Gadd45�� activity is the principal effector of Shigella mitochondria-dependent epithelial cell death in vitro and ex vivo

Modulation of death is a pathogen strategy to establish residence and promote survival in host cells and tissues. Shigella spp. are human pathogens that invade colonic mucosa, where they provoke lesions caused by their ability to manipulate the host cell responses. Shigella spp. induce various types of cell death in different cell populations. However, they are equally able to protect host cells from death. Here, we have investigated on the molecular mechanisms and cell effectors governing the balance between survival and death in epithelial cells infected with Shigella. To explore these aspects, we have exploited both, the HeLa cell invasion assay and a novel ex vivo human colon organ culture model of infection that mimics natural conditions of shigellosis. Our results definitely show that Shigella induces a rapid intrinsic apoptosis of infected cells, via mitochondrial depolarization and the ensuing caspase-9 activation. Moreover, for the first time we identify the eukaryotic stress-response factor growth arrest and DNA damage 45α as a key player in the induction of the apoptotic process elicited by Shigella in epithelial cells, revealing an unexplored role of this molecule in the course of infections sustained by invasive pathogens.     

Effect of Wild-Type Shigella Species and Attenuated Shigella Vaccine Candidates on Small Intestinal Barrier Function,Antigen Trafficking,and Cytokine Release

Bacterial dysentery due to Shigella species is a major cause of morbidity and mortality worldwide. The pathogenesis of Shigella is based on the bacteria''s ability to invade and replicate within the colonic epithelium, resulting in severe intestinal inflammatory response and epithelial destruction. Although the mechanisms of pathogenesis of Shigella in the colon have been extensively studied, little is known on the effect of wild-type Shigella on the small intestine and the role of the host response in the development of the disease. Moreover, to the best of our knowledge no studies have described the effects of apically administered Shigella flexneri 2a and S. dysenteriae 1 vaccine strains on human small intestinal enterocytes. The aim of this study was to assess the coordinated functional and immunological human epithelial responses evoked by strains of Shigella and candidate vaccines on small intestinal enterocytes. To model the interactions of Shigella with the intestinal mucosa, we apically exposed monolayers of human intestinal Caco2 cells to increasing bacterial inocula. We monitored changes in paracellular permeability, examined the organization of tight-junctions and the pro-inflammatory response of epithelial cells. Shigella infection of Caco2 monolayers caused severe mucosal damage, apparent as a drastic increase in paracellular permeability and disruption of tight junctions at the cell-cell boundary. Secretion of pro-inflammatory IL-8 was independent of epithelial barrier dysfunction. Shigella vaccine strains elicited a pro-inflammatory response without affecting the intestinal barrier integrity. Our data show that wild-type Shigella infection causes a severe alteration of the barrier function of a small intestinal cell monolayer (a proxy for mucosa) and might contribute (along with enterotoxins) to the induction of watery diarrhea. Diarrhea may be a mechanism by which the host attempts to eliminate harmful bacteria and transport them from the small to the large intestine where they invade colonocytes inducing a strong inflammatory response.     

Inhibition of death receptor signaling by bacterial gut pathogens

Gastrointestinal bacterial pathogens such as enteropathogenic Escherichia coli, Salmonella and Shigella control inflammatory and apoptotic signaling in human intestinal cells to establish infection, replicate and disseminate to other hosts. These pathogens manipulate host cell signaling through the translocation of virulence effector proteins directly into the host cell cytoplasm, which then target various signaling pathways. Death receptors such as TNFR1, FAS and TRAIL-R induce signaling cascades that are crucial to the clearance of pathogens, and as such are major targets for inhibition by pathogens. This review focuses on what is known about how bacterial gut pathogens inhibit death receptor signaling to suppress inflammation and prevent apoptosis.     

