Enteropathogenic and enterohaemorrhagic Escherichia coli: even more subversive elements

The human pathogens enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) share a unique mechanism of colonization that results from the concerted action of effector proteins translocated into the host cell by a type III secretion system (T3SS). EPEC and EHEC not only induce characteristic attaching and effacing (A/E) lesions, but also subvert multiple host cell signalling pathways during infection. Our understanding of the mechanisms by which A/E pathogens hijack host cell signalling has advanced dramatically in recent months with the identification of novel activities for many effectors. In addition to further characterization of established effectors (Tir, EspH and Map), new effectors have emerged as important mediators of virulence through activities such as mimicry of Rho guanine nucleotide exchange factors (Map and EspM), inhibition of apoptosis (NleH and NleD), interference with inflammatory signalling pathways (NleB, NleC, NleE and NleH) and phagocytosis (EspF, EspH and EspJ). The findings have highlighted the multifunctional nature of the effectors and their ability to participate in redundant, synergistic or antagonistic relationships, acting in a co-ordinated spatial and temporal manner on different host organelles and cellular pathways during infection.     

EspH,a new cytoskeleton-modulating effector of enterohaemorrhagic and enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) are closely related pathogens. During infection, EPEC and EHEC use a type III secretion system (TTSS) to translocate effector proteins into the infected cells and thereby modify specific host functions. These include transient filopodium formation which is Cdc42-dependent. Filopodia formation is followed by assembly of actin pedestals, the process enhanced by inhibition of Cdc42. We discovered that orf 18 of the enterocyte effacement locus encodes a new effector, which we termed EspH. We show that EspH is translocated efficiently into the infected cells by the TTSS and localizes beneath the EPEC microcolonies. Inactivation of espH resulted in enhanced formation of filopodia and attenuated the pedestals formation. Furthermore, overexpression of EspH resulted in strong repression of filopodium formation and heightened pedestal formation. We also demonstrate that overexpression of EspH by EHEC induces marked elongation of the typically flat pedestals. Similar pedestal elongation was seen upon infection of COS cells overexpressing EspH. EspH transiently expressed by the COS cells was localized to the membrane and disrupted the actin cytoskeletal structure. Our findings indicate that EspH is a modulator of the host actin cytoskeleton structure.     

Here,there, and everywhere: How pathogenic Escherichia coli sense and respond to gastrointestinal biogeography

Gastrointestinal (GI) pathogens enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC), and related mouse pathogen Citrobacter rodentium, are referred to as attaching and effacing (AE) pathogens for the lesions they form upon colonisation of the host epithelium. EPEC, EHEC, and C. rodentium are well known to use a type III secretion system to intimately attach to intestinal cells and secrete bacterial effectors to manipulate host cell processes. Less well known is the ability of AE pathogens to overcome significant physiological and microbial barriers and target specific gut niches for initial colonisation of the host epithelium. This review considers recent work highlighting the biogeography of the GI tract as it applies to colonisation by enteric pathogens, including environmental barriers to enteric infection, signals sensed by AE pathogens for navigation of the GI tract, and the tools AE pathogens use to respond to the changing host environment.     

Bringing down the host: enteropathogenic and enterohaemorrhagic Escherichia coli effector‐mediated subversion of host innate immune pathways

Enteric bacterial pathogens commonly use a type III secretion system (T3SS) to successfully infect intestinal epithelial cells and survive and proliferate in the host. Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC; EHEC) colonize the human intestinal mucosa, form characteristic histological lesions on the infected epithelium and require the T3SS for full virulence. T3SS effectors injected into host cells subvert cellular pathways to execute a variety of functions within infected host cells. The EPEC and EHEC effectors that subvert innate immune pathways – specifically those involved in phagocytosis, host cell survival, apoptotic cell death and inflammatory signalling – are all required to cause disease. These processes are reviewed within, with a focus on recent work that has provided insights into the functions and host cell targets of these effectors.     

The cell death response to enteropathogenic Escherichia coli infection

Given the critical roles of inflammation and programmed cell death in fighting infection, it is not surprising that many bacterial pathogens have evolved strategies to inactivate these defences. The causative agent of infant diarrhoea, enteropathogenic Escherichia coli (EPEC), is an extracellular, intestinal pathogen that blocks both inflammation and programmed cell death. EPEC attaches to enterocytes, remains in the gut lumen and utilizes a type III secretion system (T3SS) to inject multiple virulence effector proteins directly into the infected cell, many of which subvert host antimicrobial processes through the disruption of signalling pathways. Recently, T3SS effector proteins from EPEC have been identified that inhibit death receptor‐induced apoptosis. Here we review the mechanisms used by EPEC T3SS effectors to manipulate apoptosis and promote host cell survival and discuss the role of these activities during infection.     

