Hierarchical protein targeting and secretion is controlled by an affinity switch in the type III secretion system of enteropathogenic Escherichia coli.

Type III secretion (T3S), a protein export pathway common to Gram-negative pathogens, comprises a trans-envelope syringe, the injectisome, with a cytoplasm-facing translocase channel. Exported substrates are chaperone-delivered to the translocase, EscV in enteropathogenic Escherichia coli, and cross it in strict hierarchical manner, for example, first "translocators", then "effectors". We dissected T3S substrate targeting and hierarchical switching by reconstituting them in vitro using inverted inner membrane vesicles. EscV recruits and conformationally activates the tightly membrane-associated pseudo-effector SepL and its chaperone SepD. This renders SepL a high-affinity receptor for translocator/chaperone pairs, recognizing specific chaperone signals. In a second, SepD-coupled step, translocators docked on SepL become secreted. During translocator secretion, SepL/SepD suppress effector/chaperone binding to EscV and prevent premature effector secretion. Disengagement of the SepL/SepD switch directs EscV to dedicated effector export. These findings advance molecular understanding of T3S and reveal a novel mechanism for hierarchical trafficking regulation in protein secretion channels.

The Citrobacter rodentium type III secretion system effector EspO affects mucosal damage repair and antimicrobial responses.

Infection with Citrobacter rodentium triggers robust tissue damage repair responses, manifested by secretion of IL-22, in the absence of which mice succumbed to the infection. Of the main hallmarks of C. rodentium infection are colonic crypt hyperplasia (CCH) and dysbiosis. In order to colonize the host and compete with the gut microbiota, C. rodentium employs a type III secretion system (T3SS) that injects effectors into colonic intestinal epithelial cells (IECs). Once injected, the effectors subvert processes involved in innate immune responses, cellular metabolism and oxygenation of the mucosa. Importantly, the identity of the effector/s triggering the tissue repair response is/are unknown. Here we report that the effector EspO ,an orthologue of OspE found in Shigella spp, affects proliferation of IECs 8 and 14 days post C. rodentium infection as well as secretion of IL-22 from colonic explants. While we observed no differences in the recruitment of group 3 innate lymphoid cells (ILC3s) and T cells, which are the main sources of IL-22 at the early and late stages of C. rodentium infection respectively, infection with ΔespO was characterized by diminished recruitment of sub-mucosal neutrophils, which coincided with lower abundance of Mmp9 and chemokines (e.g. S100a8/9) in IECs. Moreover, mice infected with ΔespO triggered significantly lesser nutritional immunity (e.g. calprotectin, Lcn2) and expression of antimicrobial peptides (Reg3ß, Reg3γ) compared to mice infected with WT C. rodentium. This overlapped with a decrease in STAT3 phosphorylation in IECs. Importantly, while the reduced CCH and abundance of antimicrobial proteins during ΔespO infection did not affect C. rodentium colonization or the composition of commensal Proteobacteria, they had a subtle consequence on Firmicutes subpopulations. EspO is the first bacterial virulence factor that affects neutrophil recruitment and secretion of IL-22, as well as expression of antimicrobial and nutritional immunity proteins in IECs.

A type III effector antagonizes death receptor signalling during bacterial gut infection.

Successful infection by enteric bacterial pathogens depends on the ability of the bacteria to colonize the gut, replicate in host tissues and disseminate to other hosts. Pathogens such as Salmonella, Shigella and enteropathogenic and enterohaemorrhagic (EPEC and EHEC, respectively) Escherichia coli use a type III secretion system (T3SS) to deliver virulence effector proteins into host cells during infection that promote colonization and interfere with antimicrobial host responses. 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-acetylglucosamine transferase activity of NleB1, which specifically modified Arg 117 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 pathogens antagonize death-receptor-induced apoptosis of infected cells, thereby blocking a major antimicrobial host response.

The Type III Secretion System Effector SptP of Salmonella enterica Serovar Typhi.

