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Invasiveness as a putative additional virulence mechanism of some atypical Enteropathogenic Escherichia coli strains with different uncommon intimin types.

Yamamoto D, Hernandes RT, Blanco M, Greune L, Schmidt MA, Carneiro SM, Dahbi G, Blanco JE, Mora A, Blanco J, Gomes TA - BMC Microbiol. (2009)

Bottom Line: Five of six strains invaded HeLa and T84 cells in a range of 13.3%-20.9% and 5.8%-17.8%, respectively, of the total cell-associated bacteria.Invasiveness was confirmed by transmission electron microscopy.Some aEPEC strains may invade intestinal cells in vitro with varying efficiencies and independently of the intimin sub-type.

View Article: PubMed Central - HTML - PubMed

Affiliation: Universidade Federal de São Paulo, Rua Botucatu, 862, 3o andar, Vila Clementino, São Paulo, CEP 04023-062, Brazil. dyamamoto@unifesp.br

ABSTRACT

Background: Enteropathogenic Escherichia coli (EPEC) produce attaching/effacing (A/E) lesions on eukaryotic cells mediated by the outer membrane adhesin intimin. EPEC are sub-grouped into typical (tEPEC) and atypical (aEPEC). We have recently demonstrated that aEPEC strain 1551-2 (serotype O non-typable, non-motile) invades HeLa cells by a process dependent on the expression of intimin sub-type omicron. In this study, we evaluated whether aEPEC strains expressing other intimin sub-types are also invasive using the quantitative gentamicin protection assay. We also evaluated whether aEPEC invade differentiated intestinal T84 cells.

Results: Five of six strains invaded HeLa and T84 cells in a range of 13.3%-20.9% and 5.8%-17.8%, respectively, of the total cell-associated bacteria. The strains studied were significantly more invasive than prototype tEPEC strain E2348/69 (1.4% and 0.5% in HeLa and T84 cells, respectively). Invasiveness was confirmed by transmission electron microscopy. We also showed that invasion of HeLa cells by aEPEC 1551-2 depended on actin filaments, but not on microtubules. In addition, disruption of tight junctions enhanced its invasion efficiency in T84 cells, suggesting preferential invasion via a non-differentiated surface.

Conclusion: Some aEPEC strains may invade intestinal cells in vitro with varying efficiencies and independently of the intimin sub-type.

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Transmission electron microscopy of polarized and differentiated T84 cells infected via the basolateral side. A) aEPEC 1551-2. B) aEPEC 0621-6. C) prototype tEPEC E2348/69. Monolayers were infected for 6 h (aEPEC) and 3 h (tEPEC). Arrows indicate tight junction and (*) indicates a Transwell membrane pore.
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Figure 5: Transmission electron microscopy of polarized and differentiated T84 cells infected via the basolateral side. A) aEPEC 1551-2. B) aEPEC 0621-6. C) prototype tEPEC E2348/69. Monolayers were infected for 6 h (aEPEC) and 3 h (tEPEC). Arrows indicate tight junction and (*) indicates a Transwell membrane pore.

Mentions: To address a putative effect of EGTA on the invasion ability of the aEPEC strains we also cultivated T84 cells for 14 days on the lower surface of a Transwell membrane. In this manner, bacterial contact with the basolateral cell surface can be achieved without prior treatment of the T84 cells. Preparations were examined by TEM and the images suggest enhanced bacterial invasion and show bacteria within vacuoles (Fig. 5) confirming the results obtained with EGTA treated T84 cells. Regarding tEPEC E2348/69, no internalized bacteria was found in the microscope fields observed. Enteropathogens may gain access to basolateral receptors and promote host cell invasion in vivo by transcytosis through M cells [46]. Alternatively, some infectious processes can cause perturbations in the intestinal epithelium, e.g., neutrophil migration during intestinal inflammation; as a consequence, a transitory destabilization in the epithelial barrier is promoted exposing the basolateral side and allowing bacterial invasion [47]. With regard to tEPEC, it has been reported that an effector molecule, EspF is involved in tight junction disruption and redistribution of occludin with ensuing increased permeability of T84 monolayers [48,49]. Whether EspF is involved in the invasion ability of the aEPEC strains studied in vivo remains to be investigated.


