Characterization of SepL of enterohemorrhagic Escherichia coli

Journal of bacteriology, 1998

Enterohemorrhagic Escherichia coli (EHEC) exhibits a pattern of localized adherence to host cells, with the formation of microcolonies, and induces a specific histopathological phenotype collectively known as the attaching and effacing lesion. The genes encoding the products responsible for this phenotype are located on a 35-kb pathogenicity island designated the locus of enterocyte effacement, which is also shared by enteropathogenic E. coli. We have identified an open reading frame (ORF) which is located upstream of the espA, espB, and espD genes on the complementary strand and which exhibits high homology to the genes spiB from Salmonella, yscD from Yersinia, and pscD from Pseudomonas. Localization studies showed that the encoded product is present in the cytoplasmic and inner membrane fractions of EHEC. The construction and characterization of a recombinant clone containing an in-frame deletion of this ORF demonstrated that the encoded product is a putative member of a type III ...

Veterinary Microbiology, 2001

Enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli infections are characterised by the formation of attaching and effacing (AE) lesions on intestinal epithelial cells. Secretion of extracellular proteins (EspA, EspB, and EspD) via a type III secretion apparatus is necessary for the formation of the AE lesions by human EPEC. In this study, we show that bovine EPEC and EHEC are also able to secrete polypeptides homologous to the already described Esp proteins, most probably via a type III secretion system. Bovine EPEC and EHEC strains present two different secretion pro®les of Esp proteins which correlate to the pathotypes of the esp genes as determined by PCR. We also demonstrate that genes encoding secreted proteins, present in the LEE of two bovine strains, are organised in the same way as in the human EPEC strain E2348/69.

Infection and Immunity, 2005

Enteropathogenic Escherichia coli (EPEC) is a major bacterial cause of infantile diarrhea in developing countries and is the prototype for a group of gastrointestinal pathogens causing characteristic attaching and effacing (A/E) histopathology on intestinal epithelia. A/E pathogens utilize a type III secretion system (TTSS), encoded by the locus of enterocyte effacement (LEE) pathogenicity island, to deliver effector proteins into host cells. Here, we investigate sequence divergence of the LEE-encoded SepZ protein and identify it as a TTSS-secreted and -translocated molecule. SepZ is hypervariable among A/E pathogens, with sequences sharing between 60 to 81% amino acid identity with SepZ of EPEC. A SepZ-CyaA fusion was secreted and translocated into HeLa cells in a TTSS-dependent manner. Additionally, we determined that the first 20 amino acids of SepZ were sufficient to direct its translocation. In contrast to previous studies suggesting a role in invasion and the structure and/or ...

MicrobiologyOpen, 2018

Type three secretion systems (T3SSs) are virulence determinants employed by several pathogenic bacteria as molecular syringes to inject effector proteins into host cells. Diarrhea-producing enteropathogenic Escherichia coli (EPEC) uses a T3SS to colonize the intestinal tract. T3S is a highly coordinated process that ensures hierarchical delivery of three classes of substrates: early (inner rod and needle subunits), middle (translocators), and late (effectors). Translocation of effectors is triggered upon host-cell contact in response to different environmental cues, such as calcium levels. The T3S substrate specificity switch from middle to late substrates in EPEC is regulated by the SepL and SepD proteins, which interact with each other and form a trimeric complex with the chaperone CesL. In this study, we investigated the link between calcium concentration and secretion regulation by the gatekeeper SepL. We found that calcium depletion promotes late substrate secretion in a transl...

Journal of Biological Chemistry, 2008

Enterohemorrhagic Escherichia coli utilizes a type III secretion system to deliver virulent effectors into cells. The secretion apparatus comprises a membrane basal body and an external needle complex of which EspA is the major component. An l0050-deletion (ΔL50) mutation was found to impair type III secretion and bacterial adherence. These phenotypes and the localization of the gene product to the inner membrane support the hypothesis that L0050, renamed EscL, forms part of the secretion apparatus. Furthermore, in ΔL50, the amount of EspA present within the cell lysate was found to have diminished, whereas EspA's co-cistron-expressed partner protein EspB remained unaffected. The decreased EspA level appeared to result from instability of the newly synthesized EspA protein in ΔL50 rather than a decrease in EspA's mRNA. Using both biochemical co-purification and a bacterial two-hybrid interaction system, we were able to conclude that EscL is a third protein that, in addition to CesAB and CesA2, interacts with EspA and enhances the stability of intracellular EspA.