Interferon α Inhibits a Src-mediated Pathway Necessary for Shigella-induced Cytoskeletal Rearrangements in Epithelial Cells

Shigella flexneri, the causative agent of bacillary dysentery, has the ability to enter nonphagocytic cells. The interferon (IFN) family of cytokines was found to inhibit Shigella invasion of cultured epithelial cells. We show here that IFN-α inhibits a Src-dependent signaling cascade triggered by Shigella that leads to the reorganization of the host cell cytoskeleton. Immunofluorescence studies showed that IFN-α inhibits Shigella-induced actin polymerization required for bacterial entry into cells. Phosphorylation of cortactin, a Src-substrate specifically tyrosyl-phosphorylated during Shigella entry, was inhibited by IFN-α. Overexpression of a dominant interfering form of pp60c-src led to inhibition of Shigella-induced cytoskeletal rearrangements and decreased cortactin phosphorylation indicating a role for Src in Shigella entry. Also, Shigella uptake in cells that expressed constitutively active Src was unaffected by IFN-α treatment. We conclude that Src kinase activity is necessary for Shigella invasion of epithelial cells and that IFN-α inhibits this Src-dependent signaling pathway.     

Near Surface Swimming of Salmonella Typhimurium Explains Target-Site Selection and Cooperative Invasion

Targeting of permissive entry sites is crucial for bacterial infection. The targeting mechanisms are incompletely understood. We have analyzed target-site selection by S. Typhimurium. This enteropathogenic bacterium employs adhesins (e.g. fim) and the type III secretion system 1 (TTSS-1) for host cell binding, the triggering of ruffles and invasion. Typically, S. Typhimurium invasion is focused on a subset of cells and multiple bacteria invade via the same ruffle. It has remained unclear how this is achieved. We have studied target-site selection in tissue culture by time lapse microscopy, movement pattern analysis and modeling. Flagellar motility (but not chemotaxis) was required for reaching the host cell surface in vitro. Subsequently, physical forces trapped the pathogen for ∼1.5–3 s in “near surface swimming”. This increased the local pathogen density and facilitated “scanning” of the host surface topology. We observed transient TTSS-1 and fim-independent “stopping” and irreversible TTSS-1-mediated docking, in particular at sites of prominent topology, i.e. the base of rounded-up cells and membrane ruffles. Our data indicate that target site selection and the cooperative infection of membrane ruffles are attributable to near surface swimming. This mechanism might be of general importance for understanding infection by flagellated bacteria.     

The Zebrafish as a New Model for the In Vivo Study of Shigella flexneri Interaction with Phagocytes and Bacterial Autophagy

Autophagy, an ancient and highly conserved intracellular degradation process, is viewed as a critical component of innate immunity because of its ability to deliver cytosolic bacteria to the lysosome. However, the role of bacterial autophagy in vivo remains poorly understood. The zebrafish (Danio rerio) has emerged as a vertebrate model for the study of infections because it is optically accessible at the larval stages when the innate immune system is already functional. Here, we have characterized the susceptibility of zebrafish larvae to Shigella flexneri, a paradigm for bacterial autophagy, and have used this model to study Shigella-phagocyte interactions in vivo. Depending on the dose, S. flexneri injected in zebrafish larvae were either cleared in a few days or resulted in a progressive and ultimately fatal infection. Using high resolution live imaging, we found that S. flexneri were rapidly engulfed by macrophages and neutrophils; moreover we discovered a scavenger role for neutrophils in eliminating infected dead macrophages and non-immune cell types that failed to control Shigella infection. We observed that intracellular S. flexneri could escape to the cytosol, induce septin caging and be targeted to autophagy in vivo. Depletion of p62 (sequestosome 1 or SQSTM1), an adaptor protein critical for bacterial autophagy in vitro, significantly increased bacterial burden and host susceptibility to infection. These results show the zebrafish larva as a new model for the study of S. flexneri interaction with phagocytes, and the manipulation of autophagy for anti-bacterial therapy in vivo.     

Hypoxia Decreases Invasin-Mediated Yersinia enterocolitica Internalization into Caco-2 Cells

