Successful infection by enteric bacterial pathogens depends on the ability of the bacteria to colonise the gut, replicate in host tissues and disseminate to other hosts. Pathogens such as Salmonella, Shigella and enteropathogenic and enterohaemorrhagic E. coli (EPEC and EHEC), utilise a type III secretion system (T3SS) to deliver virulence effector proteins into host cells during infection that promote colonisation and interfere with antimicrobial host responses 1-3. Here we report that the T3SS effector NleB1 from EPEC binds to host cell death domain containing proteins and thereby inhibits death receptor signalling. Protein interaction studies identified FADD, TRADD and RIPK1 as binding partners of NleB1. NleB1 expressed ectopically or injected by the bacterial T3SS prevented Fas ligand or TNF-induced formation of the canonical death inducing signalling complex (DISC) and proteolytic activation of caspase-8, an essential step in death receptor induced apoptosis. This inhibition depended on the N-GlcNAc transferase activity of NleB1, which specifically modified Arg117 in the death domain of FADD. The importance of the death receptor apoptotic pathway to host defence was demonstrated using mice deficient in the FAS signalling pathway, which showed delayed clearance of the EPEC-like mouse pathogen Citrobacter rodentium and reversion to virulence of an nleB mutant. The activity of NleB suggests that EPEC and other attaching and effacing (A/E) pathogens antagonise death receptor induced apoptosis of infected cells, thereby blocking a major antimicrobial host response.

Intestinal colonization by enteropathogenic and enterohemorrhagic Escherichia coli requires the locus of enterocyte effacement-encoded type III secretion system. We report that NleC and NleD are translocated into host cells via this system. Deletion mutants induced attaching and effacing lesions in vitro, while infection of calves or lambs showed that neither gene was required for colonization.Attaching and effacing strains of Escherichia coli (AEEC) comprise a group of gastrointestinal pathogens of humans and animals that induce distinctive attaching and effacing (A/E) lesions within the host intestinal mucosa (28). A/E lesions are characterized by intimate attachment of the bacteria to the host cell surface, the localized destruction of intestinal microvilli, and the rearrangement of host cytoskeletal proteins beneath the adherent bacteria (19). A/E lesion formation is mediated by the products of the locus of enterocyte effacement (LEE) pathogenicity island (PAI) (24). The LEE of enteropathogenic E. coli (EPEC) contains 41 open reading frames (11) of which approximately half encode a type III secretion system (TTSS), which directs the secretion of several LEE-encoded translocator (EspA, EspB, and EspD) and effector (EspG, EspH, Map, Tir, EspZ, and EspF) proteins. However, only Tir plays a direct role in A/E lesion formation (12, 13). The LEE PAI is well conserved among AEEC strains, including the closely related human pathogen enterohemorrhagic E. coli (EHEC) (30) and the animal pathogens rabbit-specific enteropathogenic E. coli (35) and Citrobacter rodentium (7).Recently, several novel effector proteins that are not encoded within the LEE but are translocated into host cells by the LEE-encoded TTSS were described. Cycle inhibiting factor (designated Cif), although not required for A/E lesion formation, induces host cell cycle arrest and reorganization of the actin cytoskeleton (21). EspI/NleA (for non-LEE encoded) (Z6024) (15, 25) is also not required for A/E lesion formation but is essential for full virulence in the C. rodentium mouse model of infection and was found to be more frequently associated with EHEC strains isolated from symptomatic than from asymptomatic patients (26). EspJ (5) and TccP/EspF U (3, 14) are carried on prophage CP933U. In the murine model of infection, an espJ deletion mutant of C. rodentium showed altered colonization and clearance dynamics, although the protein was not required for A/E lesion formation (5). In contrast, TccP/EspF U is essential for A/E lesion formation by EHEC O157:H7 (3, 14) and disruption of the tight junctions (38). In EHEC O157:H7-infected cells, TccP functions as a linker between Tir and N-WASP and compensates functionally for the absence of the host adapter protein, Nck, which in EPECinfected cells is recruited to the pedestal upon tyrosine phosphorylation of Tir (1, 2). EspG2, which is encoded on the EspC PAI, shares similarity with EspG (10) and was recently shown to interact with tubulin and trigger the dissociation of microtubules beneath adherent bacter...

EspF of enteropathogenic Escherichia coli targets mitochondria and subverts a number of cellular functions. EspF consists of six putative Src homology 3 (SH3) domain binding motifs. In this study we identified sorting nexin 9 (SNX9) as a host cell EspF binding partner protein, which binds EspF via its amino-terminal SH3 region. Coimmunoprecipitation and confocal microscopy showed specific EspF-SNX9 interaction and nonmitochondrial protein colocalization in infected epithelial cells.

SummaryA key strategy in microbial pathogenesis is the subversion of the first line of cellular immune defences presented by professional phagocytes. Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC respectively) remain extracellular while colonizing the gut mucosa by attaching and effacing mechanism. EPEC use the type three secretion system effector protein EspF to prevent their own uptake into macrophages. EPEC can also block in trans the internalization of IgG-opsonized particles. In this study, we show that EspJ is the type three secretion system effector protein responsible for trans-inhibition of macrophage opsonophagocytosis by both EPEC and EHEC. While EspF plays no role in trans-inhibition of opsonophagocytosis, espJ mutants of EPEC or EHEC are unable to block uptake of opsonized sheep red blood cells (RBC), a phenotype that is rescued upon complementation with the espJ gene. Importantly, ectopic expression of EspJ EHEC in phagocytes is sufficient to inhibit internalization of both IgG-and C3bi-opsonized RBC. These results suggest that EspJ targets a basic mechanism common to these two unrelated phagocytic receptors. Moreover, EspF and EspJ target independent aspects of the phagocytic function of mammalian macrophages in vitro.

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC) are important human pathogens that rely on translocation of type III secretion system (T3SS) effectors for subversion of signal transduction pathways and colonization of the mammalian gut mucosa. While a core set of effectors is conserved between EPEC and EHEC strains, a growing number of accessory effectors that were found at various frequencies in clinical and environmental isolates have been recently identified. Recent genome projects identified espV as a pseudogene in EHEC but a putative functional gene in EPEC strains E110019 and E22 and the closely related mouse pathogen Citrobacter rodentium. The aim of this study was to determine the distribution of espV among clinical EPEC and EHEC strains and to investigate its function and role in pathogenesis. espV was found in 16% of the tested strains. While deletion of espV from C. rodentium did not affect colonization dynamics or fitness in mixed infections, expression of EspV in mammalian cells led to drastic morphological alterations, which were characterized by nuclear condensation, cell rounding, and formation of dendrite-like projections. Expression of EspV in yeast resulted in a dramatic increase in cell size and irreversible growth arrest. Although the role of EspV in infection and its target host cell protein(s) require further investigation, the data point to a novel mechanism by which the T3SS subverts cell signaling.