FEMS Microbiology Letters, 2006

Enterohaemorrhagic Escherichia coli (EHEC) causes bloody diarrhoea in humans and deploys a type III secretion system (T3SS) encoded by the locus of enterocyte effacement to elicit the formation of attaching and effacing (AE) lesions on intestinal epithelia. Here, we report the identification of a new secreted substrate of this system, z1829, which is encoded by cryptic prophage CP-933N. Elevated secretion of a b-lactamase-z1829 fusion protein was detected upon mutation of sepD in EHEC O157:H7 and the fusion protein was translocated into infected epithelial cells in a T3SS-dependent manner; accordingly, we named the protein EspK. In common with the related Salmonella enterica type III secreted effector GogB, we observed that EspK localized to the cytoplasm when transiently expressed in COS-7 cells using EspK-specific antiserum. Inactivation of espK did not impair adherence or actin nucleation during infection of HeLa cells but affected persistence of EHEC O157:H7 in the intestines of orally inoculated calves. Inactivation of an orthologue of espK in the murine AE pathogen Citrobacter rodentium did not impair intestinal colonization in mice.

Infection and Immunity, 1999

The formation of EspA-containing surface appendages in pathogenic Escherichia coli strains, both enteropathogenic E. coli (EPEC) and Shiga toxin-producing E. coli strains, is essential for critical events in the infective process, e.g., localized bacterial adherence to host cells with formation of microcolonies and induction of attaching and effacing lesions. It has been reported that EPEC mutants deficient in the production of EspD, which is encoded by the esp operon, are unable to accumulate actin underneath adherent bacteria but exhibit an attachment similar to that of the wild type. Here, we report the construction and characterization of an in-frame espD deletion mutant of the enterohemorrhagic E. coli (EHEC) strain EDL933. In contrast to what was observed in EPEC mutants, the EDL933 espD mutant not only lacked the capacity to accumulate actin but also exhibited an impaired attachment to HeLa cells. The synthesis of the EspD protein was also essential for the formation of EspA-containing filaments. Finally, localization studies demonstrated that the EspD protein is transferred to the cytoplasm and integrated into the cytoplasmic membranes of infected cells. These results help to elucidate the underlying molecular events in infections caused by EHEC.

Molecular Microbiology, 2002

to the CyaA reporter protein (EspB-CyaA), into the host cell. The translocated EspB was distributed between the membrane and the cytoplasm of the host cell. Translocation was strongly promoted by attachment of EPEC to the host cell, and both attachment factors of EPEC, intimin and the bundle-forming pili, were needed for full translocation efficiency. Translocation and secretion of EspB and EspB-CyaA were abolished in mutants deficient in components of the type III protein secretion system, including sepA and sepB mutants. EspB-CyaA was secreted but not translocated by an espB mutant. These results indicate that EspB is both translocated and required for protein translocation by EPEC.

Infection and Immunity, 2001

At least five proteins are secreted extracellularly by enteropathogenic Escherichia coli (EPEC), a leading cause of infant diarrhea in developing countries. However only one, EspC, is known to be secreted independently of the type III secretion apparatus encoded by genes located within the 35.6-kb locus of enterocyte effacement pathogenicity island. EspC is a member of the autotransporter family of proteins, and the secreted portion of the molecule is 110 kDa. Here we determine that the espC gene is located within a second EPEC pathogenicity island at 60 min on the chromosome of E. coli. We also show that EspC is an enterotoxin, indicated by rises in short-circuit current and potential difference in rat jejunal tissue mounted in Ussing chambers. In addition, preincubation with antiserum against the homologous Pet enterotoxin of enteroaggregative E. coli eliminated EspC enterotoxin activity. Like the EAF plasmid, the espC pathogenicity island was found only in a subset of EPEC, suggesting that EspC may play a role as an accessory virulence factor in some but not all EPEC strains.

Journal of Bacteriology, 2012

The virulence of many Gram-negative pathogens is associated with type III secretion systems (T3SSs), which deliver virulence effector proteins into the cytoplasm of host cells. Components of enteropathogenic Escherichia coli (EPEC) T3SS are encoded within the locus of enterocyte effacement (LEE). While most LEE-encoded T3SS proteins in EPEC have assigned names and functions, a few of them remain poorly characterized. Here, we studied a small LEE-encoded protein, Orf15, that shows no homology to other T3SS/flagellar proteins and is only present in attaching and effacing pathogens, including enterohemorrhagic E. coli and Citrobacter rodentium . Our findings demonstrated that it is essential for type III secretion (T3S) and that it is localized to the periplasm and associated with the inner membrane. Membrane association was driven by the N-terminal 19 amino acid residues, which were also shown to be essential for T3S. Consistent with its localization, Orf15 was found to interact with ...