Yersinia enterocolitica is a major cause of human yersiniosis, with enterocolitis being a typical manifestation. These bacteria can cross the intestinal mucosa, and invade eukaryotic cells by binding to host β₁ integrins, a process mediated by the bacterial effector protein invasin. This study examines the role of hypoxia on the internalization of Y. enterocolitica into intestinal epithelial cells, since the gastrointestinal tract has been shown to be physiologically deficient in oxygen levels (hypoxic), especially in cases of infection and inflammation. We show that hypoxic pre-incubation of Caco-2 cells resulted in significantly decreased bacterial internalization compared to cells grown under normoxia. This phenotype was absent after functionally blocking host β₁ integrins as well as upon infection with an invasin-deficient Y. enterocolitica strain. Furthermore, downstream phosphorylation of the focal adhesion kinase was also reduced under hypoxia after infection. In good correlation to these data, cells grown under hypoxia showed decreased protein levels of β₁ integrins at the apical cell surface whereas the total protein level of the hypoxia inducible factor (HIF-1) alpha was elevated. Furthermore, treatment of cells with the HIF-1 α stabilizer dimethyloxalylglycine (DMOG) also reduced invasion and decreased β₁ integrin protein levels compared to control cells, indicating a potential role for HIF-1α in this process. These results suggest that hypoxia decreases invasin-integrin-mediated internalization of Y. enterocolitica into intestinal epithelial cells by reducing cell surface localization of host β₁ integrins.     

The Role of Autophagy during Group B Streptococcus Infection of Blood-Brain Barrier Endothelium

Bacterial meningitis occurs when bloodborne pathogens invade and penetrate the blood-brain barrier (BBB), provoking inflammation and disease. Group B Streptococcus (GBS), the leading cause of neonatal meningitis, can enter human brain microvascular endothelial cells (hBMECs), but the host response to intracellular GBS has not been characterized. Here we sought to determine whether antibacterial autophagy, which involves selective recognition of intracellular organisms and their targeting to autophagosomes for degradation, is activated in BBB endothelium during bacterial infection. GBS infection resulted in increased punctate distribution of GFP-microtubule-associated protein 1 light chain 3 (LC3) and increased levels of endogenous LC3-II and p62 turnover, two hallmark indicators of active autophagic flux. Infection with GBS mutants revealed that bacterial invasion and the GBS pore-forming β-hemolysin/cytolysin (β-h/c) trigger autophagic activation. Cell-free bacterial extracts containing β-h/c activity induced LC3-II conversion, identifying this toxin as a principal provocative factor for autophagy activation. These results were confirmed in vivo using a mouse model of GBS meningitis as infection with WT GBS induced autophagy in brain tissue more frequently than a β-h/c-deficient mutant. Elimination of autophagy using Atg5-deficient fibroblasts or siRNA-mediated impairment of autophagy in hBMECs led to increased recovery of intracellular GBS. However, electron microscopy revealed that GBS was rarely found within double membrane autophagic structures even though we observed GBS-LC3 co-localization. These results suggest that although autophagy may act as a BBB cellular defense mechanism in response to invading and toxin-producing bacteria, GBS may actively thwart the autophagic pathway.     

ATG7 contributes to plant basal immunity towards fungal infection

Autophagy has an important function in cellular homeostasis. In recent years autophagy has been implicated in plant basal immunity and assigned negative (“anti-death”) and positive (“pro-death”) regulatory functions in controlling cell death programs that establish sufficient immunity to microbial infection. We recently showed that Arabidopsis mutants lacking the autophagy-associated (ATG) genes ATG5, ATG10 and ATG18a are compromised in their resistance towards infection with necrotrophic fungal pathogens but display an enhanced resistance towards biotrophic bacterial invaders. Thus, the function of autophagy as either being pro-death or anti-death depends critically on the lifestyle and infection strategy of invading microbes. Here we show that ATG7 contributes to resistance to fungal pathogens. Genetic inactivation of ATG7 results in elevated susceptibility towards the necrotrophic fungal pathogen, Alternaria brassicicola, with atg7 mutants developing spreading necrosis accompanied by production of reactive oxygen intermediates. Likewise, treatment with the fungal toxin fumonisin B1 causes spreading lesion formation in the atg7 mutant. We conclude that ATG7-dependent autophagy constitutes an “anti-death” (“pro-survival”) plant mechanism to control the containment of cell death and immunity to necrophic fungal infection.Key words: autophagy, ATG7, basal immunity, fungal resistance, arabidopsisPlants have evolved a bipartite plant immune system to cope with microbial infections. The first layer of defense relies on the recognition of pathogen-associated molecular patterns (PAMP) by pattern-recognition receptors (PAMP-triggered immunity, PTI).^1,2 To overcome this defense strategy, successful pathogens deliver so-called effector proteins into plant cells to modify host cellular processes and to suppress immune responses to enhance virulence. The presence or activities of these microbial effectors is sensed by plant resistance proteins and triggers the second layer of defense, the effector-triggered immunity (ETI).^1,2 In contrast to PTI, ETI is most often accompanied by programmed host cell death (PCD) at the site of attempted microbial invasion; however the molecular basis of this apoptosis-like hypersensitive response (HR) is largely unknown.In recent years evidence accumulated that a non-apoptotic form of cell death called autophagy is not only involved in animal PCD and innate immunity³ but is also an important component in the plant basal immune response.⁴ Generally, autophagy (auto, meaning “self” and phagy, “to eat”) is a cytoplasmic bulk degradation process in which cellular components are targeted to lysosomal or vacuolar degradation. This process is ubiquitous in eukaryotic organisms and is considered to aid cellular survival, differentiation, development and homeostasis by nutrient recycling or removal of damaged or toxic materials.^5–7     