Strains of the various Salmonella enterica serovars cause gastroenteritis or typhoid fever in humans, with virulence depending on the action of two type III secretion systems (Salmonella pathogenicity island 1 [SPI-1] and SPI-2). SptP is a Salmonella SPI-1 effector, involved in mediating recovery of the host cytoskeleton postinfection. SptP requires a chaperone, SicP, for stability and secretion. SptP has 94% identity between S. enterica serovar Typhimurium and S Typhi; direct comparison of the protein sequences revealed that S Typhi SptP has numerous amino acid changes within its chaperone-binding domain. Subsequent comparison of ΔsptP S Typhi and S. Typhimurium strains demonstrated that, unlike SptP in S. Typhimurium, SptP in S Typhi was not involved in invasion or cytoskeletal recovery postinfection. Investigation of whether the observed amino acid changes within SptP of S Typhi affected its function revealed that S Typhi SptP was unable to complement S. Typhimurium ΔsptP due to an absence of secretion. We further demonstrated that while S. Typhimurium SptP is stable intracellularly within S Typhi, S Typhi SptP is unstable, although stability could be recovered following replacement of the chaperone-binding domain with that of S. Typhimurium. Direct assessment of the strength of the interaction between SptP and SicP of both serovars via bacterial two-hybrid analysis demonstrated that S Typhi SptP has a significantly weaker interaction with SicP than the equivalent proteins in S. Typhimurium. Taken together, our results suggest that changes within the chaperone-binding domain of SptP in S Typhi hinder binding to its chaperone, resulting in instability, preventing translocation, and therefore restricting the intracellular activity of this effector. IMPORTANCE: Studies investigating Salmonella pathogenesis typically rely on Salmonella Typhimurium, even though Salmonella Typhi causes the more severe disease in humans. As such, an understanding of S. Typhi pathogenesis is lacking. Differences within the type III secretion system effector SptP between typhoidal and nontyphoidal serovars led us to characterize this effector within S Typhi. Our results suggest that SptP is not translocated from typhoidal serovars, even though the loss of sptP results in virulence defects in S. Typhimurium. Although SptP is just one effector, our results exemplify that the behavior of these serovars is significantly different and genes identified to be important for S. Typhimurium virulence may not translate to S Typhi.

The Enterohemorrhagic Escherichia coli Effector EspW Triggers Actin Remodeling in a Rac1-Dependent Manner.

Enterohemorrhagic Escherichia coli (EHEC) is a diarrheagenic pathogen that colonizes the gut mucosa and induces attaching-and-effacing lesions. EHEC employs a type III secretion system (T3SS) to translocate 50 effector proteins that hijack and manipulate host cell signaling pathways, which allow bacterial colonization and subversion of immune responses and disease progression. The aim of this study was to characterize the T3SS effector EspW. We found espW in the sequenced O157:H7 and non-O157 EHEC strains as well as in Shigella boydii Furthermore, a truncated version of EspW, containing the first 206 residues, is present in EPEC strains belonging to serotype O55:H7. Screening a collection of clinical EPEC isolates revealed that espW is present in 52% of the tested strains. We report that EspW modulates actin dynamics in a Rac1-dependent manner. Ectopic expression of EspW results in formation of unique membrane protrusions. Infection of Swiss cells with an EHEC espW deletion mutant induces a cell shrinkage phenotype that could be rescued by Rac1 activation via expression of the bacterial guanine nucleotide exchange factor, EspT. Furthermore, using a yeast two-hybrid screen, we identified the motor protein Kif15 as a potential interacting partner of EspW. Kif15 and EspW colocalized in cotransfected cells, while ectopically expressed Kif15 localized to the actin pedestals following EHEC infection. The data suggest that Kif15 recruits EspW to the site of bacterial attachment, which in turn activates Rac1, resulting in modifications of the actin cytoskeleton that are essential to maintain cell shape during infection.

Model of Host-Pathogen Interaction Dynamics Links In Vivo Optical Imaging and Immune Responses.

Tracking disease progression in vivo is essential for the development of treatments against bacterial infection. Optical imaging has become a central tool for in vivo tracking of bacterial population development and therapeutic response. For a precise understanding of in vivo imaging results in terms of disease mechanisms derived from detailed postmortem observations, however, a link between the two is needed. Here, we develop a model that provides that link for the investigation of Citrobacter rodentium infection, a mouse model for enteropathogenic Escherichia coli (EPEC). We connect in vivo disease progression of C57BL/6 mice infected with bioluminescent bacteria, imaged using optical tomography and X-ray computed tomography, to postmortem measurements of colonic immune cell infiltration. We use the model to explore changes to both the host immune response and the bacteria and to evaluate the response to antibiotic treatment. The developed model serves as a novel tool for the identification and development of new therapeutic interventions.

Tir Triggers Expression of CXCL1 in Enterocytes and Neutrophil Recruitment during Citrobacter rodentium Infection.

