Resumen de Generation of enteropathogenic E. coli strains lacking the repertoire of effectors translocated by the type III protein secretion system and their characterization in the infection of cultured cell lines and human intestinal biopsies
Massiel Esther Capeda Molero Molero
Although most Escherichia coli isolates are harmless commensals of the gastrointestinal tract, some strains have acquired specific virulence factors, like pathogenicity islands, insertion elements (IEs), and prophages (PPs), to become highly adapted pathogens. The enteropathogenic E. coli (EPEC) is an important category of diarrheagenic bacteria causing acute and chronic diarrhea in infants. The hallmark of EPEC infection is the formation of attachment and effacement (A/E) lesion in the intestinal mucosa surface, which is characterized by the intimate attachment of the bacteria to the enterocyte, microvilli effacement, and the formation of actin-pedestal-like structures underneath the attached bacteria. EPEC is endowed of a 35 kb pathogenicity island called the locus of enterocyte effacement (LEE) that contains all the genes necessary for the assembly of a type III secretion system (T3SS) injectisome. Through these injectisome EPEC translocates multiple effector proteins into the host cell to subvert cellular functions in benefit of the infection. The prototype strain E2348/69 of EPEC O127:H6 is endowed of six LEE encoded effectors and 17 non-LEE encoded effectors. We have engineered a set of effector mutant EPEC strains using suicide vectors to delete the whole repertoire of effector genes of this prototype EPEC strain. Genome manipulation did not affect the functionality of the T3SS injectisome. The deletion strategy was based on suicide or termosensitive plasmid integration by homologous recombination and the markerless resolution of co-integrants after I-SceI digestion. We did markerless integration of map, espH and nleC in their original locus in EPEC2 (maintain EspZ and Tir), EPEC1 (maintaining only Tir) and EPEC0 (effector-less) mutant strains. These strains were able to translocate functional effectors from chromosomal expression into HeLa cells. We infected intestinal human biopsies with the effector mutant EPEC strains to identify the effectors necessary for the induction of the A/E lesion in human intestinal tissues. We found that while EPEC2 and EPEC1 mutant strains were able to induce the actin-pedestal formation in HeLa cells in vitro, none of the biopsies infected with these strains had A/E lesion. These results demonstrated that effectors besides Tir and EspZ are essential to induce the A/E lesion formation in intestinal biopsies. We infected intestinal biopsies with several effector mutant EPEC strains and we found that effectors located outside the LEE are essential to induce efficient A/E lesion on human intestinal biopsies. Additionally we found that non-LEE effectors are characterized by having an additive effect to allow the A/E lesion development in these intestinal surfaces and that Efa1/LifA homologous proteins seem to play a major role in this process. Our results with intestinal biopsies strongly suggest that non-LEE effectors are necessary for the efficient formation of A/E lesion in the in vivo situation
