type of bacteria used for co-culture (Figure

4.5c). Conversely, A. polyphaga trophozoites commenced early encystation on the lawns

of bacteria and as a result stopped growing and multiplying.

4.3.2.2 Interaction between L. monocytogenes DRDC8 and A. polyphaga AC012

To specifically examine interactions between A. polyphaga and L. monocytogenes, a co-

culture assay that involved infection of monolayers of amoeba cells by L. monocytogenes

was developed. This assay allowed quantitative estimates the numbers of extra and intra-

amoebic bacteria, trophozoites and cysts. Furthermore, this approach allowed detailed

microscopic examination of the interaction of bacteria with individual amoeba cells (see

Section 4.3.2.5). Briefly, this assay relied on gentle centrifugation to increase the bacterial

touch with the surface of amoeba monolayers as a means of increasing the efficiency of

phagocytosis of bacterial cells. After a short incubation to allow phagocytosis of bacterial

cells, the infected amoeba monolayers were repeatedly washed with buffer to remove the

majority of extra-amoebic bacteria. Infected amoeba monolayers were then incubated at

defined temperatures for different periods of time. Numbers of intra- and extra-cellular

bacteria were estimated by plating on bacteriological media and counts of amoeba and

cysts determined using hemocytometry.

Preliminary experiments showed that washing infected amoeba monolayers 3× in AS

buffer could remove 99.99% of extra-amoebic bacteria (data not shown). This preliminary

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work confirmed similar results previously published by Bozue and Johnson (1996) and

showed washing was as effective as using gentamicin to kill extra-cellular bacteria. In

addition, this approach also reduced incubation times and ruled out any possibly adverse

effects of the antibiotic on both amoebae and internalised bacteria. Nevertheless, a small

proportion of extra-amoebic bacteria still remained associated with amoebae, and this

observation is consistent with previous reported studies.

Co-culture experiments involving L. monocytogenes and A. polyphaga AC012

demonstrated that the counts of viable intra-amoebic L. monocytogenes were significantly

reduced over a period of few hours at any of the three incubation temperatures used (15, 22

and 37°C). At 37°C and 22°C, co-cultivation was carried out for a period of 5 h without

addition of extra food to the co-cultivation media. As such, the amoebae were only able to

feed on L. monocytogenes. Counts of bacteria, trophozoites and cysts observed are

presented in Figure 4.6 and Figure 4.7. Counts of intra-amoebic bacteria decreased sharply

over the first 2 to 3 h of co-culture. The total count of bacterial cells (intra- plus extra-

amoebic bacteria) also decreased, although at a slightly slower rate during the period of co-

cultivation. In this context, the total number of bacteria represents intra-amoebic bacterial

cells plus bacterial cells that remain outside of amoeba cells during co-culture. After

incubation of amoeba monolayers in the presence of gentamicin and washing, a small

number of bacteria still remained and it is assumed that these may, in the absence of

antibiotic, grow outside cells. Some these extra-amoebic bacteria may have become prey

for amoebae so that over of the period of co-cultivation, the total number of bacteria was

decreased. Over the co-cultivation period there was no significant change in the number of

amoebae, albeit 20% of all amoeba cells were present as cysts.

Co-culture experiments conducted at 15°C required a longer incubation (4 d) to

quantitatively determine the fate of phagocytosed L. monocytogenes. To prevent starvation

stress on amoebae that might induce rapid encystation, heat-killed E. coli was added to the

co-cultivation daily for the 4 d of these experiments. As expected, the rate of decline in the

number of both total and intra-amoebic bacteria was slower than that observed when co-

cultures were incubated at higher temperatures (Figure 4.8). Intra-amoebic bacteria were

almost completely eliminated over 48 h post infection. Similarly, the total count of

bacterial cells was reduced by roughly 10 fold by the end of the co-culture experiment (4 d

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post infection). The number of amoebae in co-culture changed only slightly over the time

of experiment. Counts of amoebae decreased slightly over the first 24 h, followed by a two

fold increase over the next 24 h but remained essentially unchanged for the remainder of

the experiment. By the end of experiment, up to 25% of all amoeba cells were present as

cysts.

To show that the reduction in number of bacteria during co-culture was due to

predation by amoeba trophozoites and not due to other factors, the viability of

L. monocytogenes DRDC8 suspensions in different media with incubation at 15°C was

monitored. The suspending media used were AS buffer, AS buffer plus heat-killed E. coli

and ACM plus heat-killed E. coli. After 80 h incubation of L. monocytogenes suspensions

in AS buffer, the number of viable bacterial cells present had decreased by a factor of 100

compared to the number of viable cells at the commencement of the experiment (Figure

4.8b). However, when L. monocytogenes was suspended in AS buffer plus killed E. coli

and ACM plus killed E. coli, the number of viable L. monocytogenes increased 10 fold

relative to numbers at the commencement of the experiment. Furthermore,

L. monocytogenes grew more rapidly in ACM containing E. coli cells compared with AS

buffer containing E. coli cells. These data clearly showed that the reduction in number of

L. monocytogenes in co-culture with amoebae is not caused by loss of viability of the

bacterial cells, but rather active predation and killing by amoebae. In addition, the results

of these control experiments showed that factors released by amoebae into AS buffer can

stimulate growth of L. monocytogenes cells.

4.3.2.3 Prolonged Co-cultivation of L. monocytogenes and A. polyphaga AC012

Prolonged co-cultivation (up to 20 d at 30ºC) of bacteria with amoeba trophozoites

provided more information about the outcome of the interaction. During prolonged co-

culture, bacteria and amoeba cells compete to survive under conditions that resemble those

found in natural environments. L. monocytogenes DRDC8 and its avirulent hly mutant,

LLO17, were co-cultured with A. polyphaga cells in flasks containing AS buffer.

Amoebae were also cultured in AS buffer and AS buffer plus ACM as controls.

Figure 4.9a, b presents counts of bacteria, amoeba and cysts during co-culture of

L. monocytogenes with amoeba cells. Over the first 3 d of co-cultures, counts of bacteria

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decreased only slightly from ca. 2 × 106 CFU.mL

-1. After 4 to 6 d co-culture, counts of

both DRDC8 and LLO17 decreased (by 1,000 to 10,000 fold) to ca. 2 × 102 CFU.mL

-1.

Thereafter, the counts of bacteria decreased only marginally for the remainder of the

experiment. By contrast, counts of amoeba cells for both co-cultures gradually increased

from ca. 2 × 103 cells.mL

-1 and reached a maximum of 4.6 × 10

3 cells.mL

-1 after 6 d of co-

culture (Figure 4.9b). After 8 to 10 d, counts of amoeba decreased to ca. 2.2 × 103

cells.mL-1

and thereafter gradually decreased to ca. 1.1 × 103 cells.mL

-1. A gradual

increase in the proportion of amoeba cells converting from trophozoites to cysts also

occurred over the course of the experiment. The fact that the results of this experiment

was the same for both virulent, DRDC8, and it’s avirulent variant, LLO17, suggested that

the virulence status for mammalian cells has no obvious impact on the outcome of

interaction between L. monocytogenes and A. polyphaga.

When cells of both strains of bacteria were suspended in AS buffer alone, counts of

viable bacteria decreased from ca. 5 × 106 CFU.mL

-1 to ca. 10

2 CFU.mL

-1 over a period of

4 d (Figure 4.9b). The rate of decrease in viability was considerably greater than that

observed when these bacterial strains were co-cultured with A. polyphaga AC012 under

identical conditions. Similarly, when A. polyphaga AC012 trophozoites were suspended in

AS buffer, counts of these cells gradually decreased over the period of the experiment.

