The Type III Secretion System (T3SS)-Translocon of Atypical Enteropathogenic Escherichia coli (aEPEC) Can Mediate Adherence

Abstract

The intimin protein is the major adhesin involved in the intimate adherence of atypical enteropathogenic Escherichia coli (aEPEC) strains to epithelial cells, but little is known about the structures involved in their early colonization process. A previous study demonstrated that the type III secretion system (T3SS) plays an additional role in the adherence of an Escherichia albertii strain. Therefore, we assumed that the T3SS could be related to the adherence efficiency of aEPEC during the first stages of contact with epithelial cells. To test this hypothesis, we examined the adherence of seven aEPEC strains and their eae (intimin) isogenic mutants in the standard HeLa adherence assay and observed that all wild-type strains were adherent while five isogenic eae mutants were not. The two eae mutant strains that remained adherent were then used to generate the eae/escN double mutants (encoding intimin and the T3SS ATPase, respectively) and after the adherence assay, we observed that one strain lost its adherence capacity. This suggested a role for the T3SS in the initial adherence steps of this strain. In addition, we demonstrated that this strain expressed the T3SS at significantly higher levels when compared to the other wild-type strains and that it produced longer translocon-filaments. Our findings reveal that the T3SS-translocon can play an additional role as an adhesin at the beginning of the colonization process of aEPEC.

Keywords: EspA; EspB; EspD; adherence; atypical EPEC; gene expression; polymorphism; type III secretion system (T3SS)-translocon.

FIGURE 1

Interaction with HeLa cells of aEPEC strains 2012-1 and 3881-3 and their mutant strains, after 6 h. (A) Light microscopy images (microscopic magnification 1,000×) demonstrate the adherence patterns of the wild-type (wt) strains and their mutants. (B–D) Quantitative adherence tests. Statistical analyses of the quantitative adherence tests were performed by one-way ANOVA followed by post hoc Tukey HSD Test, ^∗∗∗p < 0.001. The 2012-1 eae::Kn maintained its adherence capacity while mutants in the escN gene (2012-1 eae::Kn ΔescN and 2012-1 ΔescN) were no longer adherent. Trans-complementation of the eae and escN mutants (2012-1 eae::Kn pINT and 2012-1ΔescN pescN, respectively) restored the wild-type adherence phenotype. The eae and escN 3881-3 mutants (3881-3 eae::Kn and 3881-3 eae::Kn ΔescN, respectively) maintained the adherence capacity.

FIGURE 2

Interaction with HeLa cells of the aEPEC 2012-1 isogenic mutants in the espD and eae genes, after 6 h. (A) The light microscopy images demonstrate that the mutant strains totally lost their ability to adhere to HeLa cells. (B) The growth curves of the wild-type and the mutant strains were similar, confirming that the mutagenesis procedure did not alter bacterial growth rates.

FIGURE 3

Relative expression of the espA, espB, and espD genes of aEPEC strains 1711-4, 2012-1, 2531-13, 3522-6, 3881-3, 4582-2 as compared to aEPEC BA4095 strain. The 2012-1 strain expresses higher levels of all three genes. Experiments were performed in biological and technical triplicates. ^*p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

FIGURE 4

Secreted protein profile of aEPEC strains. The secreted proteins contained in culture supernatants of all aEPEC strains were precipitated, resolved by 12% SDS-PAGE and stained with Coomassie Brilliant Blue R-250. (1) 1711-4 wt; (2) 2012-1 wt; (3) 2531-13 wt; (4) 3522-6 wt; (5) 3881-3 wt; (6) 4582-2 wt; and (7) BA4095 wt. (M) SeeBlue^® Plus2 Protein Standard (Invitrogen).

FIGURE 5

Analysis of T3SS filament formation by immunogold labeling (IGL) on aEPEC strains 2012-1 (wild-type), BA4095 (wild-type), and 2012-1 ΔescN (negative control). After fixation, bacterial cultures were incubated with anti-EspA antibodies, and labeled with 10 nm-gold particles. A set of preparations were firstly negatively stained with 2% uranyl acetate for general observation. In order to facilitate counting and measurement of the filaments, a second set were only immunogold-labeled. (A) EspA filament length and (B) number of filaments per bacterial cell. The strain 2012-1 produced longer EspA filaments than the strain BA4095 even though the number of T3SS-translocon per bacterial cell was the same (^∗∗p < 0.01). Bars: 0.2 μm.

FIGURE 6

Multiple alignment among the C-terminal region of the EspD coiled-coil domain from the aEPEC strains studied and the tEPEC prototype E2348/69 strain and heat map of predicted effects of 2012-1 EspD coiled-coil domain sequence variants. (A) (^*) indicates positions that have a conserved residue; (:) indicates conservation among groups of similar properties; (.) indicates conservation among groups of poorly similar properties; and (-) indicates the occurrence of gaps. The colors indicate the properties of the amino acids: (Red) small, hydrophobic and aromatic, except Y; (Blue) acids, (Magenta) basic, except H; (Green) with hydroxyl, sulfhydryl and amine groups, and Glycine (G). The gray boxes indicate the domain sequences predicted by SMART. (B) The colors showed are the predicted effect of each polymorphisms from the 2012-1 EspD coiled-coil domain amino acid sequence (x-axis) to any other amino acid (y-axis). Red indicates a strong signal for effect and blue a strong signal for neutral. Black marks indicate the wildtype residues.