Cholesterol binding by the bacterial type III translocon is essential for virulence effector delivery into mammalian cells

A ubiquitous early step in infection of man and animals by enteric bacterial pathogens like Salmonella, Shigella and enteropathogenic Escherichia coli (EPEC) is the translocation of virulence effector proteins into mammalian cells via specialized type III secretion systems (TTSSs). Translocated effectors subvert the host cytoskeleton and stimulate signalling to promote bacterial internalization or survival. Target cell plasma membrane cholesterol is central to pathogen-host cross-talk, but the precise nature of its critical contribution remains unknown. Using in vitro cholesterol-binding assays, we demonstrate that Salmonella (SipB) and Shigella (IpaB) TTSS translocon components bind cholesterol with high affinity. Direct visualization of cell-associated fluorescently labelled SipB and parallel immunogold transmission electron microscopy revealed that cholesterol levels limit both the amount and distribution of plasma membrane-integrated translocon. Correspondingly, cholesterol depletion blocked effector translocation into cultured mammalian cells by not only the related Salmonella and Shigella TTSSs, but also the more divergent EPEC system. The data reveal that cholesterol-dependent association of the bacterial TTSS translocon with the target cell plasma membrane is essential for translocon activation and effector delivery into mammalian cells.     

The pathogenic E. coli type III effector EspZ interacts with host CD98 and facilitates host cell prosurvival signalling

Enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC respectively) are diarrhoeal pathogens that cause the formation of attaching and effacing (A/E) lesions on infected host cells. These pathogens encode a type III secretion system (T3SS) used to inject effector proteins directly into host cells, an essential requirement for virulence. In this study, we identified a function for the type III secreted effector EspZ. Infection with EPEC ΔespZ caused increased cytotoxicity in HeLa and MDCK cells compared with wild‐type EPEC, and expressing espZ in cells abrogated this effect. Using yeast two‐hybrid, proteomics, immunofluorescence and co‐immunoprecipitation, it was demonstrated that EspZ interacts with the host protein CD98, which contributes to protection against EPEC‐mediated cytotoxicity. EspZ enhanced phosphorylation of focal adhesion kinase (FAK) and AKT during infection with EPEC, but CD98 only appeared to facilitate FAK phosphorylation. This study provides evidence that EspZ and CD98 promote host cell survival mechanisms involving FAK during A/E pathogen infection.     

Tandem tyrosine phosphosites in the Enteropathogenic Escherichia coli chaperone CesT are required for differential type III effector translocation and virulence

Enteropathogenic Escherichia coli (EPEC) use a type 3 secretion system (T3SS) for injection of effectors into host cells and intestinal colonization. Here, we demonstrate that the multicargo chaperone CesT has two strictly conserved tyrosine phosphosites, Y152 and Y153 that regulate differential effector secretion in EPEC. Conservative substitution of both tyrosine residues to phenylalanine strongly attenuated EPEC type 3 effector injection into host cells, and limited Tir effector mediated intimate adherence during infection. EPEC expressing a CesT Y152F variant were deficient for NleA effector expression and exhibited significantly reduced translocation of NleA into host cells during infection. Other effectors were observed to be dependent on CesT Y152 for maximal translocation efficiency. Unexpectedly, EPEC expressing a CesT Y153F variant exhibited significantly enhanced effector translocation of many CesT‐interacting effectors, further implicating phosphosites Y152 and Y153 in CesT functionality. A mouse infection model of intestinal disease using Citrobacter rodentium revealed that CesT tyrosine substitution variants displayed delayed colonization and were more rapidly cleared from the intestine. These data demonstrate genetically separable functions for tandem tyrosine phosphosites within CesT. Therefore, CesT via its C‐terminal tyrosine phosphosites, has relevant roles beyond typical type III secretion chaperones that interact and stabilize effector proteins.     

The bacterial effectors EspG and EspG2 induce a destructive calpain activity that is kept in check by the co‐delivered Tir effector

Bacterial pathogens deliver multiple effector proteins into eukaryotic cells to subvert host cellular processes and an emerging theme is the cooperation between different effectors. Here, we reveal that a fine balance exists between effectors that are delivered by enteropathogenic E. coli (EPEC) which, if perturbed can have marked consequences on the outcome of the infection. We show that absence of the EPEC effector Tir confers onto the bacterium a potent ability to destroy polarized intestinal epithelia through extensive host cell detachment. This process was dependent on the EPEC effectors EspG and EspG2 through their activation of the host cysteine protease calpain. EspG and EspG2 are shown to activate calpain during EPEC infection, which increases significantly in the absence of Tir – leading to rapid host cell loss and necrosis. These findings reveal a new function for EspG and EspG2 and show that Tir, independent of its bacterial ligand Intimin, is essential for maintaining the integrity of the epithelium during EPEC infection by keeping the destructive activity of EspG and EspG2 in check.     