That gradual decrease coincided with a concomitant increase in the proportion of amoeba

present as cysts. However, when DRDC8 and LLO17 were suspended in ACM, the counts

of viable bacteria decreased over the first 4 d in a manner similar to that observed for co-

culture experiments described above. Thereafter, counts of viable bacteria decreased

gradually from ca. 104 CFU.mL

-1 to ca. 10

3 CFU.mL

-1.

Independent research has showed that the outcome interaction of Acanthamoeba spp.

with bacteria was dependent on the strain of bacteria used. For example, it has shown that

not all bacterial strains were capable of sustaining an intra-cellular life style within amoeba

cells (Marolda et al., 1999). Thus in order to rule out the possible impact of strain specific

factors on interaction of L. monocytogenes with A. polyphaga, two different clinical

isolates of L. monocytogenes (KE504 and KE1003) and one environmental turkey isolate

(2T) were co-cultured with A. polyphaga and the results obtained compared to those for

strain DRDC8. The results of these co-culture experiments are presented as Figure 4.10.

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Significantly, no major differences in the counts of bacteria were observed during co-

culture for any strain of L. monocytogenes used. Counts of bacteria gradually decreased

from ca. 108 CFU.mL

-1 to <10

3 CFU.mL

-1 over the first 8 d of co-culture, then decreased

only marginally thereafter. Counts of amoebae however, steadily increased from ca. 1.5 ×

103 cells.mL

-1 to ca. 3.5 × 10

3 cells.mL

-1 for the first 4 to 6 d of co-culture and gradually

decreased thereafter. The steady decline in counts of amoebae was matched by a steady

increase in the percent cysts. Furthermore, the changes in counts of amoeba cells and

percent cysts were essentially the same for all strains of bacteria used in co-cultures.

Given the similarity in changes in numbers of bacteria and amoeba, the outcome of

interaction between A. polyphaga and L. monocytogenes was therefore not influenced by

any of the panel of isolates of bacteria.

4.3.2.4 The Role of Plasmid-encoded Determinants in Interactions

Nucleotide sequence data presented in Chapter 3.3.2 showed that L. monocytogenes strain

DRDC8 carries a large plasmid which encodes several cation transport genes.

Furthermore, environmental isolates of L. monocytogenes in particular, also carry large

plasmid DNA. While the role of plasmid encoded genes for the ecology of environmental

strains of L. monocytogenes is not clear, previously described data (see Chapter 3.3.4)

showed that plasmid associated determinants in L. monocytogenes had no impact on the

outcome of intra-cellular survival within mammalian cells, at least in vitro. For

completeness, the potential role of plasmid-borne determinants on the outcome of the

interaction between A. polyphaga and L. monocytogenes was assessed by experiments that

compared the fate of a wild type strain (DSE201) and a plasmidless derivative (AAC1)

during co-culture with A. polyphaga AC012. This isogenic pair of bacterial strains was

used to separately infect monolayers of A. polyphaga prepared in 24-well trays at 22°C.

After washing out extra-amoebic bacteria, infected amoebae were incubated at 22ºC for up

to 7 h post infection. The total number of bacteria (intra-amoebic and extra attached) was

counted after lysis of amoeba cells and plating the diluted lysate on BHI plates.

Counts of viable DSE201 and AAC1 cells associated with amoeba monolayers declined

at similar rates over the period of the experiment (Figure 4.11). A. polyphaga successfully

eliminated phagocytosed L. monocytogenes cells irrespective the carriage of plasmid. This

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experimental outcome was confirmed by prolonged co-culture of AAC1 and DSE201 with

A. polyphaga. These bacteria were co-cultured with amoeba trophozoites in 75 cm2 flasks

contained AS buffer for 20 d at 30ºC. The MOI of co-cultures was 1000 bacterial cells per

amoeba cell. During the co-culture experiments, counts of AAC2 and DSE201 cells,

decreased by a factor of 105 (Figure 4.12). Furthermore, counts of DSE201 and plasmid-

cured AAC1 decreased at identical rates over the 20 d period of the experiment.

The results of these experiments indicated that plasmid associated genes did not have

any effect on the interaction between L. monocytogenes DSE201 and A. polyphaga.

Furthermore, the survival a small proportion of bacteria by saprophytic feeding on material

released from amoeba cells was also the same for plasmid cured AAC1 and wild type

DSE201. This conclusion is consistent with previous work with HeLa cells (described in

Chapter 3.3.4).

4.3.2.5 Microscopy of A. polyphaga co-cultured with L. monocytogenes

Co-cultivation assays of A. polyphaga with L. monocytogenes showed that amoeba cells

effectively eliminated phagocytosed bacteria located within amoeba trophozoites.

Furthermore, bacterial virulence genes apparently did not change the outcome of their

interaction with amoeba cells. This data suggested L. monocytogenes were not able to

survive within A. polyphaga cells, nor were they able to escape from phagolysosomal

structures and gain access to the cytoplasmic compartment of this amoeba. To more

precisely identify the L. monocytogenes - amoeba trophozoite cell interactions following

phagocytosis, the fate of internalised bacteria was determined using fluorescence

microscopy and TEM.

4.3.2.5.1 Fluorescence Microscopy

Fluorescently stained L. monocytogenes cells were internalized by most amoeba

trophozoites within a period of 2 h post inoculation of amoeba monolayers with bacteria

(Figure 4.13). The number of immuno-labelled intra-amoebic bacteria per amoeba cell

varied widely from one or two bacteria, to as many as fifty. A small percentage of

amoebae contained large numbers of immuno-labelled bacteria. While some immuno-

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labelled bacteria were apparently attached to amoeba surfaces, most intra-amoebic bacteria

were distributed as either single cells or clusters of cells apparently located in vacuoles.

Over a period of 5 h post co-cultivation, the number of immuno-labelled intra-amoebic

bacteria gradually reduced. By 5 h after commencement of the incubation period, no intact

intra-amoebic bacterial cells were observed (Figure 4.13). After 5 to 6 h, most amoebae

showed morphological changes characteristic of progression towards encystation. No

bacteria were observed within the cyst forms of amoeba cells (data not shown). These

results directly correlate with the bacteriological data described in Section 4.3.2.2.

4.3.2.5.2 Transmission Electron Microscopy

The results obtained by fluorescence microscopy showed that L. monocytogenes cells were

phagocytosed by amoeba trophozoites and apparently degraded intra-cellularly 2 to 5 h

post phagocytosis. A more detailed study of the fate of internalised L. monocytogenes cells

was obtained by examination the sections of infected amoeba cells by Transmission

Electron Microscopy (TEM). Micrographs typical of infected amoeba at different times

post infection are shown in Figure 4.14.

TEM of sections of amoeba cells infected with L. monocytogenes DRDC8 2 h post

infection, showed the bacteria were confined within tight vacuoles within the host amoeba

cytoplasm (Figure 4.14). Control sections are shown as Figure 4.14a. Each vacuole in

infected cells contained a single bacterial cell and in each case, the vacuole membrane was

in close juxtaposition to the bacterial cells (Figure 4.14c, d). Typically, vacuoles were

surrounded by a few mitochondria and a number of other vesicles, presumably lysosomes.

