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).