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.     

Shigella enterotoxin-2 is a type III effector that participates in Shigella-induced interleukin 8 secretion by epithelial cells

We have previously described a protein termed Shigella enterotoxin 2 (ShET-2), which induces rises in short-circuit current in rabbit ileum mounted in the Ussing chamber. Published reports have postulated that ShET-2 may be secreted by the Shigella type III secretion system (T3SS). In this study, we show that ShET-2 secretion into the extracellular space requires the T3SS in Shigella flexneri 2a strain 2457T and a ShET-2-TEM fusion was translocated into epithelial cells in a T3SS-dependent manner. The ShET-2 gene, sen, is encoded downstream of the ospC1 gene of S. flexneri, and we show that sen is cotranscribed with this T3SS-secreted product. Considering that T3SS effectors have diverse roles in Shigella infection and that vaccine constructs lacking ShET-2 are attenuated in volunteers, we asked whether ShET-2 has a function other than its enterotoxic activity. We constructed a ShET-2 mutant in 2457T and tested its effect on epithelial cell invasion, plaque formation, guinea pig keratoconjunctivitis and interleukin 8 (IL-8) secretion from infected monolayers. Although other phenotypes were not different compared with the wild-type parent, we found that HEp-2 and T84 cells infected with the ShET-2 mutant exhibited significantly reduced IL-8 secretion into the basolateral compartment, suggesting that ShET-2 might participate in the Shigella-induced inflammation of epithelial cells.     

Modulation of innate immune responses by Yersinia type III secretion system translocators and effectors

The innate immune system of mammals responds to microbial infection through detection of conserved molecular determinants called ‘pathogen‐associated molecular patterns’ (PAMPs). Pathogens use virulence factors to counteract PAMP‐directed responses. The innate immune system can in turn recognize signals generated by virulence factors, allowing for a heightened response to dangerous pathogens. Many Gram‐negative bacterial pathogens encode type III secretion systems (T3SSs) that translocate effector proteins, subvert PAMP‐directed responses and are critical for infection. A plasmid‐encoded T3SS in the human‐pathogenic Yersinia species translocates seven effectors into infected host cells. Delivery of effectors by the T3SS requires plasma membrane insertion of two translocators, which are thought to form a channel called a translocon. Studies of the Yersinia T3SS have provided key advances in our understanding of how innate immune responses are generated by perturbations in plasma membrane and other signals that result from translocon insertion. Additionally, studies in this system revealed that effectors function to inhibit innateimmune responses resulting from insertion of translocons into plasma membrane. Here, we review these advances with the goal of providing insight into how a T3SS can activate and inhibit innate immune responses, allowing a virulent pathogen to bypass host defences.     

Yeast functional genomic screens lead to identification of a role for a bacterial effector in innate immunity regulation

Numerous bacterial pathogens manipulate host cell processes to promote infection and ultimately cause disease through the action of proteins that they directly inject into host cells. Identification of the targets and molecular mechanisms of action used by these bacterial effector proteins is critical to understanding pathogenesis. We have developed a systems biological approach using the yeast Saccharomyces cerevisiae that can expedite the identification of cellular processes targeted by bacterial effector proteins. We systematically screened the viable yeast haploid deletion strain collection for mutants hypersensitive to expression of the Shigella type III effector OspF. Statistical data mining of the results identified several cellular processes, including cell wall biogenesis, which when impaired by a deletion caused yeast to be hypersensitive to OspF expression. Microarray experiments revealed that OspF expression resulted in reversed regulation of genes regulated by the yeast cell wall integrity pathway. The yeast cell wall integrity pathway is a highly conserved mitogen-activated protein kinase (MAPK) signaling pathway, normally activated in response to cell wall perturbations. Together these results led us to hypothesize and subsequently demonstrate that OspF inhibited both yeast and mammalian MAPK signaling cascades. Furthermore, inhibition of MAPK signaling by OspF is associated with attenuation of the host innate immune response to Shigella infection in a mouse model. These studies demonstrate how yeast systems biology can facilitate functional characterization of pathogenic bacterial effector proteins.     

The versatility of Shigella effectors

When Shigella infect the intestinal epithelium, they deliver several effectors through the type III secretion system (T3SS) into the surrounding space and directly into the host-cell cytoplasm, where they can mimic and usurp host cellular functions or subvert host-cell signalling pathways and the immune response. Although bacterial strategies and mechanisms of infection vary greatly, recent studies of Shigella effectors have revealed that Shigella possess a highly evolved strategy for infection.     