Invasiveness as a putative additional virulence mechanism of some atypical Enteropathogenic Escherichia coli strains with different uncommon intimin types.

Yamamoto D, Hernandes RT, Blanco M, Greune L, Schmidt MA, Carneiro SM, Dahbi G, Blanco JE, Mora A, Blanco J, Gomes TA - BMC Microbiol. (2009)

Transmission electron microscopy of polarized and differentiated T84 cells infected via the basolateral side. A) aEPEC 1551-2. B) aEPEC 0621-6. C) prototype tEPEC E2348/69. Monolayers were infected for 6 h (aEPEC) and 3 h (tEPEC). Arrows indicate tight junction and (*) indicates a Transwell membrane pore.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2724384&req=5

Figure 5: Transmission electron microscopy of polarized and differentiated T84 cells infected via the basolateral side. A) aEPEC 1551-2. B) aEPEC 0621-6. C) prototype tEPEC E2348/69. Monolayers were infected for 6 h (aEPEC) and 3 h (tEPEC). Arrows indicate tight junction and (*) indicates a Transwell membrane pore.
Mentions: To address a putative effect of EGTA on the invasion ability of the aEPEC strains we also cultivated T84 cells for 14 days on the lower surface of a Transwell membrane. In this manner, bacterial contact with the basolateral cell surface can be achieved without prior treatment of the T84 cells. Preparations were examined by TEM and the images suggest enhanced bacterial invasion and show bacteria within vacuoles (Fig. 5) confirming the results obtained with EGTA treated T84 cells. Regarding tEPEC E2348/69, no internalized bacteria was found in the microscope fields observed. Enteropathogens may gain access to basolateral receptors and promote host cell invasion in vivo by transcytosis through M cells [46]. Alternatively, some infectious processes can cause perturbations in the intestinal epithelium, e.g., neutrophil migration during intestinal inflammation; as a consequence, a transitory destabilization in the epithelial barrier is promoted exposing the basolateral side and allowing bacterial invasion [47]. With regard to tEPEC, it has been reported that an effector molecule, EspF is involved in tight junction disruption and redistribution of occludin with ensuing increased permeability of T84 monolayers [48,49]. Whether EspF is involved in the invasion ability of the aEPEC strains studied in vivo remains to be investigated.

Bottom Line: Five of six strains invaded HeLa and T84 cells in a range of 13.3%-20.9% and 5.8%-17.8%, respectively, of the total cell-associated bacteria.Invasiveness was confirmed by transmission electron microscopy.Some aEPEC strains may invade intestinal cells in vitro with varying efficiencies and independently of the intimin sub-type.

View Article: PubMed Central - HTML - PubMed

Affiliation: Universidade Federal de São Paulo, Rua Botucatu, 862, 3o andar, Vila Clementino, São Paulo, CEP 04023-062, Brazil. dyamamoto@unifesp.br

ABSTRACT

Background: Enteropathogenic Escherichia coli (EPEC) produce attaching/effacing (A/E) lesions on eukaryotic cells mediated by the outer membrane adhesin intimin. EPEC are sub-grouped into typical (tEPEC) and atypical (aEPEC). We have recently demonstrated that aEPEC strain 1551-2 (serotype O non-typable, non-motile) invades HeLa cells by a process dependent on the expression of intimin sub-type omicron. In this study, we evaluated whether aEPEC strains expressing other intimin sub-types are also invasive using the quantitative gentamicin protection assay. We also evaluated whether aEPEC invade differentiated intestinal T84 cells.

Results: Five of six strains invaded HeLa and T84 cells in a range of 13.3%-20.9% and 5.8%-17.8%, respectively, of the total cell-associated bacteria. The strains studied were significantly more invasive than prototype tEPEC strain E2348/69 (1.4% and 0.5% in HeLa and T84 cells, respectively). Invasiveness was confirmed by transmission electron microscopy. We also showed that invasion of HeLa cells by aEPEC 1551-2 depended on actin filaments, but not on microtubules. In addition, disruption of tight junctions enhanced its invasion efficiency in T84 cells, suggesting preferential invasion via a non-differentiated surface.

Conclusion: Some aEPEC strains may invade intestinal cells in vitro with varying efficiencies and independently of the intimin sub-type.

Show MeSH
Related in: MedlinePlus