Real-time analysis of effector translocation by the type III secretion system of enteropathogenic Escherichia coli

Bacteria use type III secretion systems (TTSS) to translocate effector proteins into host cells. Better understanding of the TTSS and its effectors' functions will require assays to measure their activities in vivo and in real time. We designed a real-time, high-throughput translocation assay that utilizes fusions of effector genes to the beta-lactamase reporter gene, positioned under the effector's native promoter and chromosomal location. Using this assay, we simultaneously and quantitatively analyzed the translocation kinetics of six core enteropathogenic E. coli effectors, EspF, EspG, EspH, EspZ, Map, and Tir. A distinct order in the efficiencies of effector translocation was observed. Translocation efficiency was determined by multiple factors, including the intrabacterial effector concentration, effector-chaperone interactions, the efficiency of bacterial attachment to the host cells, and possibly also by a translocation autoinhibition mechanism. The described real-time translocation assay could be easily adapted for varied applications in the study of bacterial pathogenesis.     

The Salmonella Typhimurium effector protein SopE transiently localizes to the early SCV and contributes to intracellular replication

Salmonella enterica serovar Typhimurium (S. Tm) is a facultative intracellular pathogen that induces entry into non‐phagocytic cells by a Type III secretion system (TTSS) and cognate effector proteins. Upon host cell entry, S. Tm expresses a second TTSS and subverts intracellular trafficking to create a replicative niche – the Salmonella‐containing vacuole (SCV). SopE, a guanidyl exchange factor (GEF) for Rac1 and Cdc42, is translocated by the TTSS‐1 upon host cell contact and promotes entry through triggering of actin‐dependent ruffles. After host cell entry, the bulk of SopE undergoes proteasomal degradation. Here we show that a subfraction is however detectable on the nascent SCV membrane up to ～ 6 h post infection. Membrane localization of SopE and the closely related SopE2 differentially depend on the Rho‐GTPase‐binding GEF domain, and to some extent involves also the unstructured N‐terminus. SopE localizes transiently to the early SCV, dependent on continuous synthesis and secretion by the TTSS‐1 during the intracellular state. Mutant strains lacking SopE or SopE2 are attenuated in early intracellular replication, while complementation restores this defect. Hence, the present study reveals an unanticipated role for SopE and SopE2 in establishing the Salmonella replicative niche, and further emphasizes the importance of entry effectors in later stages of host‐cell manipulation.     

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.     

EspJ effector in enterohemorrhagic E. coli translocates into host mitochondria via an atypical mitochondrial targeting signal

EHEC is a bacterial pathogen causing diarrhea and hemorrhagic colitis in humans. To exert virulence, EHEC exploits a subset of effectors that are translocated into host cells via the type III secretion system. EspJ, which was recently identified as a type III secreted effector, is conserved in related pathogens such as EPEC and Citrobacter rodentium. However, the exact function of EspJ remains unclear. In the present study, we found that EspJ was unstable in host cells, which might be attributable to the N‐terminal part beginning from amino acid number 59. Using stable forms of EspJ derivatives, we demonstrated for the first time that EspJ has the ability to translocate into mitochondria via an atypical mitochondrial targeting signal at the N terminus (1–36 a.a.) of EspJ. It has been reported that a mitochondrial targeting effector, EspF, disrupts the mitochondrial membrane potential, resulting in an induction of host cell death. To further investigate EspJ function in mitochondria, HeLa cells were infected with wild‐type EPEC, an isogenic EspJ‐mutant or an EspJ‐overexpressing strain. The result of LDH release assay using an EspJ‐mutant showed that the EspJ effector appears not to be involved in cytotoxicity.     

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 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 of intimin with Tir on the bacterial surface is prerequisite for the barrier disruption induced by enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) infects intestinal epithelial cells and perturbs the intestinal barrier that limits the paracellular movement of molecules. The disruption of the barrier is mediated by the effectors translocated into the host cells through the bacterial type III secretion system (TTSS). A previous report has described the importance of a bacterial outer membrane protein, intimin, in EPEC-mediated disruption of the barrier, and proposed that intimin, in concert with a host intimin receptor, controls the activity of the translocated barrier-disrupting effectors [P. Dean, B. Kenny, Intestinal barrier dysfunction by enteropathogenic Escherichia coli is mediated by two effector molecules and a bacterial surface protein, Mol. Microbiol. 54 (2004) 665-675]. In this study, we found that the importance of intimin is in its ability to bind a bacterial intimin receptor, Tir. Additionally, the impaired ability of an intimin-negative mutant was not restored by co-infection with intimin-expressing TTSS mutants. Collectively, the results in this study favor an alternative scenario explaining the importance of intimin, that the binding of intimin with Tir on the bacterial surface triggers or promotes the translocation of factors required for the efficient disruption of the barrier. Thus, the interaction of intimin with Tir may serve as a molecular switch that controls the delivery of virulence factors into the host cells.     