GEF-H1 Mediated Control of NOD1 Dependent NF-κB Activation by Shigella Effectors

Shigella flexneri has evolved the ability to modify host cell function with intracellular active effectors to overcome the intestinal barrier. The detection of these microbial effectors and the initiation of innate immune responses are critical for rapid mucosal defense activation. The guanine nucleotide exchange factor H1 (GEF-H1) mediates RhoA activation required for cell invasion by the enteroinvasive pathogen Shigella flexneri. Surprisingly, GEF-H1 is requisite for NF-κB activation in response to Shigella infection. GEF-H1 interacts with NOD1 and is required for RIP2 dependent NF-κB activation by H-Ala-D-γGlu-DAP (γTriDAP). GEF-H1 is essential for NF-κB activation by the Shigella effectors IpgB2 and OspB, which were found to signal in a NOD1 and RhoA Kinase (ROCK) dependent manner. Our results demonstrate that GEF-H1 is a critical component of cellular defenses forming an intracellular sensing system with NOD1 for the detection of microbial effectors during cell invasion by pathogens.     

Saccharomyces boulardii interferes with Shigella pathogenesis by postinvasion signaling events

Saccharomyces boulardii is gaining in popularity as a treatment for a variety of diarrheal diseases as well as inflammatory bowel disease. This study was designed to examine the effect of this yeast on infection by Shigella flexneri, a highly infectious and human host-adapted enteric pathogen. We investigated key interactions between the bacteria and host cells in the presence of the yeast in addition to a number of host responses including proinflammatory events and markers. Although the presence of the yeast during infection did not alter the number of bacteria that was able to attach or invade human colon cancer-derived T-84 cells, it did positively impact the tight junction protein zonula occluden-2 and significantly increase the barrier integrity of model epithelia. The yeast also decreased ERK, JNK, and NF-kappaB activation in response to S. flexneri, events likely responsible for the observed reductions in IL-8 secretion and the transepithelial migration of polymorphonuclear leukocytes across T-84 monolayers. These results, suggesting that the yeast allowed for a dampened inflammatory response, were confirmed in vivo utilizing a highly relevant model of human fetal colonic tissue transplanted into scid mice. Furthermore, a cell-free S. boulardii culture supernatant was also capable of reducing IL-8 secretion by infected T-84 cells. These data suggest that although the use of S. boulardii during infection with S. flexneri may alleviate symptoms associated with the inflammatory response of the host, it would not prevent infection.     

The Shigella dysenteriae serotype 1 proteome,profiled in the host intestinal environment,reveals major metabolic modifications and increased expression of invasive proteins

Shigella dysenteriae serotype 1 (SD1) causes the most severe form of epidemic bacillary dysentery. We present the first comprehensive proteome analysis of this pathogen, profiling proteins from bacteria cultured in vitro and bacterial isolates from the large bowel of infected gnotobiotic piglets (in vivo). Overall, 1061 distinct gene products were identified. Differential display analysis revealed that SD1 cells switched to an anaerobic energy metabolism in vivo. High in vivo abundances of amino acid decarboxylases (GadB and AdiA) which enhance pH homeostasis in the cytoplasm and protein disaggregation chaperones (HdeA, HdeB and ClpB) were indicative of a coordinated bacterial survival response to acid stress. Several type III secretion system effectors were increased in abundance in vivo, including OspF, IpaC and IpaD. These proteins are implicated in invasion of colonocytes and subversion of the host immune response in S. flexneri. These observations likely reflect an adaptive response of SD1 to the hostile host environment. Seven proteins, among them the type III secretion system effectors OspC2 and IpaB, were detected as antigens in Western blots using piglet antisera. The outer membrane protein OmpA, the heat shock protein HtpG and OspC2 represent novel SD1 subunit vaccine candidates and drug targets.     