The hallmarks of enteropathogenic Escherichia coli (EPEC) infection are formation of attaching and effacing (A/E) lesions on mucosal surfaces and actin-rich pedestals on cultured cells, both of which are dependent on the type III secretion system effector Tir. Following translocation into cultured cells and clustering by intimin, Tir Y474 is phosphorylated, leading to recruitment of Nck, activation of N-WASP, and actin polymerization via the Arp2/3 complex. A secondary, weak, actin polymerization pathway is triggered via an NPY motif (Y454). Importantly, Y454 and Y474 play no role in A/E lesion formation on mucosal surfaces following infection with the EPEC-like mouse pathogen Citrobacter rodentium. In this study, we investigated the roles of Tir segments located upstream of Y451 and downstream of Y471 in C. rodentium colonization and A/E lesion formation. We also tested the role that Tir residues Y451 and Y471 play in host immune responses to C. rodentium infection. We found that deletion of amino acids 382 to 462 or 478 to 547 had no impact on the ability of Tir to mediate A/E lesion formation, although deletion of amino acids 478 to 547 affected Tir translocation. Examination of enterocytes isolated from infected mice revealed that a C. rodentium strain expressing Tir_Y451A/Y471A recruited significantly fewer neutrophils to the colon and triggered less colonic hyperplasia on day 14 postinfection than the wild-type strain. Consistently, enterocytes isolated from mice infected with C. rodentium expressing Tir_Y451A/Y471A expressed significantly less CXCL1. These result show that Tir-induced actin remodeling plays a direct role in modulation of immune responses to C. rodentium infection.

Fermented dairy products modulate Citrobacter rodentium-induced colonic hyperplasia.

We evaluated the protective effects of fermented dairy products (FDPs) in an infection model, using the mouse pathogen Citrobacter rodentium (CR). Treatment of mice with FDP formulas A, B, and C or a control product did not affect CR colonization, organ specificity, or attaching and effacing lesion formation. Fermented dairy product A (FDP-A), but neither the supernatant from FDP-A nor ß-irradiated (IR) FDP-A, caused a significant reduction in colonic crypt hyperplasia and CR-associated pathology. Profiling the gut microbiota revealed that IR-FDP-A promoted higher levels of phylotypes belonging to Alcaligenaceae and a decrease in Lachnospiraceae (Ruminococcus) during CR infection. Conversely, FDP-A prevented a decrease in Ruminococcus and increased Turicibacteraceae (Turicibacter). Importantly, loss of Ruminococcus and Turicibacter has been associated with susceptibility to dextran sodium sulfate-induced colitis. Our results demonstrate that viable bacteria in FDP-A reduced CR-induced colonic crypt hyperplasia and prevented the loss of key bacterial genera that may contribute to disease pathology.

The enteropathogenic E. coli effector EspH promotes actin pedestal formation and elongation via WASP-interacting protein (WIP).

Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC) are diarrheagenic pathogens that colonize the gut mucosa via attaching-and-effacing lesion formation. EPEC and EHEC utilize a type III secretion system (T3SS) to translocate effector proteins that subvert host cell signalling to sustain colonization and multiplication. EspH, a T3SS effector that modulates actin dynamics, was implicated in the elongation of the EHEC actin pedestals. In this study we found that EspH is necessary for both efficient pedestal formation and pedestal elongation during EPEC infection. We report that EspH induces actin polymerization at the bacterial attachment sites independently of the Tir tyrosine residues Y474 and Y454, which are implicated in binding Nck and IRSp53/ITRKS respectively. Moreover, EspH promotes recruitment of neural Wiskott-Aldrich syndrome protein (N-WASP) and the Arp2/3 complex to the bacterial attachment site, in a mechanism involving the C-terminus of Tir and the WH1 domain of N-WASP. Dominant negative of WASP-interacting protein (WIP), which binds the N-WASP WH1 domain, diminished EspH-mediated actin polymerization. This study implicates WIP in EPEC-mediated actin polymerization and pedestal elongation and represents the first instance whereby N-WASP is efficiently recruited to the EPEC attachment sites independently of the Tir:Nck and Tir:IRTKS/IRSp53 pathways. Our study reveals the intricacies of Tir and EspH-mediated actin signalling pathways that comprise of distinct, convergent and synergistic signalling cascades.

The WxxxE effector EspT triggers expression of immune mediators in an Erk/JNK and NF-κB-dependent manner.

Enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic E. coli (EHEC) and Citrobacter rodentium colonize their respective hosts while forming attaching and effacing lesions. Their infection strategy relies on translocation of a battery of type III secretion system effectors, including Map, EspM and EspT, which belong to the WxxxE/SopE family of guanine nucleotide exchange factors. Using the C. rodentium mouse model we found that EspT triggers expression of KC and TNFα in vivo. Indeed, a growing body of evidence suggests that, in addition to subversion of actin dynamics, the SopE and the WxxxE effectors activate signalling pathways involved in immune responses. In this study we found that EspT induces expression of the pro-inflammatory mediators cyclooxygenase-2 (COX-2) an enzyme involved in production of prostaglandin E(2) (PGE2), interleukin (Il)-8 and Il-1ß in U937 human macrophages by activating the nuclear factor kappa-B (NF-κB), the extracellular signal-regulated kinases 1 and 2 (Erk1/2) and c-Jun N-terminal kinase (JNK) pathways. Since EspT modulates the activation of Cdc42 and Rac1, which mediates bacterial invasion into epithelial cells, we investigated the involvement of these Rho GTPases and bacterial invasion on pro-inflammatory responses and found that (i) Rac1, but not Cdc42, is involved in EspT-induced Il-8 and Il-1ß secretion and (ii) cytochalasin D inhibits EspT-induced EPEC invasion into U937 but not Il-8 or Il-1ß secretion. These results suggest that while EPEC translocates a number of effectors (i.e. NleC, NleD, NleE, NleH) that inhibit inflammation, a subset of strains, which encode EspT, employ an infection strategy that also involves upregulation of immune mediators.

Dissecting the role of the Tir:Nck and Tir:IRTKS/IRSp53 signalling pathways in vivo.

Attaching and effacing (A/E) lesions and actin polymerization, the hallmark of enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and Citrobacter rodentium (CR) infections, are dependent on the effector Tir. Phosphorylation of Tir(EPEC/CR) Y474/1 leads to recruitment of Nck and neural Wiskott-Aldrich syndrome protein (N-WASP) and strong actin polymerization in cultured cells. Tir(EPEC/CR) also contains an Asn-Pro-Tyr (NPY(454/1)) motif, which triggers weak actin polymerization. In EHEC the NPY(458) actin polymerization pathway is amplified by TccP/EspF(U), which is recruited to Tir via IRSp53 and/or insulin receptor tyrosine kinase substrate (IRTKS). Here we used C. rodentium to investigate the different Tir signalling pathways in vivo. Following infection with wild-type C. rodentium IRTKS, but not IRSp53, was recruited to the bacterial attachment sites. Similar results were seen after infection of human ileal explants with EHEC. Mutating Y471 or Y451 in Tir(CR) abolished recruitment of Nck and IRTKS respectively, but did not affect recruitment of N-WASP or A/E lesion formation. This suggests that despite their crucial role in actin polymerization in cultured cells the Tir:Nck and Tir:IRTKS pathways are not essential for N-WASP recruitment or A/E lesion formation in vivo. Importantly, wild-type C. rodentium out-competed the tir tyrosine mutants during mixed infections. These results uncouple the Tir:Nck and Tir:IRTKS pathways from A/E lesion formation in vivo but assign them an important in vivo role.

The T3SS effector EspT defines a new category of invasive enteropathogenic E. coli (EPEC) which form intracellular actin pedestals.

Enteropathogenic Escherichia coli (EPEC) strains are defined as extracellular pathogens which nucleate actin rich pedestal-like membrane extensions on intestinal enterocytes to which they intimately adhere. EPEC infection is mediated by type III secretion system effectors, which modulate host cell signaling. Recently we have shown that the WxxxE effector EspT activates Rac1 and Cdc42 leading to formation of membrane ruffles and lamellipodia. Here we report that EspT-induced membrane ruffles facilitate EPEC invasion into non-phagocytic cells in a process involving Rac1 and Wave2. Internalized EPEC resides within a vacuole and Tir is localized to the vacuolar membrane, resulting in actin polymerization and formation of intracellular pedestals. To the best of our knowledge this is the first time a pathogen has been shown to induce formation of actin comets across a vacuole membrane. Moreover, our data breaks the dogma of EPEC as an extracellular pathogen and defines a new category of invasive EPEC.

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.

N-terminal type III secretion signal of enteropathogenic Escherichia coli translocator proteins.