The Shigella Type Three Secretion System Effector OspG Directly and Specifically Binds to Host Ubiquitin for Activation

The genus Shigella infects human gut epithelial cells to cause diarrhea and gastrointestinal disorders. Like many other Gram-negative bacterial pathogens, the virulence of Shigella spp. relies on a conserved type three secretion system that delivers a handful of effector proteins into host cells to manipulate various host cell physiology. However, many of the Shigella type III effectors remain functionally uncharacterized. Here we observe that OspG, one of the Shigella effectors, interacted with ubiquitin conjugates and poly-ubiquitin chains of either K48 or K63 linkage in eukaryotic host cells. Purified OspG protein formed a stable complex with ubiquitin but showed no interactions with other ubiquitin-like proteins. OspG binding to ubiquitin required the carboxyl terminal helical region in OspG and the canonical I44-centered hydrophobic surface in ubiquitin. OspG and OspG-homologous effectors, NleH1/2 from enteropathogenic E coli (EPEC), contain sub-domains I-VII of eukaryotic serine/threonine kinase. GST-tagged OspG and NleH1/2 could undergo autophosphorylation, the former of which was significantly stimulated by ubiquitin binding. Ubiquitin binding was also required for OspG functioning in attenuating host NF-κB signaling. Our data illustrate a new mechanism that bacterial pathogen like Shigella exploits ubiquitin binding to activate its secreted virulence effector for its functioning in host eukaryotic cells.     

Molecular mechanisms of cell–cell spread of intracellular bacterial pathogens

Several bacterial pathogens, including Listeria monocytogenes, Shigella flexneri and Rickettsia spp., have evolved mechanisms to actively spread within human tissues. Spreading is initiated by the pathogen-induced recruitment of host filamentous (F)-actin. F-actin forms a tail behind the microbe, propelling it through the cytoplasm. The motile pathogen then encounters the host plasma membrane, forming a bacterium-containing protrusion that is engulfed by an adjacent cell. Over the past two decades, much progress has been made in elucidating mechanisms of F-actin tail formation. Listeria and Shigella produce tails of branched actin filaments by subverting the host Arp2/3 complex. By contrast, Rickettsia forms tails with linear actin filaments through a bacterial mimic of eukaryotic formins. Compared with F-actin tail formation, mechanisms controlling bacterial protrusions are less well understood. However, recent findings have highlighted the importance of pathogen manipulation of host cell–cell junctions in spread. Listeria produces a soluble protein that enhances bacterial protrusions by perturbing tight junctions. Shigella protrusions are engulfed through a clathrin-mediated pathway at ‘tricellular junctions’—specialized membrane regions at the intersection of three epithelial cells. This review summarizes key past findings in pathogen spread, and focuses on recent developments in actin-based motility and the formation and internalization of bacterial protrusions.     