The cell wall of internalised bacteria was still intact with the thick peptidoglycan layer

typical of Gram-positive bacteria clearly visible (Figure 4.14d). No section of bacterial

cells within amoeba showed evidence of cell division. Further, none of the thin sections of

infected amoebae examined showed any evidence of electron dense material indicative of

actin polymerisation at the pole of bacterial cells. After 4 to 5 h post co-cultivation,

sections showed bacterial cells with degraded cell walls and loss of electron dense material

from the bacterial cytoplasm (Figure 4.14e, f). Furthermore, these sections showed large

numbers of lysosome-like vesicles surrounding vacuoles containing bacterial cells. Some

of the lysosome-like vesicles appeared to be in the process of fusing with the bacteria

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containing vacuoles (Figure 4.14e, f). These results indicated there was no evidence of

intra-amoebic survival or replication of L. monocytogenes in trophozoites or cystic forms

of A. polyphaga.

4.3.2.6 Use of GFP Expressing L. monocytogenes to examine Cell Interactions

Co-culture assay results described above showed that A. polyphaga eliminated intra-

amoebic bacteria within a few hours post infection. This occurred irrespective of whether

the strain of L. monocytogenes used had potential to express virulence genes such as that

encoding the listeriolysin protein necessary for invasion of the cytoplasmic compartment

of host cells. Further, immuno-fluorescence and TEM confirmed that phagocytosed

bacteria were degraded rapidly within amoeba cells. However, it was not clear whether

L. monocytogenes expressed virulence genes during the phagocytosis by amoeba cells. For

example, it was of interest to know whether the positive regulatory factor, PrfA, required

for virulence of L. monocytogenes in mammalian cells, was expressed during co-cultures

with amoebae. Similarly the expression of stationary phase genes was also of interest to

show the intra-amoebic bacteria.

To determine whether particular genes were expressed by the bacteria during different

stages of co-culture with amoebae, L. monocytogenes DRDC8 strains ADGP and ADGA

that carry two promoter::gfp transcriptional fusion constructs that allowed expression of

GFP under control of the prfA or the abrB gene promoters were used for co-cultures with

A. polyphaga. Expression of GFP by the recombinant L. monocytogenes could then be

detected using standard fluorescence microscopy without the need for indirect immuno-

fluorescence staining methods.

4.3.2.6.1 GFP Expression from promoter::gfp fusions

To first demonstrate that the promoter::gfp fusions were expressed by L. monocytogenes

strains ADGP and ADGA, cultures of these constructs were used to infect HeLa cells. The

L. monocytogenes-HeLa infection model has been characterised and expression of PrfA

dependent virulence genes required for establishment of an intra-cellular lifestyle in HeLa

cells has also been described (Francis and Thomas, 1996). Consequently expression of

GFP from the PprfA::gfp fusion was expected using this model, in which transcription of

121

prfA is required for expression of virulence genes such as hly, actA, plcA, plcB. As

expression of abrB by L. monocytogenes during infection of HeLa cells is unknown,

expression of GFP from the PabrB::gfp fusion was uncertain.

The ADGP and ADGA bacterial strains were used to infect HeLa cell monolayers

prepared in 25 cm2 tissue culture flasks and also in 24-well tissue culture trays as described

in Section 2.9.2. After washing to remove extra-cellular bacteria, the monolayers were

examined by an inverted microscope using a blue filter that allowed the fluorescent light

produced by excited GFP to be visualised (Figure 4.15). As expected, the bacteria inside

the cytoplasm of infected HeLa cells expressed GFP from both the abrB and prfA

promoters. GFP expressing cells emitted characteristic green fluorescence when excited

by UV. Location of GFP expressing cells within pseudopodia and evaluation of ability to

engage in intercellular spread was not evaluated. Similarly, formation of actin-based tails

by GFP expressing cells was not evaluated.

Interestingly, only a proportion of L. monocytogenes cells were found to express GFP.

Furthermore, GFP expressing cells that emitted fluorescence had atypical cell

morphologies; these cells were usually longer in length than non-GFP expressing cells.

Nevertheless, this result showed that bacteria expressed both prfA and abrB genes during

survival within HeLa cells.

4.3.2.6.2 GFP Expression by L. monocytogenes infected A. polyphaga AC012

L. monocytogenes strains ADGP and ADGA were also co-cultured with A. polyphaga

AC012 monolayers in 24 well trays and in 25 cm2 flasks. In contrast to the HeLa cell

infection model, neither strain expressed GFP within amoeba cells (Figure 4.16). This data

suggested that under the conditions of co-cultures used, neither prfA or abrB genes were

expressed. Apparently, L. monocytogenes cells were inactivated by A. polyphaga AC012

before the bacteria were able to express virulence genes necessary for access to the

cytoplasm of host cells. These results directly supported the data obtained from previous

co-cultivation experiments described in Section 4.3.2 as well as the results obtained by

microscopic examination of infected amoeba monolayers described in Section 4.3.2.5.

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4.3.3 Co-culture of A. polyphaga AC012 with S. Typhimurium C5

In view of the fact that A. polyphaga can rapidly kill phagocytosed L. monocytogenes cells,

it was of interest to determine whether this amoeba could also kill other types of intra-

cellular bacterial pathogens. Previous research has indicated that the intra-cellular

pathogen, Salmonella enterica serotype Typhimurium, is capable of survival and

multiplication within A. polyphaga (Gaze et al., 2003). Thus, to compare the fate of this

intra-cellular pathogen to L. monocytogenes during co-culture with A. polyphaga, S.

Typhimurium strain C5 was used in co-culture with monolayers of amoeba trophozoites.

Figure 4.17 shows the changes in numbers of viable intra-amoebic S. Typhimurium

during co-culture of this bacterium with A. polyphaga AC012. Counts of intra- amoebic

bacteria in co-culture with amoeba monolayers almost doubled over the 6 h duration of the

experiment. During the first 2 h post incubation, a slight reduction in counts of bacteria

was observed, which could reflect the initial killing of phagocytosed bacteria. Thereafter,

however, the number of bacteria gradually increased (by 3 fold) to the end of experiment.

This observation indicated S. Typhimurium C5 was capable of growth and multiplication

within amoeba.

Immuno-fluorescence microscopy of co-cultures of amoeba monolayers and S.

Typhimurium provided additional evidence to support the co-culture results. Micrographs

of amoeba at different times post infection are shown as Figure 4.18a–f. After 2 h of co-

culture, bacteria were internalised by amoeba trophozoites; an outcome similar to that

observed for L. monocytogenes. However, 30 h post inoculation of amoeba monolayers,

most amoeba cells contained clusters of internalised S. Typhimurium cells (Figure 4.18f).

This finding indicated that amoebae were not able to kill and clear internalised S.

Typhimurium cells. Conversely, these bacteria apparently resisted the killing mechanism

of amoebae and were able to multiply within amoeba host cells. This outcome is in direct

contrast to results obtained for co-culture experiments using L. monocytogenes and A.

polyphaga.

