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The gene tia, harbored by the SE-PAI, is involved in the ability of LEE-negative STEC strains to invade monolayers of epithelial cells
P. Chiani, R. Bondì, V. Michelacci, F. Minelli, S. Morabito
European Union Reference Laboratory for E. coli, Istituto Superiore di Sanità, Rome Infect Immun. 2017 Nov 17;85(12). pii: e00613-17. doi: 10.1128/IAI.00613-17. Print 2017 Dec.
INTRODUCTION Shiga toxin-producing Escherichia coli (STEC) are pathogens causing severe diseases in humans such as hemorrhagic colitis and hemolytic uremic syndrome. The production
of Shiga toxin (Stx), an AB5 toxin, is the main virulence factor of STEC, but other pathogenic mechanisms are essential for causing disease, such as the effective colonization of
the intestine (1). The majority of STEC usually associated with severe disease induce the attaching and effacing lesion on the host intestinal mucosa, through the action of
effectors encoded by the LEE locus. Nevertheless, some STEC, which do not possess the LEE locus, have been isolated from patients with severe clinical manifestations (8). It
has been proposed that LEE-negative STEC can use a mechanism of intracellular localization to limit the elimination from the gastro-intestinal tract of reservoir animals and
human host (2). This mechanism is still not clear. The presence of the Subtilase-encoding pathogenicity island (SE-PAI) has been described in LEE-negative STEC strains
isolated from human cases of diarrhea and small ruminants (7). This PAI encodes the Subtilase, an AB5 toxin similar to Stx (5) (Figure 2) and Tia, previously described as an invasion determinant in Enterotoxigenic E. coli (ETEC) strains (6). We evaluated the role of the gene tia in the invasion of monolayers of cultured cells by this group of STEC.
MATERIALS AND METHODS
Invasion assay:
• HEp-2 monolayers in 96 wells plates incubated with 106 CFUs of E. coli in the logarithmic phase for 3 hours at 37 °C and 5 % CO2.
• Wells were washed with TSB and Gentamicin (100 μg/ml for) was subsequently added and incubated for 2 hours to eliminate
bacteria located outside the Hep-2 cells (4).
• HEp-2 cells were lysed incubating for 5 minutes with 0.5 ml of PBS/Triton 1 %, in order to release the internalized bacteria.
• Serial dilutions of the lysate have been titled on TSA. The invasive capacity of each strain was evaluated as survival residual, by
counting the colony forming units (CFU).
1) Caprioli A, et al. 2005. Vet Res. 36(3):289-311.
2) Cordeiro F, et al 2013. Microbiology. 159 (Pt 8):1683-1694.
3) Datsenko KA and Wanner BL. 2000. Prc.Natl. Acad Sci USA 97:6640-6645.
4) Elsinghorst EA. 1994. Methods Enzymol. 236:405-420.
Inactivation of the gene tia in strain ED32:
To determine the role of tia in the invasive capacity knockout experiments of this gene
were performed.
The mutant strain ED 32 Δtia was generated by deletion of 509 bp out of 747 bp of the
gene tia and replacement with a KM resistant cassette, as previously described (3).
Figure 1. Invasion of Caco-2 (A) and HEp-2 (B) cell
monolayers.
The level of invasion of both the Caco-2 and HEp-2cell
monolayers is expressed as the number of CFU per
milliliter.
The difference between strain ED32 Δtia and both the
ED32 and ED32 Δtia/pGEM_tia strains was statistically
significant with a P value of 0.01, indicated by the
asterisk.
Quantitative analysis of tia expression:
To compare the expression of the gene tia, we extracted the total RNA from the bacteria inside or outside the cell monolayers and performed a quantitative
PCR experiment targeting tia gene. The results were analysed calculating the 2-ΔΔCT.
CONCLUSIONS
tia gene is involved in the ability to invade cultured monolayers of epithelial cells shown by SE-PAI-positive E. coli, including
STEC.
The presence of tia gene was not sufficient to confer invasion ability to E. coli K12 JM109 strain, suggesting that at least another
factor must be involved in the mechanism leading to intracellular localization.