Roles for T and NK cells in the innate immune response to Shigella flexneri

Shigella flexneri, an enteroinvasive Gram-negative bacterium, is responsible for the worldwide endemic form of bacillary dysentery. The host response to primary infection is characterized by the induction of an acute inflammation, which is accompanied by polymorphonuclear cell (PMN) infiltration, resulting in massive destruction of the colonic mucosa. However, PMN play a major role in the recovery from primary infection, by restricting the bacterial infection at the intestinal mucosa. In this study, we assessed the roles for T and NK cells in the control of primary S. flexneri infection, using an alymphoid mouse strain (Rag null gamma(c) null) devoid of B, T, and NK cells. Using the mouse pulmonary model of Shigella infection, we showed that alymphoid Rag null gamma(c) null mice were highly susceptible to S. flexneri infection in comparison with wild-type (wt) mice. Whereas PMN recruitment upon infection was similar, macrophage recruitment and production of proinflammatory cytokines were significantly decreased in Rag null gamma(c) null mice compared with wt mice. Upon selective engraftment of Rag null gamma(c) null mice with polyclonal alphabeta T cells, but not with alphabeta T cells from IFN-gamma null , S. flexneri infection could be subsequently controlled. Rag null mice devoid of B and T cells but harboring NK cells could control infection. Local IFN-gamma production by T and NK cells recruited to the lung was demonstrated in S. flexneri-infected wt mice. These data demonstrate that both alphabeta T cells and NK cells contribute to the early control of S. flexneri infection through amplification of an inflammatory response. This cellular lymphocyte redundancy assures IFN-gamma production, which is central to innate immunity against Shigella infection.     

A human pathogenic bacterium Shigella proliferates in plants through adoption of type III effectors for shigellosis

Shigella, which infects primates, can be transmitted via fresh vegetables; however, its molecular interactions with plants have not been elucidated. Here, we show that four Shigella strains, Shigella boydii, Shigella sonnei, Shigella flexneri 2a, and S. flexneri 5a, proliferate at different levels in Arabidopsis thaliana. Microscopic studies revealed that these bacteria were present inside leaves and damaged plant cells. Green fluorescent protein (GFP)‐tagged S. boydii and S. flexneri 5a colonized leaves only, whereas S. flexneri 2a colonized both leaves and roots. Using Shigella mutants lacking type III secretion systems (T3SSs), we found that T3SSs that regulate the pathogenesis of shigellosis in humans also play a central role in bacterial proliferation in Arabidopsis. Strikingly, the immunosuppressive activity of two T3S effectors, OspF and OspG, was required for proliferation of Shigella in Arabidopsis. Of note, delivery of OspF or OspG effectors inside plant cells upon Shigella inoculation was confirmed using a split GFP system. These findings demonstrate that the human pathogen Shigella can proliferate in plants by adapting immunosuppressive machinery used in the original host human.     

Secretion of type III effectors into host cells in real time

Type III secretion (T3S) systems are key features of many gram-negative bacteria that translocate T3S effector proteins directly into eukaryotic cells. There, T3S effectors exert many effects, such as cellular invasion or modulation of host immune responses. Studying spatiotemporal orchestrated secretion of various effectors has been difficult without disrupting their functions. Here we developed a new approach using Shigella flexneri T3S as a model to investigate bacterial translocation of individual effectors via multidimensional time-lapse microscopy. We demonstrate that direct fluorescent labeling of tetracysteine motif-tagged effectors IpaB and IpaC is possible in situ without loss of function. Studying the T3S kinetics of IpaB and IpaC ejection from individual bacteria, we found that the entire pools of IpaB and IpaC were released concurrently upon host cell contact, and that 50% of each effector was secreted in 240 s. This method allows an unprecedented analysis of the spatiotemporal events during T3S.     

Modulation of phosphoinositide metabolism by pathogenic bacteria

Phosphoinositide metabolism plays a pivotal role in the regulation of receptor-mediated signal transduction, actin remodelling and membrane dynamics. Phosphoinositides co-ordinate these processes by recruiting protein effectors to distinct cellular membranes in a time- and organelle-dependent manner. Intracellular bacterial pathogens interfere with phosphoinositide metabolism to direct their entry into eukaryotic cells, form replication-permissive vacuoles, modulate apoptosis, or trigger fluid secretion. Gram-negative pathogens such as Legionella pneumophila, Shigella flexneri, or Salmonella enterica employ secretion systems to invade host cells by 'pathogen-triggered phagocytosis' and thereby bypass a requirement for phosphatidylinositol 3-kinases [PI(3)Ks]. Contrarily, 'receptor-mediated phagocytosis' of Yersinia spp., Listeria monocytogenes and other pathogenic bacteria depends on PI(3)Ks. Secreted effector proteins have been found to directly bind to and modify host cell phosphoinositides, thus modulating phagocytosis and intracellular survival of the pathogens. These effectors include L. pneumophila proteins that specifically attach to phosphatidylinositol 4-phosphate [PI(4)P] on the Legionella-containing vacuole, and phosphoinositide phosphatases produced by S. flexneri, S. enterica or Mycobacterium tuberculosis. This review covers current knowledge about subversion of host cell phosphoinositide metabolism by intracellular bacterial pathogens with an emphasis on recently identified secreted effector proteins directly engaging phosphoinositides.     