4.4 Discussion

Published research has shown that A. polyphaga can promote the intra-cellular growth of

Legionella pneumophila, S. Typhimurium, Simkania negevensis, Burkholderia cepacia and

123

Mycobacterium avium (Gao et al., 1997; Gaze et al., 2003; Kahane et al., 2001; Marolda et

al., 1999; Steinert et al., 1998). However, there are few studies of the interaction of L.

monocytogenes and protozoa, such as amoebae. Work published by Ly and Müller (1990)

is the only comprehensive study of co-cultivation of L. monocytogenes with two types of

amoebae, Acanthamoeba sp. (unknown species) and Tetrahymena pyriformis. They

concluded that L. monocytogenes could invade, and either survive, or may replicate within

infected amoeba cells. That work suggested that amoebae may act as a natural reservoir

for L. monocytogenes. However, the authors did not describe the fate of internalised

bacteria within amoeba cell at the cellular and sub cellular levels and did not control for the

effect of bacterial growth in the presence of conditioned media. The work described in this

chapter therefore, represents the first detailed study to clarify the role of A. polyphaga as a

potential environmental reservoir for L. monocytogenes and the interaction of

L. monocytogenes with free-living amoeba cells.

The substantial capability of A. polyphaga to grow and replicate on lawns of different

strains of clinical and environmental isolates of L. monocytogenes provided the first

evidence that amoeba trophozoites are capable of phagocytosing and killing different

isolates of bacteria. These results suggested that the outcome of bacterial interaction with

A. polyphaga is apparently independent on the strain of L. monocytogenes used. No

obvious changes to amoeba morphology occurred during growth on lawns of live or dead

bacterial cells. Also, no change in the size or number of cytoplasmic vacuoles was

apparent and cellular debris typical of dead amoeba cells was absent. Together, these

observations indicated internalised L. monocytogenes cells do not interfere with either the

growth or encystation of amoeba trophozoites.

The fact that amoeba grew on lawns of L. monocytogenes as well as viable and heat-

killed E. coli cells at any incubation temperature tested, suggested L. monocytogenes is

apparently not able to kill or stop the growth of amoeba trophozoites. Consequently it is

likely that A. polyphaga AC012 at least can predate L. monocytogenes cells and use these

bacteria as a food source. However amoeba trophozoites underwent early encystation,

without obvious growth, during co-culture with L. monocytogenes or E. coli (live and heat-

killed) at 37°C. This phenomenon is not due to bacterial activity on co-culture plates and

124

their probable effect on amoeba cells; rather it is related to physiological growth

requirement of amoeba cells.

These plate co-cultures observations are different from a report describing the co-

culture of Burkholderia cepacia with A. polyphaga (Marolda et al, 1999). These

researchers reported that during co-culture on plates, A. polyphaga grew on lawns of B.

cepacia, but amoebae delayed encystation because of interactions with bacteria. However,

the amoeba trophozoites developed large cytoplasmic vacuoles that contained live motile

bacteria. This research also showed the B. cepacia was capable of survival and

multiplication within large vacuoles in amoeba cytoplasm. Thus, the way B. cepacia and

L. monocytogenes interact with A. polyphaga cells on plates in different ways.

Similar work has also shown that S. Typhimurium in co-culture with A. polyphaga on

plates could survive and replicate within amoeba vacuoles. This work also reported that

bacteria secreted from vacuoles by amoeba trophozoites, formed colonies on the non-

nutrient co-culture medium, by utilizing nutrients released from lysed amoebae (Gaze et

al., 2003). These observations for S. Typhimurium on plate co-culture are clearly different

from the results obtained from palate co-culture of L. monocytogenes with A. polyphaga.

This data indicated, in contrast to S. Typhimurium, L. monocytogenes is not able to survive

or replicate within A. polyphaga trophozoites.

Quantitative assays that tracked the fate of L. monocytogenes following co-culture with

monolayers of A. polyphaga AC012, provided good evidence to corroborate the plate co-

culture data. These assays showed A. polyphaga was able to eliminate intra-amoebic

L. monocytogenes cells within 2 to 5 h post co-cultivation. Thus A. poluphaga AC012

trophozoites can kill L. monocytogenes shortly after internalisation, probably before these

bacteria can express proteins critical for access to host cell cytoplasmic compartments.

This contention is supported by data that showed that the avirulent listeriolysin mutant

(LLO17), is killed at the same rate as wild type virulent strains. Indeed A. polyphaga

AC012 was able to kill clinical and environmental strains of L. monocytogenes equally

well. Furthermore, the fact that total numbers of amoeba cells do not decline over the

duration of short term co-culture experiments (5 h) is additional strong evidence to show

amoeba trophozoites are not killed/lysed by L. monocytogenes following phagocytosis of

125

these bacteria. These results therefore directly contradict those published by Ly and

Müller (1990).

This conclusion is also supported by direct observation of the fate of L. monocytogenes

cells internalised by grazing A. polyphaga cells as well as other microbiological evidence.

The survival of L. monocytogenes in ACM suggested that the killing of bacteria by

amoebae in co-cultures is not due to the toxicity effects of materials released by amoeba

cells. Furthermore, the fact that L. monocytogenes loses viability only slowly when

suspended in non-nutrient medium indicated that the observed reduction of viable cells

during co-culture represents an active intra-cellular killing process. In addition, immuno-

fluorescence microscopy clearly showed loss of labelled bacterial cells after 4 to 5 h of co-

culture. This observation was confirmed by TEM. Indeed after extended co-culture, L.

monocytogenes cells could not be found within either trophozoites or cysts. These

observations therefore provide strong evidence to indicate that A. polyphaga trophozoites

can rapidly kill internalised L. monocytogenes cells over 2 to 5 h post co-cultivation.

In view of the strong bactericidal response of A. polyphaga AC012 trophozoites for

L. monocytogenes, it was of interest to determine whether this response was typical for co-

cultivations with other intra-cellular pathogenic bacteria. When S. Typhimurium was co-

cultivated with A. polyphaga, this Gram-negative pathogen was shown to be able to

survive within vacuoles following phagocytosis. Clearly S. Typhimurium possesses

molecular mechanisms that allow this bacterium to avoid killing by amoeba trophozoites

during both short and long term co-cultivation.

The results obtained following examination of thin sections of infected amoebae by

TEM provided a detailed account of the interaction of the bacteria with amoeba cell at

cellular and sub cellular levels. Phagocytosed bacteria are confined within phagosome in

amoeba cytosol; never escape into the cytosol as is the case for infected mammalian cells;

nor do they apparently undergo replication within the phagosomal vacuole. Rather, the

loss of the integrity of cell wall material indicated phagocytosed bacteria are degraded in

vacuoles within a short period of time (2 to 4 h) post phagocytosis. Interestingly,

phagosomes carrying L. monocytogenes cells were found to be surrounded by

mitochondria and small lysosome-like vesicles. The fact that several vesicles were noted

126

to be in the process of fusing with the phagosome, suggested these structures may play an

active role in degradation of internalised bacteria.

Prolonged co-culture of L. monocytogenes with amoeba cells in flasks provided

interesting information concerning outcome of interaction between bacteria and amoeba

cells. Given the significantly longer survival time of bacteria in co-cultures with amoebae

compared with cultures suspended in AS buffer, L. monocytogenes cells apparently benefit

from materials released from amoeba cells during encystation, non-microbial induced lysis

and growth. In fact L. monocytogenes is able to grow and multiply in ACM as reported for

B. cepacia and Mycobacterium ovium during co-culture with Acanthamoeba spp. (Marolda

et al., 1999; Steinert et al., 1998). Thus there is potential for L. monocytogenes to grow

and survive in the extra-cellular environment of grazing amoeba populations as long as

numbers of bacteria do not exceed critical levels needed for efficient predation by amoeba.

Consequently equilibrium populations of L. monocytogenes and amoeba may exist in the

environment.