The level of intracellular colonization of SE-PAI-positive E. coli is lower when compared to typical Enteroinvasive E. coli,
suggesting that their invasion ability may represent a mechanism to escape the host defense operating on the intestinal
mucosal surface, rather than a virulence mechanism.
Expression of tia in JM109 /pGEM_tia:
To exclude the possibility that in strain JM109 pGEM_tia the gene tia was not
sufficiently expressed to confer the invasive phenotype, we retrotranscribed the total
RNA from strains JM109/pGEM_tia and ED 32 and used the same amount of cDNA
as template in RT-PCR with primers amplifying a 571-bp fragment within the coding
sequence (8)
RESULTS
Figure 2. Amplification of the cDNA of
the gene tia.
Expression of the gene tia by strain ED32
(A) and by the K-12 strain
JM109/pGEM_tia (B) by RT-PCR.
The gene tia is overexpressed in
JM109 pGEM_tia strain.
The RT-PCR confirmed the
expression of tia in pGEM-tia
strain, excluding the possibility that
the lower invasion capacity of this
strain was due to the lack or to a
poor expression of the gene.
The gene tia is overexpressed in
strains ED32 and ED97 within
HEp-2 cells.
To further investigate the role of
the gene tia in conferring the
invasive phenotype to these two
strains, we performed a
quantitative real-time PCR on the
total RNA extracted from the
bacteria recovered from inside the
HEp-2 cells and from the bacteria
that were incubated with the HEp-
2 cell monolayer but that did not
invade the bacteria.
Our results showed that the gene
tia was overexpressed in both
ED32 and ED97 strains recovered
from inside the HEp_2 monolayers
with respect to the bacteria
recovered from the medium
outside the monolayer with a fold
change of +48.5 and +47.0
respectively (p value <0.01).
HEp-2
Monolayers
MATERIALS:
RPMI 1640 added with 10 %
FBS, 1 % Glutamine, 1 %
sodium pyruvate, 1 % amino
acids not essential in absence
of streptomycin and penicillin
MATERIALS:
TSB
E. coli strains
logarithmic culture
ED 32 Wild-type strain: stx negative,
LEE negative, and SE-PAI
positive
ED 32 Δtia ED32 strain in which the gene tia
has been inactivated by the
partial replacement of the gene
with Km resistance cassette
ED32 Δtia/pGEM_tia
ED32 Δtia strain carrying the
plasmid pGEM_tia to
complement the tia mutation
ED 97 Wild-type strain: stx positive,
LEE negative and SE-PAI positive
JM109 E. coli K-12 Laboratory strain
JM109 pGEM_tia E. coli K-12 Laboratory strain
with pGEM-T Easy containing tia
gene
JM109 pGEM-T
Easy
E. coli K-12 Laboratory strain
containing pGEM-T Easy
(Promega, Madison, WI, USA)
5) Fleckenstein JM, et al. 1996. Infect Immun. 64(6):2256-2265.
6) Kaper JB, et al. 2004. Nat Rev Microbiol. 2(2):123-140.
7) Michelacci V, et al. 2013. Clin Microbiol lnfect. 19(3):E149-156.
8) Paton AW, et al. 1999. J Clin Microbiol. Oct;37(10):3357-3361.
References
SE-PAI-positive E. coli strains
ED32 and ED97 showed the
ability to invade Caco-2 and
HEp-2 cells.
The inactivation of the gene tia
in strain ED32 Δtia strongly
reduced the invasion ability on
Caco-2 and HEp-2 cell
monolayers.
Complementation of the tia
mutation in the strain ED32
pGEM_tia restored the invasive
phenotype.
The strain JM109 pGEM-T Easy
was used to control the effect of
the presence of the plasmid pGEM
in the absence of tia gene.
The expression of tia in the
laboratory strain JM109
conferred only a moderate
ability to invade the Caco-2 cell
monolayers (A) but did not
confer any ability to invade the
HEp-2 cell monolayers (B).
SE-PAI-positive E. coli strains
Invade at a lower level than
typical EIEC (not shown).