Vying for the control of inflammasomes: The cytosolic frontier of enteric bacterial pathogen–host interactions

Enteric pathogen–host interactions occur at multiple interfaces, including the intestinal epithelium and deeper organs of the immune system. Microbial ligands and activities are detected by host sensors that elicit a range of immune responses. Membrane‐bound toll‐like receptors and cytosolic inflammasome pathways are key signal transducers that trigger the production of pro‐inflammatory molecules, such as cytokines and chemokines, and regulate cell death in response to infection. In recent years, the inflammasomes have emerged as a key frontier in the tussle between bacterial pathogens and the host. Inflammasomes are complexes that activate caspase‐1 and are regulated by related caspases, such as caspase‐11, ‐4, ‐5 and ‐8. Importantly, enteric bacterial pathogens can actively engage or evade inflammasome signalling systems. Extracellular, vacuolar and cytosolic bacteria have developed divergent strategies to subvert inflammasomes. While some pathogens take advantage of inflammasome activation (e.g. Listeria monocytogenes, Helicobacter pylori), others (e.g. E. coli, Salmonella, Shigella, Yersinia sp.) deploy a range of virulence factors, mainly type 3 secretion system effectors, that subvert or inhibit inflammasomes. In this review we focus on inflammasome pathways and their immune functions, and discuss how enteric bacterial pathogens interact with them. These studies have not only shed light on inflammasome‐mediated immunity, but also the exciting area of mammalian cytosolic immune surveillance.     

The enteropathogenic E. coli effector EspB facilitates microvillus effacing and antiphagocytosis by inhibiting myosin function

Enteropathogenic Escherichia coli (EPEC) destroys intestinal microvilli and suppresses phagocytosis by injecting effectors into infected cells through a type III secretion system (TTSS). EspB, a component of the TTSS, is also injected into the cytoplasm of host cells. However, the physiological functions of EspB within the host cell cytoplasm remain unclear. We show that EspB binds to myosins, which are a superfamily of proteins that interact with actin filaments and mediate essential cellular processes, including microvillus formation and phagocytosis. EspB inhibits the interaction of myosins with actin, and an EspB mutant that lacks the myosin-binding region maintained its TTSS function but could not induce microvillus effacing or suppress phagocytosis. Moreover, the myosin-binding region of EspB is essential for Citrobacter rodentium, an EPEC-related murine pathogen, to efficiently infect mice. These results suggest that EspB inhibits myosin functions and thereby facilitates efficient infection by EPEC.     

Enteropathogenic and enterohaemorrhagic Escherichia coli deliver a novel effector called Cif, which blocks cell cycle G2/M transition

Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) are closely related pathogens. Both use a type III secretion system (TTSS) encoded by the 'locus of enterocyte effacement' (LEE) to subvert and attach to epithelial cells through the injection of a repertoire of effector molecules. Here, we report the identification of a new TTSS translocated effector molecule called Cif, which blocks cell cycle G2/M transition and induces the formation of stress fibres through the recruitment of focal adhesions. Cif is not encoded by the LEE but by a lambdoid prophage present in EPEC and EHEC. A cif mutant causes localized effacement of microvilli and intimately attaches to the host cell surface, but is defective in the ability to block mitosis. When expressed in TTSS competent LEE-positive pathogens, Cif is injected into the infected epithelial cells. These cells arrested at the G2/M phase displayed accumulation of inactive phosphorylated Cdk1. In conclusion, Cif is a new member of a growing family of bacterial cyclomodulins that subvert the host eukaryotic cell cycle.     

Genomic mining type III secretion system effectors in Pseudomonas syringae yields new picks for all TTSS prospectors

Many bacterial pathogens of plants and animals use a type III secretion system (TTSS) to deliver virulence effector proteins into host cells. Because effectors are heterogeneous in sequence and function, there has not been a systematic way to identify the genes encoding them in pathogen genomes, and our current inventories are probably incomplete. A pre-closure draft sequence of Pseudomonas syringae pv. tomato DC3000, a pathogen of tomato and Arabidopsis, has recently supported five complementary studies which, collectively, identify 36 TTSS-secreted proteins and many more candidate effectors in this strain. These studies demonstrate the advantages of combining experimental and computational approaches, and they yield new insights into TTSS effectors and virulence regulation in P. syringae, potential effector targeting signals in all TTSS-dependent pathogens, and strategies for finding TTSS effectors in other bacteria that have sequenced genomes.