Inhibition of Shigella flexneri-induced transepithelial migration of polymorphonuclear leucocytes by cadaverine

Dysentery caused by Shigella species is characterized by infiltration of polymorphonuclear leucocytes (PMNs) into the colonic mucosa. Shigella spp. evolved into pathogens by the acquisition of virulence genes and by the deletion of 'antivirulence' genes detrimental to its pathogenic lifestyle. An example is cad A (encoding lysine decarboxylase), which is uniformly absent in Shigella spp., whereas it is present in nearly all isolates of the closely related non-pathogen Escherichia coli . Here, using monolayers of T84 cells to model the human intestinal epithelium, we determined that the introduction of cad A into S. flexneri and the expression of lysine decarboxylase attenuated the bacteria's ability to induce PMN influx across model intestinal epithelium. Such inhibition was caused by cadaverine generated from the decarboxylation of lysine. Cadaverine treatment of model intestinal epithelia specifically inhibited S. flexneri induction of PMN transepithelial migration, while having no effect on the ability of Salmonella or enteropathogenic E. coli (EPEC) to induce PMN migration. These observations not only provide insight into mechanisms of S. flexneri pathogen evolution and pathogenesis, but also suggest a potential for the use of cadaverine in the treatment of dysentery.     

Multiple lessons from the multiple functions of a regulator of type III secretion system assembly in the plant pathogen Pseudomonas syringae

The assembly of type III secretion systems (T3SSs), which inject bacterial effector proteins into the cytosol of animal and plant hosts, is a highly regulated process. Animal pathogens use a length-control protein to produce T3SS needles of fixed length and then a second regulator, such as YopN in Yersinia spp, to mediate host contact-dependent effector delivery. For Pseudomonas syringae and other plant pathogens, regulation of the assembly process differs because the T3SS pilus must grow through variably thick plant cell walls before contacting the host plasma membrane. In this issue of Molecular Microbiology, Crabill et al. (2012) report evidence that the YopN homologue HrpJ is a multifunctional regulator of T3SS assembly in DC3000. A hrpJ mutant hyper-secretes pilus protein and no longer secretes four translocator proteins in culture, and it fails to inject effectors in planta. As with other proteins in this class, HrpJ is itself a T3SS substrate, but secretion-incompetent forms retain regulatory function. However, HrpJ is unusual in suppressing innate immune responses within host cells, as demonstrated with transgenic plants. The multiple capabilities of HrpJ appear to couple host contact sensing with pilus length control and translocator secretion while also contributing to immunity suppression early in the interaction.     

Shigella chromosomal IpaH proteins are secreted via the type III secretion system and act as effectors

Shigella possess 220 kb plasmid, and the major virulence determinants, called effectors, and the type III secretion system (TTSS) are exclusively encoded by the plasmid. The genome sequences of S. flexneri strains indicate that several ipaH family genes are located on both the plasmid and the chromosome, but whether their chromosomal IpaH cognates can be secreted from Shigella remains unknown. Here we report that S. flexneri strain, YSH6000 encodes seven ipaH cognate genes on the chromosome and that the IpaH proteins are secreted via the TTSS. The secretion kinetics of IpaH proteins by bacteria, however, showed delay compared with those of IpaB, IpaC and IpaD. Expression of the each mRNA of ipaH in Shigella was increased after bacterial entry into epithelial cells, and the IpaH proteins were secreted by intracellular bacteria. Although individual chromosomal ipaH deletion mutants showed no appreciable changes in the pathogenesis in a mouse pulmonary infection model, the DeltaipaH-null mutant, whose chromosome lacks all ipaH genes, was attenuated to mice lethality. Indeed, the histological examination for mouse lungs infected with the DeltaipaH-null showed a greater inflammatory response than induced by wild-type Shigella, suggesting that the chromosomal IpaH proteins act synergistically as effectors to modulate the host inflammatory responses.     