We report that the N terminus of the type III secretion system translocator proteins EspB, EspD, and EspA mediate protein secretion and translocation from wild-type enteropathogenic Escherichia coli and hypersecretion from sepL and sepD mutants. EspA containing the translocation signal of Map and Tir containing the secretion signal of EspA are biologically active.

The Citrobacter rodentium genome sequence reveals convergent evolution with human pathogenic Escherichia coli.

Citrobacter rodentium (formally Citrobacter freundii biotype 4280) is a highly infectious pathogen that causes colitis and transmissible colonic hyperplasia in mice. In common with enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively), C. rodentium exploits a type III secretion system (T3SS) to induce attaching and effacing (A/E) lesions that are essential for virulence. Here, we report the fully annotated genome sequence of the 5.3-Mb chromosome and four plasmids harbored by C. rodentium strain ICC168. The genome sequence revealed key information about the phylogeny of C. rodentium and identified 1,585 C. rodentium-specific (without orthologues in EPEC or EHEC) coding sequences, 10 prophage-like regions, and 17 genomic islands, including the locus for enterocyte effacement (LEE) region, which encodes a T3SS and effector proteins. Among the 29 T3SS effectors found in C. rodentium are all 22 of the core effectors of EPEC strain E2348/69. In addition, we identified a novel C. rodentium effector, named EspS. C. rodentium harbors two type VI secretion systems (T6SS) (CTS1 and CTS2), while EHEC contains only one T6SS (EHS). Our analysis suggests that C. rodentium and EPEC/EHEC have converged on a common host infection strategy through access to a common pool of mobile DNA and that C. rodentium has lost gene functions associated with a previous pathogenic niche.

Bacterial guanine nucleotide exchange factors SopE-like and WxxxE effectors.

Subversion of Rho family small GTPases, which control actin dynamics, is a common infection strategy used by bacterial pathogens. In particular, Salmonella enterica serovar Typhimurium, Shigella flexneri, enteropathogenic Escherichia coli (EPEC), and enterohemorrhagic Escherichia coli (EHEC) translocate type III secretion system (T3SS) effector proteins to modulate the Rho GTPases RhoA, Cdc42, and Rac1, which trigger formation of stress fibers, filopodia, and lamellipodia/ruffles, respectively. The Salmonella effector SopE is a guanine nucleotide exchange factor (GEF) that activates Rac1 and Cdc42, which induce "the trigger mechanism of cell entry." Based on a conserved Trp-xxx-Glu motif, the T3SS effector proteins IpgB1 and IpgB2 of Shigella, SifA and SifB of Salmonella, and Map of EPEC and EHEC were grouped together into a WxxxE family; recent studies identified the T3SS EPEC and EHEC effectors EspM and EspT as new family members. Recent structural and functional studies have shown that representatives of the WxxxE effectors share with SopE a 3-D fold and GEF activity. In this minireview, we summarize contemporary findings related to the SopE and WxxxE GEFs in the context of their role in subverting general host cell signaling pathways and infection.

The mechanisms used by enteropathogenic Escherichia coli to control filopodia dynamics.

Enteropathogenic Escherichia coli (EPEC) subverts actin dynamics in eukaryotic cells by injecting effector proteins via a type III secretion system. First, WxxxE effector Map triggers transient formation of filopodia. Then, following recovery from the filopodial signals, EPEC triggers robust actin polymerization via a signalling complex comprising Tir and the adaptor proteins Nck. In this paper we show that Map triggers filopodia formation by activating Cdc42; expression of dominant-negative Cdc42 or knock-down of Cdc42 by siRNA impaired filopodia formation. In addition, Map binds PDZ1 of NHERF1. We show that Map-NHERF1 interaction is needed for filopodia stabilization in a process involving ezrin and the RhoA/ROCK cascade; expression of dominant-negative ezrin and RhoA or siRNA knock-down of RhoA lead to rapid elimination of filopodia. Moreover, we show that formation of the Tir-Nck signalling complex leads to filopodia withdrawal. Recovery from the filopodial signals requires phosphorylation of a Tir tyrosine (Y474) residue and actin polymerization pathway as both infection of cells with EPEC expressing TirY474S or infection of Nck knockout cells with wild-type EPEC resulted in persistence of filopodia. These results show that EPEC effectors modulate actin dynamics by temporal subverting the Rho GTPases and other actin polymerization pathways for the benefit of the adherent pathogen.