Escape of intracellular Shigella from autophagy requires binding to cholesterol through the type III effector,IcsB

Type III secretion systems are present in many pathogenic bacteria and mediate the translocation of bacterial effectors into host cells. Identification of host targets of these effectors is crucial for understanding bacterial virulence. IcsB, a type III secretion effector, helps Shigella to evade the host autophagy defense system by binding to the autophagy protein, Atg5. Here, we show that IcsB is able to interact specifically with cholesterol. The cholesterol binding domain (CBD) of IcsB is located between residues 288 and 351. Specific mutations of single tyrosine residues Y297 or Y340 of IcsB by phenylalanine (F) slightly reduced cholesterol binding, whereas deletion of the entire CBD or double mutation Y297F-Y340F strongly abolished interactions with cholesterol. To determine whether Shigella expressing IcsB variants could evade autophagy as effectively as the wild-type Shigella, we infected MDAMC cells stably expressing the autophagy marker LC3 fused to GFP and bacterial autophagosome formation was quantified using fluorescence microscopy. Mutation Y297F or Y340F slightly impaired IcsB function, whereas complete removal of CBD or mutation Y297F-Y340F significantly impaired autophagy evasion. Furthermore, we report that BopA, the counterpart of IcsB in Burkholderia pseudomallei with similar autophagy-evading properties, contains the CBD domain and is also able to bind cholesterol.     

ARHGEF26 enhances Salmonella invasion and inflammation in cells and mice

Salmonella hijack host machinery in order to invade cells and establish infection. While considerable work has described the role of host proteins in invasion, much less is known regarding how natural variation in these invasion-associated host proteins affects Salmonella pathogenesis. Here we leveraged a candidate cellular GWAS screen to identify natural genetic variation in the ARHGEF26 (Rho Guanine Nucleotide Exchange Factor 26) gene that renders lymphoblastoid cells susceptible to Salmonella Typhi and Typhimurium invasion. Experimental follow-up redefined ARHGEF26’s role in Salmonella epithelial cell infection. Specifically, we identified complex serovar-by-host interactions whereby ARHGEF26 stimulation of S. Typhi and S. Typhimurium invasion into host cells varied in magnitude and effector-dependence based on host cell type. While ARHGEF26 regulated SopB- and SopE-mediated S. Typhi (but not S. Typhimurium) infection of HeLa cells, the largest effect of ARHGEF26 was observed with S. Typhimurium in polarized MDCK cells through a SopB- and SopE2-independent mechanism. In both cell types, knockdown of the ARHGEF26-associated protein DLG1 resulted in a similar phenotype and serovar specificity. Importantly, we show that ARHGEF26 plays a critical role in S. Typhimurium pathogenesis by contributing to bacterial burden in the enteric fever murine model, as well as inflammation in the colitis infection model. In the enteric fever model, SopB and SopE2 are required for the effects of Arhgef26 deletion on bacterial burden, and the impact of sopB and sopE2 deletion in turn required ARHGEF26. In contrast, SopB and SopE2 were not required for the impacts of Arhgef26 deletion on colitis. A role for ARHGEF26 on inflammation was also seen in cells, as knockdown reduced IL-8 production in HeLa cells. Together, these data reveal pleiotropic roles for ARHGEF26 during infection and highlight that many of the interactions that occur during infection that are thought to be well understood likely have underappreciated complexity.     

Autophagy and the cytoskeleton

Actin-based motility is used by various pathogens such as Listeria and Shigella for dissemination within cells

and tissues, yet host factors counteracting this process have not been identified. We have recently discovered that infected host cells can prevent actin-based motility of Shigella by compartmentalizing bacteria inside ‘septin cages,’ revealing a novel mechanism of host defense that restricts dissemination. Because bacterial proteins controlling actin-based motility also regulate the autophagy process, we hypothesized and then established a link between septin caging and autophagy. Together, these results unveiled the first cellular mechanism that counteracts pathogen dissemination. Understanding the role of septins, a so far poorly characterized component of the cytoskeleton, will thus provide new insights into bacterial infection and autophagy.     