Although different strains of B. cepacia interact with A. polyphaga in different ways,

not all strains of these bacteria are able to survive equally well within amoeba cells

(Marolda et al., 1999; Taylor et al., 2003). This is not the case for L. monocytogenes

however. To assess this phenomenon, a number of clinical and environmental strains of

L. monocytogenes were used in co-culture with amoeba under different culture conditions.

However, no difference in the rate of killing of different strains of bacteria by amoeba

trophozoites was observed. This data suggested A. polyphaga AC012 can kill

L. monocytogenes irrespective of strain characteristics. Furthermore, plasmid associated

genes do not change the outcome of bacterial interaction with A. polyphaga as this amoeba

is able to kill both plasmid-cured and wild type L. monocytogenes. Nevertheless, plasmid

encoded determinants do not provide significant benefit for bacteria. However, in the

extreme environmental conditions plasmid associated genes may play significant roles for

survival of bacteria. This speculation needs to be investigated under conditions that

resemble natural amoeba environments.

Use of GFP expressing strains of L. monocytogenes provided an additional means of

tracking the fate of these bacteria during co-culture. Furthermore, the use of strains in

which GFP expression was placed under control of different Listeria promoters provided

127

an opportunity to determine whether key genes were expressed during co-culture.

Importantly, when GFP expression was placed under control of the promoter responsible

for expression of the regulatory protein (PrfA) needed for expression of virulence factors

involved in intra-cellular spread of L. monocytogenes in mammalian cells, no GFP was

expressed by these strains during co-culture with amoeba cells. This outcome was in

contrast to results obtained when the same strain was used to infect HeLa cells. A similar

result was obtained when GFP expression was placed under control of the abrB stationary

phase gene promoter. These experimental outcomes provided strong evidence that protein

expression in L. monocytogenes cells is limited during co-culture with A. polyphaga

AC012. Indeed, it is possible that the strongly bactericidal effect of the amoeba

phagosomal environment disrupts the bacterial cell so quickly that any response of the

bacterial cell to the vacuolar environment is inadequate and comes too late to be of benefit

for the bacterium.

4.5 Conclusions

This study has for the first time provided detailed data describing the interaction of

L. monocytogenes with the free-living A. polyphaga. The main conclusions arising from

the work are:

1. A. polyphaga AC012 rapidly kill phagocytosed L. monocytogenes cells during co-

culture at temperatures from 15ºC to 37ºC. Consequently, it is unlikely that

A. polyphaga AC012 at least can act as an environmental reservoir for

L. monocytogenes.

2. A. polyphaga AC012 cells are able to phagocytose and kill clinical and

environmental strains of L. monocytogenes equally well.

3. The outcome of co-culture is not influenced by plasmid encoded genes carried by

L. monocytogenes strains.

4. Phagocytosed L. monocytogenes cells never escape from amoeba phagocytic

vacuoles into the cytoplasmic compartment of amoeba host cells.

5. Phagocytosed bacterial cells are rapidly degraded by A. polyphaga.

128

6. Killing of bacterial cells following phagocytosis is so rapid that L. monocytogenes

cells are apparently unable to express the major virulence gene regulator PrfA.

Expression of this regulator is required for production of proteins that may assist

survival within phagocytic vacuoles as is the case when these bacteria infect

mammalian cells.

129

Table 4.1: Characteristics of bacteria used for co-culture with A. polyphaga

AC012 on NNA plates.

Bacterial strain Characteristics

E. coli DH5Non invasive. Used either as a live

culture or as a heat killed suspension.

L. monocytogenes DRDC8Virulent for mice, ctpA positive,

contains plasmid DNA. Serotype 4.

L. monocytogenes DSE 201 ctpA mutant, contains plasmid DNA.

Serotype 4.

L. monocytogenes 2T No plasmid DNA, environmental isolate

L. monocytogenes KE 504 No plasmid DNA, clinical isolate

L. monocytogenes LLO 17 hly mutant, contains plasmid DNA.

Serotype 4.

L. monocytogenes ING 30 Contains plasmid DNA, ctpA negative.

130

1

pREP-GFP6551bp

Sp-Ori

erm

Ec-Ori

Cml

gfpMCS

iI

atpIabrBprfA

400bp

200bp

1

pREP-atp6950 bp

Sp-Ori

erm

Ec-Ori

cml

gfp

BamHI

SphI

1

pREP-abr6850bp

Sp-Ori

erm

Ec-Ori

cml

gfp

BamHISphI

Double digestion with

BamHI and SphI

Ligation

PRC products

purified, digested with

BamHI and SphI

PabrBPatpI

SphI BamHI

BamHI BamHI

1

pREP-prf6700bp

Sp-Ori

erm

Ec-Ori

cml

gfp

BamHISphI

PprfA

BamHISphISphI SphI

1

pREP-GFP6551bp

Sp-Ori

erm

Ec-Ori

Cml

gfpMCS

iI

atpIabrBprfA

400bp

200bp

1

pREP-atp6950 bp

Sp-Ori

erm

Ec-Ori

cml

gfp

BamHI

SphI

1

pREP-abr6850bp

Sp-Ori

erm

Ec-Ori

cml

gfp

BamHISphI


BamHI and SphI

Ligation

PRC products

purified, digested with

BamHI and SphI

PabrBPatpI

SphI BamHI

BamHI BamHI

1

pREP-prf6700bp

Sp-Ori

erm

Ec-Ori

cml

gfp

BamHISphI

PprfA

BamHISphISphI SphI

Figure 4.1: Construction of plasmids pREP-abr, pREP-atp and pREP-prf.

DNA encoding promoters for prfA (Pprf = 261 bp), atpI (Patp = 408 bp) and abrB

(Pabr = 309 bp) was PCR amplified from L. monocytogenes strain DRDC8. The PCR

products were purified, digested with BamHI and SphI and ligated to similarly digested

plasmid pREP-GFP to create promoter::gfp transcriptional fusions within plasmids

pREP-abr, pREP-atp and pREP-prf.

131

Figure 4.2: Nucleotide sequence of promoter::gfp transcriptional fusions.

DNA for the abrB, atpI and prfA genes promoters was amplified from L. monocytogenes

DRDC8. Each amplicon was directionally cloned into a HindIII site upstream of a

promoterless gfp gene cassette. The sequence for the cloned promoter region and the

HindIII fusion joint with gfp is shown. The position of the abrB, atpI and prfA start codons

and the gfp start codon are marked. The ribosome binding site upstream of gfp is also

marked.

Panel A: atpI::gfp fusion

Panel B: abrB::gfp fusion

Panel C: prfA::gfp fusion. The known -35 and -10 regions characterising the prfA

promoter are underlined and labelled. An inverted repeat characteristic of

the prfA promoter region is identified by arrows beneath the nucleotide

sequence.