Modelling of infection by enteropathogenic Escherichia coli strains in lineages 2 and 4 ex vivo and in vivo by using Citrobacter rodentium expressing TccP.

Enteropathogenic Escherichia coli (EPEC) strains colonize the human gut mucosa via attaching-and-effacing (A/E) lesion formation, while in vitro they employ diverse strategies to trigger actin polymerization. Strains belonging to the EPEC-1 lineage trigger strong actin polymerization via tyrosine phosphorylation of the type III secretion system (T3SS) effector Tir, recruitment of Nck, and activation of N-WASP. Strains belonging to EPEC-2 and EPEC-4 can trigger strong actin polymerization by dual mechanisms, since while employing the Tir-Nck pathway they can additionally activate N-WASP via the T3SS effectors TccP2 and TccP, respectively. It is currently not known if the ability to trigger actin polymerization by twin mechanisms increases in vivo virulence or fitness. Since mice are resistant to EPEC infection, in vivo studies are frequently done using the murine model pathogen Citrobacter rodentium, which shares with EPEC-1 strains the ability to induce A/E lesions and trigger strong actin polymerization via the Tir:Nck pathway. In order to model infections with EPEC-2 and EPEC-4, we constructed C. rodentium strains expressing TccP. Using a mouse intestinal in vitro organ culture model and oral gavage into C57BL/6 mice, we have shown that TccP can cooperate with Tir of C. rodentium. The recombinant strains induced typical A/E lesions ex vivo and in vivo. Expression of TccP did not alter C. rodentium colonization dynamics or pathology. In competition with the wild-type strain, expression of TccP in C. rodentium did not confer a competitive advantage.

Subversion of actin dynamics by EspM effectors of attaching and effacing bacterial pathogens.

Rho GTPases are common targets of bacterial toxins and type III secretion system effectors. IpgB1 and IpgB2 of Shigella and Map of enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli were recently grouped together on the basis that they share a conserved WxxxE motif. In this study, we characterized six WxxxE effectors from attaching and effacing pathogens: TrcA and EspM1 of EPEC strain B171, EspM1 and EspM2 of EHEC strain Sakai and EspM2 and EspM3 of Citrobacter rodentium. We show that EspM2 triggers formation of global parallel stress fibres, TrcA and EspM1 induce formation of localized parallel stress fibres and EspM3 triggers formation of localized radial stress fibres. Using EspM2 and EspM3 as model effectors, we report that while substituting the conserved Trp with Ala abolished activity, conservative Trp to Tyr or Glu to Asp substitutions did not affect stress-fibre formation. We show, using dominant negative constructs and chemical inhibitors, that the activity of EspM2 and EspM3 is RhoA and ROCK-dependent. Using Rhotekin pull-downs, we have shown that EspM2 and EspM3 activate RhoA; translocation of EspM2 and EspM3 triggered phosphorylation of cofilin. These results suggest that the EspM effectors modulate actin dynamics by activating the RhoA signalling pathway.

The S. Typhi effector StoD is an E3/E4 ubiquitin ligase which binds K48- and K63-linked diubiquitin.

Salmonella enterica (e.g., serovars Typhi and Typhimurium) relies on translocation of effectors via type III secretion systems (T3SS). Specialization of typhoidal serovars is thought to be mediated via pseudogenesis. Here, we show that the Salmonella Typhi STY1076/t1865 protein, named StoD, a homologue of the enteropathogenic Escherichia coli/enterohemorrhagic E. coli/Citrobacter rodentium NleG, is a T3SS effector. The StoD C terminus (StoD-C) is a U-box E3 ubiquitin ligase, capable of autoubiquitination in the presence of multiple E2s. The crystal structure of the StoD N terminus (StoD-N) at 2.5 Å resolution revealed a ubiquitin-like fold. In HeLa cells expressing StoD, ubiquitin is redistributed into puncta that colocalize with StoD. Binding assays showed that StoD-N and StoD-C bind the same exposed surface of the ß-sheet of ubiquitin, suggesting that StoD could simultaneously interact with two ubiquitin molecules. Consistently, StoD interacted with both K63- (KD = 5.6 ± 1 µM) and K48-linked diubiquitin (KD = 15 ± 4 µM). Accordingly, we report the first S. Typhi-specific T3SS effector. We suggest that StoD recognizes and ubiquitinates pre-ubiquitinated targets, thus subverting intracellular signaling by functioning as an E4 enzyme.