Binding to Na(+) /H(+) exchanger regulatory factor 2 (NHERF2) affects trafficking and function of the enteropathogenic Escherichia coli type III secretion system effectors Map, EspI and NleH.

Enteropathogenic Escherichia coli (EPEC) strains are diarrhoeal pathogens that use a type III secretion system to translocate effector proteins into host cells in order to colonize and multiply in the human gut. Map, EspI and NleH1 are conserved EPEC effectors that possess a C-terminal class I PSD-95/Disc Large/ZO-1 (PDZ)-binding motif. Using a PDZ array screen we identified Na(+)/H(+) exchanger regulatory factor 2 (NHERF2), a scaffold protein involved in tethering and recycling ion channels in polarized epithelia that contains two PDZ domains, as a common target of Map, EspI and NleH1. Using recombinant proteins and co-immunoprecipitation we confirmed that NHERF2 binds each of the effectors. We generated a HeLa cell line stably expressing HA-tagged NHERF2 and found that Map, EspI and NleH1 colocalize and interact with intracellular NHERF2 via their C-terminal PDZ-binding motif. Overexpression of NHERF2 enhanced the formation and persistence of Map-induced filopodia, accelerated the trafficking of EspI to the Golgi and diminished the anti-apoptotic activity of NleH1. The binding of multiple T3SS effectors to a single scaffold protein is unique. Our data suggest that NHERF2 may act as a plasma membrane sorting site, providing a novel regulatory mechanism to control the intracellular spatial and temporal effector protein activity.

The type III effectors NleE and NleB from enteropathogenic E. coli and OspZ from Shigella block nuclear translocation of NF-kappaB p65.

Many bacterial pathogens utilize a type III secretion system to deliver multiple effector proteins into host cells. Here we found that the type III effectors, NleE from enteropathogenic E. coli (EPEC) and OspZ from Shigella, blocked translocation of the p65 subunit of the transcription factor, NF-kappaB, to the host cell nucleus. NF-kappaB inhibition by NleE was associated with decreased IL-8 expression in EPEC-infected intestinal epithelial cells. Ectopically expressed NleE also blocked nuclear translocation of p65 and c-Rel, but not p50 or STAT1/2. NleE homologues from other attaching and effacing pathogens as well OspZ from Shigella flexneri 6 and Shigella boydii, also inhibited NF-kappaB activation and p65 nuclear import; however, a truncated form of OspZ from S. flexneri 2a that carries a 36 amino acid deletion at the C-terminus had no inhibitory activity. We determined that the C-termini of NleE and full length OspZ were functionally interchangeable and identified a six amino acid motif, IDSY(M/I)K, that was important for both NleE- and OspZ-mediated inhibition of NF-kappaB activity. We also established that NleB, encoded directly upstream from NleE, suppressed NF-kappaB activation. Whereas NleE inhibited both TNFalpha and IL-1beta stimulated p65 nuclear translocation and IkappaB degradation, NleB inhibited the TNFalpha pathway only. Neither NleE nor NleB inhibited AP-1 activation, suggesting that the modulatory activity of the effectors was specific for NF-kappaB signaling. Overall our data show that EPEC and Shigella have evolved similar T3SS-dependent means to manipulate host inflammatory pathways by interfering with the activation of selected host transcriptional regulators.

The innate immune molecule, NOD1, regulates direct killing of Helicobacter pylori by antimicrobial peptides.

The cytosolic innate immune molecule, NOD1, recognizes peptidoglycan (PG) delivered to epithelial cells via the Helicobacter pylori cag pathogenicity island (cagPAI), and has been implicated in host defence against cagPAI(+)H. pylori bacteria. To further clarify the role of NOD1 in host defence, we investigated NOD1-dependent regulation of human beta-defensins (DEFBs) in two epithelial cell lines. Our findings identify that NOD1 activation, via either cagPAI(+) bacteria or internalized PG, was required for DEFB4 and DEFB103 expression in HEK293 cells. To investigate cell type-specific induction of DEFB4 and DEFB103, we generated stable NOD1'knockdown' (KD) and control AGS cells. Reporter gene assay and RT-PCR analyses revealed that only DEFB4 was induced in an NOD1-/cagPAI-dependent fashion in AGS cells. Moreover, culture supernatants from AGS control, but not AGS NOD1 KD cells, stimulated with cagPAI(+)H. pylori, significantly reduced H. pylori bacterial numbers. siRNA studies confirmed that human beta-defensin 2 (hBD-2), but not hBD-3, contributes to the antimicrobial activity of AGS cell supernatants against H. pylori. This study demonstrates, for the first time, the involvement of NOD1 and hBD-2 in direct killing of H. pylori bacteria by epithelial cells and confirms the importance of NOD1 in host defence mechanisms against cagPAI(+)H. pylori infection.

Secretion of flagellin by the LEE-encoded type III secretion system of enteropathogenic Escherichia coli.

BACKGROUND: Enteropathogenic Escherichia coli (EPEC) is an attaching and effacing (A/E) pathogen that possesses a type III secretion system (T3SS) encoded within the locus of enterocyte effacement (LEE). The LEE is essential for A/E lesion formation and directs the secretion and translocation of multiple LEE-encoded and non-LEE encoded effector proteins into the cytosol of infected cells. In this study we used proteomics to compare proteins exported to the culture supernatant by wild type EPEC E2348/69, a DeltaespADB mutant and a DeltaescF T3SS mutant. RESULTS: We observed that flagellin was consistently and strongly present in the secretome of wild type EPEC and the DeltaespADB mutant but present only weakly in the secretome of the DeltaescF mutant. Given the ancestral relationship between the flagella export apparatus and virulence associated T3SSs, we investigated whether FliC could utilise the LEE-encoded T3SS for export. In the absence of a functional flagella export apparatus, we showed that FliC could be secreted by the LEE-encoded T3SS and stimulate (Toll-like receptor 5) TLR5 signalling but could not confer motility. CONCLUSION: Since the secretion of FliC during A/E lesion formation would presumably be disadvantageous for the pathogen, we propose that virulence associated T3SSs and flagella T3SSs have evolved through a system of chaperones and complex regulatory pathways to be functional at different times to ensure that FliC secretion does not occur during T3SS effector translocation.