132

A. atpI promoter

1 AATAATTCTG GCGTGGCAAT CTAAGAAACA GATAGATTTT GAAATCAAAA

51 TAGGTTTTCT CAACGATTAC GGAGGTGTTG GTTGCTGTTA AATGGACAAA

101 AAATCTCAAA AATAATAGGC TATTCTTTTA TGGATGGGTC CGATTGTGGT

151 ACTATTAGTA ATAGATGTAC TTACAATTTG TGAAAGTGTA TTGACTTTTC

201 TCTTTTGGCT GTTGTATGCT AAGAACTGAC ACGCTGATAA GGCTTTCATG

251 TGTGTTTGGG ATTGGGATGA ATTGGGGAAA ATGTGCTTGT TTTCACAAAC

301 CAAACTTACT CCCTGAGATT TCAAAATGGT CATCTGGAAA GAAAAATCTT

351 TTGAAAAAGA AAGGATTGCG CCCCGAATGT TAGAATCGCT AATTGGCATG

Start of atpI

401 GATCCCAAGA AGGAGAATAC ATATGAGTAA AGGAGAAGAA CTTT

HindIII RBS Start of gfp

B. abrB promoter

1 TCCGATTGCG AAAGCTAGTG CGCCGAATGT TGTACCAATG ATTTGCTGCC

51 TCGGTTTACC GCCAATTTTT GAAACAACTA ATGGCATAAT CCCCCACATC

101 ACTGCCGGAA TTAATGCGAT AACTATGTTC ATTTTTATTC CCCCTAATTC

151 TTCTGCTTAT GGTCTATACC CTTTTCCTCA TGAAATCATT CCTATTCCCG

201 TAATTATCTG TTAAAATGAA TTAAAATTAT CAAGAAGGTG AGAAAATGAA

Start of abrB

251 ATCAACTGGA ATGGTAAGAA AAATCGACGA ACTTGGTCGT GTGGTTATAC

301 CGATTGGATC CCAAGAAGGA GAATACATAT GAGTAAAGGA GAAGAACTTT


C. prfA promoter

1 CGAGCAACCA TCGGAACCAT ATACTAACCC TATTTCAATT TTAACATCTA

51 AATAAATCCG TTTTTAAATA TGTATGCATT TCTTTTGCGA AATCAAAATT

101 TGTATAATAA AATCCTATAT GTAAAAAATA TTATTTAGCG TGACTTTCTT

151 TCAACAGCTA ACAATTGTTG TTACTGCCTA ATGTTTTTAG GGTATTTTAA

Palindromic sequences -35 -10

201 AAAAGGGCGA TAAAAAACGA TTGGGGGATG AGACATGAAC GCTCAAGCAG

Start of prfA

251 AAGGATCCCA AGAAGGAGAA TACATATGAG TAAAGGAGAA GAACTTT


133

M

1500 bp

1000 bp

1

pAT18

6.6 Kb

orfE

oriRpAMB1

oripR pUC

erm

PstIKpnI

1

pAT-abrB

6.6 Kb erm

oriRpAMB1

oripR pUC

orfE

PstIKpnI

1

pAT-atpI

6.6 Kberm

oriRpAMB1

oripR pUC

orfE

PstIKpnI

1

pAT-prfA

6.6 Kberm

oriRpAMB1

oripR pUC

orfE

PstIKpnI


KpnI and PstI

Ligation

PCR products, purified,

digested with KpnI and PstI

lacZ

lacZlacZlacZ

prfA::gfpabrB::gfp atpI::gfp

abrB::gfp atpI::gfp prfA::gfp

PstIKpnI

PstIKpnI KpnI KpnI

PstIPstI

M

1500 bp

1000 bp

1

pAT18

6.6 Kb

orfE

oriRpAMB1

oripR pUC

erm

PstIKpnI

1

pAT-abrB

6.6 Kb erm

oriRpAMB1

oripR pUC

orfE

PstIKpnI

1

pAT-atpI

6.6 Kberm

oriRpAMB1

oripR pUC

orfE

PstIKpnI

1

pAT-prfA

6.6 Kberm

oriRpAMB1

oripR pUC

orfE

PstIKpnI


KpnI and PstI

Ligation

PCR products, purified,

digested with KpnI and PstI

lacZ

lacZlacZlacZ

prfA::gfpabrB::gfp atpI::gfp

abrB::gfp atpI::gfp prfA::gfp

PstIKpnI

PstIKpnI KpnI KpnI

PstIPstI

Figure 4.3: Construction of plasmids pAT-abrB, pAT-atpI and pAT-prfA.

PCR was used to amplify the promoter::gfp transcriptional fusions (PabrB::gfp, PatpI::gfp

and PprfA::gfp) from plasmids pREP-abr, pREP-atp and pREP-prf. Each PCR product was

purified, digested with KpnI and PstI and ligated with similarly digested pAT18 shuttle

vector to create plasmids pAT-abrB, pAT-atpI and pAT-prfA.

134

SP

PI (E

coR

I)

6.11

7.358.51

4.84

3.59

2.81

1.951.861.511.39

1.16

0.98

6.11

7.358.51

4.84

3.59

2.81

1.951.861.511.39

1.16

0.98

6.11

7.358.51

4.84

3.59

2.81

1.951.861.511.39

1.16

0.98

pA

T-a

br

(KpnI/P

stI)

ab

rB::gfp

prf

A::

gfp

pA

T-p

rf(K

pnI/P

stI)

SP

PI (E

coR

I)

Figure 4.4: Confirmation of transformants carrying pAT-arbB and pAT –prfA.

Plasmids pAT-arbB and pAT –prfA were isolated from L. monocytogenes DRDC8

transformants, digested with KpnI and PstI to release a vector fragment (~7 Kbp) and

insert. PCR amplified DNA specific for the promoter::gfp fusions (PabrB::gfp, PatpI::gfp

and PprfA :: gfp) carried by these plasmids are also shown.

135

Figure 4.5: Growth of A. polyphaga on NNA with L. monocytogenes strains.

L. monocytogenes strains used were: DRDC8 (Dairy isolate), 2T (Turkey isolate), KE504

(Clinical isolate), ING30 (Poultry processing plant isolate) and LLO17 (avirulent DRDC8

mutant) E. coli DH5 and heat-killed E. coli DH5 were used as control bacterial cultures.

Growth of A. polyphaga AC012 is reported as the diameter of amoeba growth zones on co-

culture plates. Data shown are the mean of 3 observations.

Panel A: Incubation at 22 ºC.

Panel B: Incubation at 30 ºC.

Panel C: Incubation at 37 ºC.

136

1 2 3 1 2 30

15

30

45

60

75

90E. coli

DRDC8

2T

KE504

Killed E. coli

ING30

LLO17

0

25

50

75

100

A: 22 °C

1 2 3 1 2 30

15

30

45

60

75

90

0

25

50

75

100

B: 30 °C

1 2 3 1 2 30

15

30

45

60

75

90

0

25

50

75

100C: 37 °C

Duration of co-culture (d)

Dia

mete

r of am

oebic

gro

wth

zone (m

m)

Perc

ent c

ysts

137

Figure 4.6: Counts L. monocytogenes and A. polyphaga during co-culture at 22°C.

Monolayers of A. polyphaga AC012 cells in wells of tissue culture trays were infected with

bacteria (MOI=50 bacterial cells per amoeba cell), followed by incubation at 22°C for 1 to

2 h. After removal of extra-amoebic bacteria by washing with AS buffer, the infected

amoebae were incubated at 22°C for up to 5 h. Individual wells were processed to

determine the total viable count of bacteria, numbers of amoebae and percentage of

amoeba cysts. Counts of amoeba cells and cysts were carried out using hemocytometer to

determine numbers of amoebae per mL culture and percent cysts respectively. Counts of

bacteria were determined by plating on BHI medium. Data shown are the mean of 3

replicates. Error bars represent the standard deviation about the mean counts.

Panel A: Counts of the total number of viable bacteria (intra- and extra-amoebic

bacteria) and intra-amoebic bacteria.

Panel B: Counts of amoeba and percent cysts.