Structurally Distinct Bacterial TBC-like GAPs Link Arf GTPase to Rab1 Inactivation to Counteract Host Defenses

Rab GTPases are frequent targets of vacuole-living bacterial pathogens for appropriate trafficking of the vacuole. Here we discover that bacterial effectors including VirA from nonvacuole Shigella flexneri and EspG from extracellular Enteropathogenic Escherichia coli (EPEC) harbor TBC-like dual-finger motifs and exhibits potent RabGAP activities. Specific inactivation of Rab1 by VirA/EspG disrupts ER-to-Golgi trafficking. S.?flexneri intracellular persistence requires VirA TBC-like GAP activity that mediates bacterial escape from autophagy-mediated host defense. Rab1 inactivation by EspG severely blocks host secretory pathway, resulting in inhibited interleukin-8 secretion from infected cells. Crystal structures of VirA/EspG-Rab1-GDP-aluminum fluoride complexes highlight TBC-like catalytic role for the arginine and glutamine finger residues and reveal?a 3D architecture distinct from that of the TBC domain. Structure of Arf6-EspG-Rab1 ternary complex illustrates a pathogenic signaling complex that rewires host Arf signaling to Rab1 inactivation. Structural distinctions of VirA/EspG further predict a possible extensive presence of TBC-like RabGAP effectors in counteracting various host defenses.     

Structural and functional studies on the N-terminal domain of the Shigella type III secretion protein MxiG

MxiG is a single-pass membrane protein that oligomerizes within the inner membrane ring of the Shigella flexneri type III secretion system (T3SS). The MxiG N-terminal domain (MxiG-N) is the predominant cytoplasmic structure; however, its role in T3SS assembly and secretion is largely uncharacterized. We have determined the solution structure of MxiG-N residues 6-112 (MxiG-N(6-112)), representing the first published structure of this T3SS domain. The structure shows strong structural homology to forkhead-associated (FHA) domains. Canonically, these cell-signaling modules bind phosphothreonine (Thr(P)) via highly conserved residues. However, the putative phosphate-binding pocket of MxiG-N(6-112) does not align with other FHA domain structures or interact with Thr(P). Furthermore, mutagenesis of potential phosphate-binding residues has no effect on S. flexneri T3SS assembly and function. Therefore, MxiG-N has a novel function for an FHA domain. Positioning of MxiG-N(6-112) within the EM density of the S. flexneri needle complex gives insight into the ambiguous stoichiometry of the T3SS, supporting models with 24 MxiG subunits in the inner membrane ring.     

Shigella effector IpaH4.5 targets 19S regulatory particle subunit RPN13 in the 26S proteasome to dampen cytotoxic T lymphocyte activation

Subversion of antigen‐specific immune responses by intracellular pathogens is pivotal for successful colonisation. Bacterial pathogens, including Shigella, deliver effectors into host cells via the type III secretion system (T3SS) in order to manipulate host innate and adaptive immune responses, thereby promoting infection. However, the strategy for subverting antigen‐specific immunity is not well understood. Here, we show that Shigella flexneri invasion plasmid antigen H (IpaH) 4.5, a member of the E3 ubiquitin ligase effector family, targets the proteasome regulatory particle non‐ATPase 13 (RPN13) and induces its degradation via the ubiquitin–proteasome system (UPS). IpaH4.5‐mediated RPN13 degradation causes dysfunction of the 19S regulatory particle (RP) in the 26S proteasome, inhibiting guidance of ubiquitinated proteins to the proteolytically active 20S core particle (CP) of 26S proteasome and thereby suppressing proteasome‐catalysed peptide splicing. This, in turn, reduces antigen cross‐presentation to CD8+ T cells via major histocompatibility complex (MHC) class I in vitro. In RPN13 knockout mouse embryonic fibroblasts (MEFs), loss of RPN13 suppressed CD8+ T cell priming during Shigella infection. Our results uncover the unique tactics employed by Shigella to dampen the antigen‐specific cytotoxic T lymphocyte (CTL) response.