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.

Genetic and Virulence Characteristics of a Hybrid Atypical Enteropathogenic and Uropathogenic Escherichia coli (aEPEC/UPEC) Strain

“…These data suggested that this specific cluster could harbor strains with an attenuated diarrheagenic phenotype, which is reinforced by the few FAS positive signals produced by UPEC 252 in HeLa cells mentioned above. However, more studies are necessary to confirm this hypothesis because ST10 bears other EPEC clusters in which several pathogenic EPEC strains that had been isolated from diarrhea were detected (Ferdous et al, 2016 ; Arais et al, 2018 ; Santos et al, 2019 ). Besides the EPEC related genes, other virulence factors were identified in the genome of UPEC 252, among which genes that are involved in bacterial adherence to host cells, like E. coli laminin-binding fimbriae (ELF), E. coli common pilus (ECP), and Type 1 fimbriae.…”

“…Secreted proteins were extracted as described by Zarivach et al ( 2007 ), and an immunoblotting assay using an anti-EspB primary antibody (Guirro et al, 2013 ) and anti-rabbit secondary IgG antibody (whole molecule)-Peroxidase (Sigma, Saint Louis, EUA) was employed to evaluate the ability of UPEC 252 to produce T3SS. A protein extract from the typical EPEC prototype strain E2348/69 was used as a positive control (Santos et al, 2019 ).…”

“…The adherence pattern was determined as described (Santos et al, 2019 ) using HeLa cells in 6 h of interaction in the presence of 2% D-mannose (Sigma - USA) to prevent Type 1 fimbria-mediated adhesion. Bacteria previously grown in LB for 18 h at 37°C were inoculated in 1 mL of cell media, at a 1:50 dilution, and incubated at 37°C for 3 h. The preparation was then washed with phosphate-buffered saline (PBS), fresh medium was added, and incubation proceeded for an additional 3 h. After the 6 h period, the cells were washed, fixed with methanol, stained with May Grünwald and Giemsa (Merck, New Jersey, USA) and evaluated by light microscopy (Hernandes et al, 2013 ).…”

“…Adherence assays were performed as described above but in 3 h and the absence of D-mannose. For the quantitative adhesion assays, after the 3 h incubation period, cells were incubated for ~30 min with 1 mL sterile distilled water for cell lysis (Santos et al, 2019 ). Then, the suspension containing lysed cells and bacteria was subjected to serial dilutions and seeding onto MacConkey Agar to determine the number of colony-forming units (CFU) associated with the cells.…”

Virulence Potential of a Multidrug-Resistant Escherichia coli Strain Belonging to the Emerging Clonal Group ST101-B1 Isolated from Bloodstream Infection

“…and bacterial count, was used as inoculum. After incubation and washes, the cells were lysed by adding 1 mL of sterile bi-distilled water, which was recovered, diluted, and plated onto MacConkey agar for quantification as previously described [77]. The assays were performed in biological duplicates and experimental triplicates, and the data were expressed as the mean and standard error of the mean (SEM).…”