138

A

0 1 2 3 4 51.0×101

5.1×102

1.0×103

1.5×103

2.0×103

2.5×103

Total number of bacteria

Intra-amoebic bacteria

CF

U m

L-1

B

0 1 2 3 4 51.0×104

4.0×104

7.0×104

1.0×105

1.3×105

1.6×105

Number of Amoebae

0

25

50

75

100

Percent Cysts

Duration of co-culture (h)

Am

oe

ba

e m

L-1

Pe

rce

nt c

ysts

139

Figure 4.7: Counts of L. monocytogenes and A. polyphaga during co-culture at

37°C.


L. monocytogenes DRDC8 (MOI= 50 bacterial cells per amoeba cell), followed by

incubation at 37°C for 1 to 2 h. After removal of extra-amoebic bacteria by washing with

AS buffer, the infected amoebae were incubated at 37°C for up to 5 h. Individual wells

were processed to determine the total viable count of bacteria, numbers of amoebae and

percentage of amoeba cysts. Counts of amoeba cells and cysts were carried out using

hemocytometer to determine numbers of amoebae per mL culture and percent cysts

respectively. Counts of bacteria were determined by plating on BHI medium. Data shown

are the mean of 3 replicates. Error bars are the standard deviation about the mean counts.

Panel A: Counts of total number of viable bacteria (intra- and extra-amoebic bacteria)

and intra-amoebic bacteria.

Panel B: Counts of amoeba and percent cysts.

140

A

0 1 2 3 4 51.0×101

2.0×103

4.0×103

6.0×103

8.0×103



CF

U m

L-1

B

0 1 2 3 4 51.0×104

6.0×104

1.1×105

1.6×105

2.1×105

Number of amoebae

0

25

50

75

100

Percent cyst


Am

oeb

ae m

L-1

Perc

en

t cysts

141


15°C.


L. monocytogenes DRDC8 (MOI= 100 bacterial cells per amoeba cell), followed by


AS buffer, the infected amoebae were incubated at 15°C for up to 100 h. Individual wells

were processed to determine the total viable count of bacteria, numbers of amoebae and

percentage of amoeba cysts. Counts of amoeba cells and cysts were carried out using

hemocytometer to determine numbers of amoebae per mL culture and percent cysts

respectively. Counts of bacteria were determined by plating on BHI medium.

Panel A: Mean counts of bacteria, amoebae and percent cysts during co-culture. Data

shown are the mean of 3 replicates. Error bars are the standard deviation

about the mean counts.

Panel B: Viability of L. monocytogenes DRDC8 suspensions in AS buffer, AS buffer

containing heat killed E. coli DH5α and, AS buffer plus heat killed E. coli plus ACM. Counts of bacteria were determined over a period of 144 h post

inoculation. Data shown are the mean of 3 replicates. Error bars are the

standard deviation about the mean counts of bacteria.

142

A

0 20 40 60 80 100100

101

102

103

104

105

106



Number of amoebae

Percent cysts

CF

U m

L-1

B

0 24 48 72 96 120 144102

103

104

105

106

107DRDC8 in AS+ACM+Killed E.coli

DRDC8 in AS+Killed E.coli

DRDC8 in AS


CF

U m

L-1

143


22ºC.

L. monocytogenes DRDC8 and the avirulent variant LLO17, were cultured in flasks

containing 25 mL AS buffer and A. polyphaga AC012 trophozoites at MOI of 1000

bacterial cells per amoeba cell. Identical suspensions of bacteria were prepared in AS and

AS plus ACM as controls. Counts of viable bacteria were determined by plating on BHI

agar. Counts of amoeba cells and cysts in 10 to 20 microscopic fields (100 times

magnification) was used to determine numbers of amoebae per mL culture and percent

cysts respectively.

Panel A: Counts of viable L. monocytogenes DRDC8 and LLO17 during co-culture.

The viability of suspensions of each strain of bacterium in AS buffer alone

was used as a control. Counts shown are the mean of 3 replicates. Error

bars represent the standard deviation about the mean counts of bacteria.

Panel B: Mean counts of amoeba cells and percent cysts during co-culture with

L. monocytogenes strains DRDC8 and LLO17. Suspensions of amoebae in

AS buffer were used as a control. Error bars represent the standard deviation

about the mean counts of trophozoites and percent cysts. Closed symbols

represent the number of amoeba cells and open symbols represent the

precent of encysted amoebae.

144

A

0 4 8 12 16 20101

102

103

104

105

106

107

DRDC8 in coculture

DRDC8 in AS

DRDC8 in ACM

LLO17 in coculture

LLO17 in AS

LLO17 in ACM

CF

U m

L-1

B

0 4 8 12 16 201.0×102

1.1×103

2.1×103

3.1×103

4.1×103

5.1×103

AC012 in AS

AC012 in DRDC8 co-culture

AC012 in LLO17 co-culture

Cysts in AS

Cysts in DRDC8 co-culture

Cysts in LLO17 co-culture

0

20

40

60

80

100


Am

oe

ba

e m

L-1

Pre

ce

nt c

ysts

145

Figure 4.10: Counts of L. monocytogenes and A. polyphaga during co-cultivation at

22ºC.

L. monocytogenes strains DRDC8, KE504, KE1003 and 2T were added to flasks

containing 25 mL AS buffer and A. polyphaga AC012 trophozoites at a MOI of 1000

bacteria per amoeba cell. Counts of viable bacteria were determined by plating on BHI

agar. Counts of amoeba cells and cysts in 10 to 20 microscopic fields (100 times

magnification) was used to determine numbers of amoebae per mL culture and percent

cysts respectively.

Panel A: Counts of viable L. monocytogenes during co-culture. Counts shown are the

mean of 3 replicates. Error bars represent the standard deviation about the

mean counts of bacteria.

Panel B: Mean counts of amoeba cells and percent cysts during co-culture with

L. monocytogenes strains. Error bars represent the standard deviation about

the mean counts of trophozoites and percent cysts. Closed symbols represent

the number of amoeba cells and open symbols represents the precent of

encysted amoebae.

146

A

0 4 8 12 16 20102

103

104

105

106

107

108

109

DRDC8 in co-culture

KE504 in co-culture

KE1003 in co-culture

2T in co-cultureCF

U m

L-1

B

0 4 8 12 16 200

1.0×103

2.0×103

3.0×103

4.0×103

AC012 in DRDC8 co-culture

AC012 in KE504 co-culture

AC012 in KE1003 co-culture

AC012 in 2T co-culture

Cysts in DRDC8 co-culture

Cysts in KE504 co-culture

Cysts in KE1003 co-culture

Cysts in 2T co-culture

0

25

50

75

100


Am

oe

ba

e m

L-1

Pe

rce

nt c

ysts

147

1 2 3 4 5 6 71.0×105

2.0×105

3.0×105

4.0×105

5.0×105

6.0×105

7.0×105

8.0×105

DSE201

AAC1


CF

U m

L-1

Figure 4.11: Counts of plasmid cured L. monocytogenes during co-culture with

A. polyphaga at 22°C.

Monolayers of A. polyphaga AC012 in 24 well trays were infected with L. monocytogenes

DSE201 and the plasmid cured variant AAC1, at MOI of 100 bacterial cells per amoeba

cell. The trays were incubated at 22°C for 1 h followed by washing with AS buffer to

remove extra-amoebic bacteria. Infected amoebae were incubated at 22°C. Total viable

counts of L. monocytogenes (intra and extra-amoebic bacteria) were determined by plating

on BHI. Data shown represents the mean of 3 replicates. Error bars represent the standard

deviation about the mean counts of bacteria.

