Analysis of the Virulence of an Atypical Enteropathogenic Escherichia coli Strain In Vitro and In Vivo and the Influence of Type Three Secretion System

Atypical enteropathogenic Escherichia coli (aEPEC) inject various effectors into intestinal cells through a type three secretion system (T3SS), causing attaching and effacing (A/E) lesions. We investigated the role of T3SS in the ability of the aEPEC 1711-4 strain to interact with enterocytes in vitro (Caco-2 cells) and in vivo (rabbit ileal loops) and to translocate the rat intestinal mucosa in vivo. A T3SS isogenic mutant strain was constructed, which showed marked reduction in the ability to associate and invade but not to persist inside Caco-2 cells. After rabbit infection, only aEPEC 1711-4 was detected inside enterocytes at 8 and 24 hours pointing to a T3SS-dependent invasive potential in vivo. In contrast to aEPEC 1711-4, the T3SS-deficient strain no longer produced A/E lesions or induced macrophage infiltration. We also demonstrated that the ability of aEPEC 1711-4 to translocate through mesenteric lymph nodes to spleen and liver in a rat model depends on a functional T3SS, since a decreased number of T3SS mutant bacteria were recovered from extraintestinal sites. These findings indicate that the full virulence potential of aEPEC 1711-4 depends on a functional T3SS, which contributes to efficient adhesion/invasion in vitro and in vivo and to bacterial translocation to extraintestinal sites

<i>Escherichia albertii</i>, a novel human enteropathogen, colonizes rat enterocytes and translocates to extra-intestinal sites

<div><p>Diarrhea is the second leading cause of death of children up to five years old in the developing countries. Among the etiological diarrheal agents are atypical enteropathogenic <i>Escherichia coli</i> (aEPEC), one of the diarrheagenic <i>E</i>. <i>coli</i> pathotypes that affects children and adults, even in developed countries. Currently, genotypic and biochemical approaches have helped to demonstrate that some strains classified as aEPEC are actually <i>E</i>. <i>albertii</i>, a recently recognized human enteropathogen. Studies on particular strains are necessary to explore their virulence potential in order to further understand the underlying mechanisms of <i>E</i>. <i>albertii</i> infections. Here we demonstrated for the first time that infection of fragments of rat intestinal mucosa is a useful tool to study the initial steps of <i>E</i>. <i>albertii</i> colonization. We also observed that an <i>E</i>. <i>albertii</i> strain can translocate from the intestinal lumen to Mesenteric Lymph Nodes and liver in a rat model. Based on our finding of bacterial translocation, we investigated how <i>E</i>. <i>albertii</i> might cross the intestinal epithelium by performing infections of M-like cells <i>in vitro</i> to identify the potential <i>in vivo</i> translocation route. Altogether, our approaches allowed us to draft a general <i>E</i>. <i>albertii</i> infection route from the colonization till the bacterial spreading <i>in vivo</i>.</p></div

Bacterial Translocation through the M-like cells.

<p>Caco-2 cells were cultured for 10 days on Millicell filter. One group was co-cultured with Raji-B cell and control group were maintained in monoculture for 6 days. Then, upper chambers containing Caco-2 cells only or M-like cells were infected with <i>E</i>. <i>albertii</i> 1551–2, isogenic mutants or complemented strains for 6 h, or with tEPEC E2348/69 for 3 h. Media from the lower chamber were collected in three different periods during the incubation and seeded onto MacConkey agar for CFU counting. (A) Translocation of <i>E</i>. <i>albertii</i> and tEPEC through the Caco-2 and M-like cells: (***) indicates statistical differences between the two lineages infected with <i>E</i>. <i>albertii</i> strain (P<0.05). (##) indicates differences between M-like cells infected with 1551–2 and E2348/69 strains (P<0.05). (B) Translocation of <i>E</i>. <i>albertii</i>, isogenic mutants and complemented strains: (**) indicates differences between M-like cells infected with wt and T3SS mutant (P<0.05). (#) indicates difference between M-like cells infected with the T3SS mutant and its complemented strain (P<0.05). No statistical differences were found between in the transcytosis of wt and T1P mutant or between T1P mutant and its complement strains. <i>eae</i>, intimin mutant; <i>tir</i>, Tir mutant; <i>escN</i>, T3SS mutant; pEscN, complemented T3SS mutant; Δ<i>fimA</i>, T1P mutant. pFimA, complemented T1P mutant.</p

Bacterial strains.

<p>Bacterial strains.</p

IVOC and <i>E</i>. <i>albertii</i> colonization assays.

<p>Ileal fragments collected from 3 Wistar-EPM rats were inoculated with 10¹⁰ CFU of each bacterial strain and incubated for 2 h at 37°C. SEM: (A) wild type <i>E</i>. <i>albertii</i> strain 1551–2 (black arrow), (B) T3SS mutant (white arrow), (C) non-infected control. TEM: (D) AE lesion underneath the wild type strain adherence site (#), (E) absence of AE lesion under the T3SS mutant interaction and (F) Non-infected control. (#) indicates preserved brush borders. Bars, 2 μm. (G) Quantification assay. After the incubation period, preparations were washed, macerated, and bacterial suspensions were diluted for seeding and CFU counting. (**) Significantly less adherent than wild type (p = 0.013), (#) significantly more adherent than T3SS mutant (P = 0.02). <i>eae</i>, intimin mutant; Δ<i>tir</i>, Tir mutant; <i>esc</i>N, T3SS mutant; pEscN, complemented T3SS mutant; Δ<i>fimA</i>, T1P mutant.</p

Quantitative assessment of association and invasion of <i>E</i>. <i>albertii</i> 1551–2 and its isogenic mutants to differentiated Caco-2 cells.

<p>Cells were infected with 10⁷ CFU for 6 h, monolayers were washed and one set of monolayer was lysed while another set was submitted to the gentamicin protection assay. The invasion indexes were calculated as the percentage of the total number of cell-associated bacteria that were located in the intracellular compartment. Assays were carried out in triplicate, and the results from at least three independent experiments were expressed as the percentage of invasion (mean ± standard error). A) Association. No statistical differences in the association of the wild type, mutant and complemented mutant strains were found. B) Invasion: (**) indicates statistical differences between 1551–2 and the intimin mutant (P = 0.0030). (***) indicates statistical differences between 1551–2 and Tir (P = 0.0002) or T3SS (P = 0.0004) mutants. (#) indicates statistical differences between T3SS mutant and complemented T3SS (P = 0.0238) strains.</p

TEM of M-like and Caco-2 cells infected with <i>E</i>. <i>albertii</i>.

<p>M-like cells (A) showed few microvilli and a disorganized morphology, while on apical surface of differentiated Caco-2 cells (B) microvilli were still present. After <i>E</i>. <i>albertii</i> infection, M-like cells (C) allowed bacterial transcytosis (note bacteria crossing the filter membrane pore—white arrow), while in Caco-2 cells (D), <i>E</i>. <i>albertii</i> remained on the cells surface (black arrow). Bars, 10 μm.</p

Bacterial translocation (BT) <i>in vivo</i>.

<p><i>E</i>. <i>albertii</i> 1551–2 was recovered from the mesenteric lymph nodes (MLN) and liver. The T3SS mutant strain was not recovered from any tested organs. <i>E</i>. <i>coli</i> R6 (BT-positive control) was recovered from all examined organs while <i>E</i>. <i>coli</i> HB101 (BT-negative control) was not. <i>escN</i>, T3SS mutant.</p