The NleE/OspZ family of effector proteins is required for polymorphonuclear transepithelial migration, a characteristic shared by enteropathogenic Escherichia coli and Shigella flexneri infections.

Enteropathogenic Escherichia coli (EPEC) and Shigella flexneri are human host-specific pathogens that infect intestinal epithelial cells. However, each bacterial species employs a different infection strategy within this environmental niche. EPEC attaches to the apical surface of small intestine enterocytes, causing microvillus effacement and rearrangement of the host cell cytoskeleton beneath adherent bacteria. In contrast, S. flexneri invades the large intestine epithelium at the basolateral membrane, replicates, and spreads cell to cell. Both EPEC and S. flexneri rely on type three secretion systems (T3SS) to secrete effectors into host cells, and both pathogens recruit polymorphonuclear leukocytes (PMNs) from the submucosa to the lumen of the intestine. In this report, we compared the virulence functions of the EPEC T3SS effector NleE and the homologous Shigella protein Orf212. We discovered that Orf212 was secreted by the S. flexneri T3SS and renamed this protein OspZ. Infection of polarized T84 intestinal epithelial cells with an ospZ deletion mutant of S. flexneri resulted in reduced PMN transepithelial migration compared to infection by the wild type. An nleE deletion mutant of EPEC showed a similar reduction of PMN migration. The ability to induce PMN migration was restored in both mutants when either ospZ or nleE was expressed from a plasmid. An infection of T84 cells with the delta ospZ mutant resulted in reduced extracellular signal-related kinase phosphorylation and NF-kappaB activation compared to infection with the wild type. Therefore, we conclude that OspZ and NleE have similar roles in the upstream induction of host signaling pathways required for PMN transepithelial migration in Shigella and EPEC infections.

Contribution of FliC to epithelial cell invasion by enterohemorrhagic Escherichia coli O113:H21.

Enterohemorrhagic Escherichia coli (EHEC) O113:H21 can invade epithelial cells. In this study, we found that invasion but not adherence was inhibited by anti-FliC(H21) specific antibodies. In addition, deletion of fliC(H21) from EHEC O113:H21 resulted in an eightfold decrease in invasion that was restored upon transcomplementation with fliC(H21) but not with fliC(H6). These results suggested that FliC plays an important role in the pathogenesis of infections caused by EHEC O113:H21 by allowing bacteria to penetrate the intestinal epithelium.

Essential role of the type III secretion system effector NleB in colonization of mice by Citrobacter rodentium.

Attaching and effacing (A/E) pathogens are a significant cause of gastrointestinal illness in humans and animals. All A/E pathogens carry a large pathogenicity island, termed the locus for enterocyte effacement (LEE), which encodes a type III secretion system that translocates several effector proteins into host cells. To identify novel virulence determinants in A/E pathogens, we performed a signature-tagged mutagenesis screen in C57BL/6 mice by using the mouse A/E pathogen Citrobacter rodentium. Five hundred seventy-six derivatives of C. rodentium were tested in pools of 12 mutants. One attenuated mutant carried a transposon insertion in nleB, which encodes a putative effector of the LEE-encoded type III secretion system (T3SS). nleB is present in a genomic pathogenicity island that also encodes another putative effector, NleE, immediately downstream. Using translational fusions with beta-lactamase (TEM-1), we showed that both NleB and NleE were translocated into host cells by the LEE-encoded T3SS of enteropathogenic Escherichia coli. In addition, deletion of the gene encoding NleB in C. rodentium resulted in reduced colonization of mice in single infections and reduced colonic hyperplasia. In contrast, the deletion of other non-LEE-encoded effector genes in C. rodentium, nleC, nleD, or nleE, had no effect on host colonization or disease. These results suggest that nleB encodes an important virulence determinant of A/E pathogens.

Contribution of Efa1/LifA to the adherence of enteropathogenic Escherichia coli to epithelial cells.

Enteropathogenic E. coli(EPEC) is an important diarrhoeal pathogen that induces characteristic lesions on the host intestine termed attaching and effacing (A/E) lesions. In this study we have examined the contribution of a large gene, efa1, which is present in all A/E pathogens, to the adherence phenotype of EPEC. An efa- derivative of EPEC JPN15 was constructed and this mutant was significantly less adherent to epithelial cells than the parent strain. The JPN15 efa- derivative was FAS-positive, produced EspA filaments and showed comparable levels of EspA secretion to JPN15. In addition, polyclonal antibodies raised to Efa1 partially inhibited the adherence of JPN15 to cultured epithelial cells. In further work, we showed that human and rabbit hosts infected with an A/E pathogen produced antibodies to Efa1 and we observed that the truncated form of efa1 present in EHEC O157:H7 was specific to that serotype. Generally efa1 was present in its entirety in the genomes of other A/E pathogens. Overall our data suggest that Efa1 has host cell binding activity, at least in tissue culture, and that it is produced during infection. These findings suggest that Efa1 may play a direct role in the pathogenesis of infections caused by A/E pathogens.