148

0 4 8 12 16 20102

103

104

105

106

107

108

AAC1 in co-culture

DSE201 in co-culture


CF

U m

L-1

Figure 4.12: Counts of L. monocytogenes DSE201 and AAC1 during prolonged co-

culture with A. polyphaga at 22ºC.

of

L. monocytogenes DRDC8 and plasmid cured AAC1 were added to flasks containing 25

mL AS buffer and A. polyphaga AC012 trophozoites at a MOI of 1000 bacteria per

amoeba cell. Counts of viable bacteria were determined by plating on BHI agar. Counts

shown are the mean of 3 replicates. Error bars represent the standard deviation about the

mean counts of bacteria.

149

Figure 4.13: Localisation and survival of L. monocytogenes within A. polyphaga

during incubation at 22°C.

Monolayers of A. polyphaga AC012 were infected with L. monocytogenes DRDC8 at an

MOI of 100. After washing to remove extra-amoebic bacteria, the preparations were

incubated at 22ºC for 0, 1, 2, 3, 4 and 5 h. Monolayers were fixed and FITC immuno-

labelled using antibodies specific for bacterial cells. Immuno-labelled preparations were

examined with a fluorescent microscope. Bar marker = 20 µm.

Panel A: Time = 0 h. Immuno-labelled bacteria attached to or located within amoeba

cells.

Panel B: Time = 1 h. Two amoeba cells, with one cell containing clusters of immuno-

labelled bacterial cells.

Panel C: Time = 2 h. Immuno-labelled bacterial cells within vacuoles in amoeba

cells.

Panel D: Time = 3 h. Density of immuno-labelled cells is reduced compared with

preparations examined after 1 or 2 h incubation (Panels B and C). Note the

indistinct margins of the bacterial cells compared with micrographs shown

in Panel C.

Panel E: Time = 4 h. Most amoeba cells are clear of immuno-labelled bacterial cells.

Panel F: Time = 5 h. As for Panel E.

150

A B

DC

E F

A B

DC

E F

151

Figure 4.14: TEM of L. monocytogenes cells within vacuoles of A. polyphaga.

Monolayers of A. polyphaga AC012 were infected with L. monocytogenes DRDC8 cells at

an MOI of 50 bacteria per amoeba cell and incubated at 22ºC for1 h. Extra-amoebic

bacteria were removed washing followed by incubation at 22ºC for 0, 1, 2 and 4 h.

Infected amoeba cells were prepared for examination by TEM.

Panel A: Control amoeba cells 2 h post washing. Cells were processed identically

except that no bacteria were added. Note the large number of vacuoles

located within the amoeba cell.

Panel B: Infected amoebae 2 h post washing. Note the bacterial cell located within

vacuole (arrow).

Panel C: Infected amoebae 2 h post washing. Note the intact bacterium (B) located

within a vacuole.

Panel D: As for Panel C except at higher magnification. Note the intra-cellular

bacterial cells (B) with intact bacterial cell wall (BW) and the vacuolar

membrane (M).

Panel E: Phagocytosed bacteria (B) within vacuoles 4 h post washing. Note the loss

of a distinct cell wall compared with that shown in Panels C and D.

Panel F: As for Panel E except at higher magnification. Note the small lysosome-like

vacuolar structures (L) surrounding the vacuole containing a bacterial cell.

152

2 µm

1 µm

A

0.5 µm 0.5 µm

0.5 µm

2 µm

B

C D

E F

V

B

B B

BB

B

B

M

M

BW

ML

L

M

2 µm

1 µm

A

0.5 µm 0.5 µm

0.5 µm

2 µm

B

C D

E F

2 µm

1 µm

A

0.5 µm 0.5 µm

0.5 µm

2 µm

B

C D

E F

VV

B

B B

BB

B

B

M

M

BW

ML

L

M

153

Figure 4.15: Expression of GFP by L. monocytogenes strains ADGP and ADGA

when grown in cell culture medium and HeLa cells.

The monolayers of HeLa cells were infected by bacteria at MOI of 100, followed by a brief

centrifugation and incubation at 37ºC for 3 h. After the removal extra-cellular bacteria by

washing in PBS buffer the cells were fixed with formalin and examined by fluorescent

microscopy.

Panel A: Uninfected HeLa cells (control) by fluorescence microscopy.

Panel B: Uninfected HeLa cells (control) by bright light microscopy.

Panel C: Expression of GFP by ADGA bacteria in cell culture medium.

Panel D: Expression of GFP by ADGP bacteria in cell culture medium.

Panel E: Expression of GFP by ADGA in co-culture with HeLa.

Panel F: Expression of GFP by ADGP in co-culture with HeLa.

154

A B

C D

E F

A B

C D

E F

155

Figure 4.16: Expression of GFP by L monocytogenes strains ADGP and ADGA

during co-culture with A. polyphaga.

Monolayers of A. polyphaga AC012 were infected by L monocytogenes strains ADGP and

ADGA for 1 h at 22°C, washed to remove free bacteria and incubated at 30°C for 4 h.

Samples of infected amoebae were examined by phase contrast (left) and fluorescence

microscopy (right). GFP expression by L. monocytogenes cells was not observed for any

preparation examined. Bar = 20 µm.

Panel A: Amoebae co-cultured with ADGP (prfA::gfp) expression)

Panel B: As above

Panel C: Amoebae co-cultured with ADGA (abrB::gfp)

Panel D: As above

156

A B

DC

A B

DC

157

0 1 2 3 4 5 61.0×103

1.0×106

2.0×106

3.0×106

4.0×106

Number of bacteria


CF

U m

L-1

Figure 4.17: Counts of S. Typhimurium C5 during co-culture with A. polyphaga at

22°C.


S. Typhimurium C5 cells (MOI = 100 bacterial cells per amoeba cell) followed by


AS buffer, the infected amoebae were incubated at 22°C for up to 6 h. Total viable counts

of bacteria were determined for each monolayer preparation. Results shown are the mean

of 3 replicates. Error bars represent the standard deviation about the mean counts obtained.

158

Figure 4.18: Localisation and survival of S. Typhimurium within A. polyphaga.

Monolayers of A. polyphaga AC012 on cover-slips were infected with S. Typhimurium C5

cells at an MOI of 100. After washing to remove of extra-amoebic bacteria, the monolayers

were incubated at 30ºC for 0, 2, 4, 6 and 30 h post washing. Monolayers were fixed and

FITC immuno-labelled using antibodies specific for S. Typhimurium cells. Immuno-

labelled preparations were examined with a fluorescent microscope. Bar marker=20 µm

Panel A: Time = 0 h. Low magnification micrograph of amoeba cells with associated

S. Typhimurium cells.

Panel B: As for Panel A. Note the S. Typhimurium cells on the surface of the

amoeba cells and also the larger number located with in the amoeba cells.

Panel C: Time = 2 h. Many S. Typhimurium cells are located within cell of the

amoeba host cell.

Panel D: Time = 4 h. Note the clumps of S. Typhimurium cells within the amoeba

cell.

Panel E: Time = 6 h. Clumps of S. Typhimurium within an amoeba cell.

Panel F: Time = 30 h. Most amoeba cells are filled with S. Typhimurium cells.

159

A B

C D

E F

A B

C D

E F

chapter 4: co-cultivation of a. polyphaga and l. monocytogenes

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