Host and Bacterial Proteins That Repress Recruitment of LC3 to Shigella Early during Infection

Shigella spp. are intracytosolic gram-negative pathogens that cause disease by invasion and spread through the colonic mucosa, utilizing host cytoskeletal components to form propulsive actin tails. We have previously identified the host factor Toca-1 as being recruited to intracellular S. flexneri and being required for efficient bacterial actin tail formation. We show that at early times during infection (40 min.), the type three-secreted effector protein IcsB recruits Toca-1 to intracellular bacteria and that recruitment of Toca-1 is associated with repression of recruitment of LC3, as well as with repression of recruitment of the autophagy marker NDP52, around these intracellular bacteria. LC3 is best characterized as a marker of autophagosomes, but also marks phagosomal membranes in the process LC3-associated phagocytosis. IcsB has previously been demonstrated to be required for S. flexneri evasion of autophagy at late times during infection (4–6 hr) by inhibiting binding of the autophagy protein Atg5 to the Shigella surface protein IcsA (VirG). Our results suggest that IcsB and Toca-1 modulation of LC3 recruitment restricts LC3-associated phagocytosis and/or LC3 recruitment to vacuolar membrane remnants. Together with published results, our findings suggest that IcsB inhibits innate immune responses in two distinct ways, first, by inhibiting LC3-associated phagocytosis and/or LC3 recruitment to vacuolar membrane remnants early during infection, and second, by inhibiting autophagy late during infection.     

Pseudomonas aeruginosa Transmigrates at Epithelial Cell-Cell Junctions,Exploiting Sites of Cell Division and Senescent Cell Extrusion

To achieve systemic infection, bacterial pathogens must overcome the critical and challenging step of transmigration across epithelial barriers. This is particularly true for opportunistic pathogens such as Pseudomonas aeruginosa, an agent which causes nosocomial infections. Despite extensive study, details on the mechanisms used by this bacterium to transmigrate across epithelial tissues, as well as the entry sites it uses, remain speculative. Here, using real-time microscopy and a model epithelial barrier, we show that P. aeruginosa employs a paracellular transmigration route, taking advantage of altered cell-cell junctions at sites of cell division or when senescent cells are expelled from the cell layer. Once a bacterium transmigrates, it is followed by a cohort of bacteria using the same entry point. The basal compartment is then invaded radially from the initial penetration site. Effective transmigration and propagation require type 4 pili, the type 3 secretion system (T3SS) and a flagellum, although flagellum-deficient bacteria can occasionally invade the basal compartment from wounded areas. In the basal compartment, the bacteria inject the T3SS toxins into host cells, disrupting the cytoskeleton and focal contacts to allow their progression under the cells. Thus, P. aeruginosa exploits intrinsic host cell processes to breach the epithelium and invade the subcellular compartment.     

Ca2+ signals triggered by bacterial pathogens and microdomains

Recent reports have highlighted the pivotal role of Ca²⁺ during host cell infection by bacterial pathogens. Here, we review how bacterial pore-forming toxins (PFTs) trigger global Ca²⁺ signals to regulate cell adhesion-, inflammatory- or death processes. We comment recent reports describing the role of bacterial effectors injected by a type III secretion system (T3SS) as well as host cell players in the formation of Ca²⁺ microdomains during Shigella invasion and Chlamydia extrusion of host cells. We discuss how modeling and comparison between bacterial-induced and physiological Ca²⁺ microdomains provides insight into the critical parameters shaping the duration of local Ca²⁺ responses.     

Intracellular Staphylococcus aureus employs the cysteine protease staphopain A to induce host cell death in epithelial cells

Staphylococcus aureus is a major human pathogen, which can invade and survive in non-professional and professional phagocytes. Uptake by host cells is thought to contribute to pathogenicity and persistence of the bacterium. Upon internalization by epithelial cells, cytotoxic S. aureus strains can escape from the phagosome, replicate in the cytosol and induce host cell death. Here, we identified a staphylococcal cysteine protease to induce cell death after translocation of intracellular S. aureus into the host cell cytoplasm. We demonstrated that loss of staphopain A function leads to delayed onset of host cell death and prolonged intracellular replication of S. aureus in epithelial cells. Overexpression of staphopain A in a non-cytotoxic strain facilitated intracellular killing of the host cell even in the absence of detectable intracellular replication. Moreover, staphopain A contributed to efficient colonization of the lung in a mouse pneumonia model. In phagocytic cells, where intracellular S. aureus is exclusively localized in the phagosome, staphopain A did not contribute to cytotoxicity. Our study suggests that staphopain A is utilized by S. aureus to exit the epithelial host cell and thus contributes to tissue destruction and dissemination of infection.