Regulation of Type III Secretion Hierarchy of Translocators and Effectors in Attaching and Effacing Bacterial Pathogens

Abstract

Human enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and the mouse pathogen Citrobacter rodentium (CR) belong to the family of attaching and effacing (A/E) bacterial pathogens. They possess the locus of enterocyte effacement (LEE) pathogenicity island, which encodes a type III secretion system. These pathogens secrete a number of proteins into culture media, including type III effector proteins and translocators that are required for the translocation of effectors into host cells. Preliminary evidence indicated that the LEE-encoded SepL and Rorf6/SepD may form a molecular switch that controls the secretion of translocators and effectors in CR. Here, we show that SepL and SepD indeed perform this function in A/E pathogens such as EHEC and EPEC. Their sepL and sepD mutants do not secrete translocators but exhibit enhanced secretion of effectors. We demonstrate that SepL and SepD interact with each other and that both SepL and SepD are localized to the bacterial membranes. Furthermore, we demonstrate that culture media influence the type III secretion profile of EHEC, EPEC, and CR and that low-calcium concentrations inhibit secretion of translocators but promote the secretion of effectors, similar to effects on type III secretion by mutations in sepL and sepD. However, the secretion profile of the sepD and sepL mutants is not affected by these culture conditions. Collectively, our results suggest that SepL and SepD not only are necessary for efficient translocator secretion in A/E pathogens but also control a switch from translocator to effector secretion by sensing certain environmental signals such as low calcium.

Hierarchical Delivery of an Essential Host Colonization Factor in Enteropathogenic Escherichia coli

“…Recently, we demonstrated that the efficient secretion of many other EPEC type III effector proteins also required CesT (32,33), suggesting a possible interaction of these proteins with CesT. To identify new CesT interacting type III effector proteins, we took advantage of a ⌬sepD strain of EPEC that is known to hypersecrete type III effector proteins but not translocator proteins (38). ⌬sepD was cultured under conditions that promote type III effector secretion, and the culture supernatant was collected and passed over a CesT affinity column.…”

Quantitative Proteomic Analysis of Type III Secretome of Enteropathogenic Escherichia coli Reveals an Expanded Effector Repertoire for Attaching/Effacing Bacterial Pathogens

“…The primer pairs argH-F/ argH-R and lysA-F/lysA-R were used to screen by PCR for the ⌬argH and ⌬lysA mutants, respectively. The pRE-⌬lysA and pRE-⌬argH deletion constructs were sequentially introduced into an EPEC ⌬sepD⌬escN double mutant (25) to generate EPEC ⌬lysA-⌬argH⌬sepD⌬escN. Deletion constructs for EPEC escN (26) and sepD (25) in pRE112 were introduced into EPEC ⌬lysA⌬argH to generate EPEC ⌬lysA⌬argH⌬escN and ⌬lysA⌬argH⌬sepD, respectively.…”

“…The pRE-⌬lysA and pRE-⌬argH deletion constructs were sequentially introduced into an EPEC ⌬sepD⌬escN double mutant (25) to generate EPEC ⌬lysA-⌬argH⌬sepD⌬escN. Deletion constructs for EPEC escN (26) and sepD (25) in pRE112 were introduced into EPEC ⌬lysA⌬argH to generate EPEC ⌬lysA⌬argH⌬escN and ⌬lysA⌬argH⌬sepD, respectively. The four EPEC mutant strains, ⌬lysA⌬argH, ⌬lysA⌬argH⌬escN, ⌬lysA⌬argH⌬sepD, and ⌬lysA⌬argH⌬sepD⌬escN, were then used for SILAC analysis of their secreted proteins.…”

Analysis of the expression, regulation and export of NleA–E in Escherichia coli O157 : H7

“…Our understanding of EPEC and EHEC pathogenesis has benefited from the study of the mouse pathogen Citrobacter rodentium, which also expresses a T3SS encoded from a homologous locus of enterocyte effacement (LEE), and this pathogen forms characteristic attaching and effacing lesions in vivo and in eukaryotic HeLa cells Gruenheid et al, 2004;Mundy et al, 2004b). Through an understanding of the regulation of LEE expression in C. rodentium, a number of putative type III (T3)-secreted effector proteins have been identified (Deng et al, 2005). As the genes for these proteins are not encoded on the LEE, they are termed non-LEE-encoded effector (Nle) proteins.…”