Shigella promotes major alteration of gut epithelial physiology and tissue invasion by shutting off host intracellular transport.

Intracellular trafficking pathways in eukaryotic cells are essential to maintain organelle identity and structure, and to regulate cell communication with its environment. Shigella flexneri invades and subverts the human colonic epithelium by the injection of virulence factors through a type 3 secretion system (T3SS). In this work, we report the multiple effects of two S. flexneri effectors, IpaJ and VirA, which target small GTPases of the Arf and Rab families, consequently inhibiting several intracellular trafficking pathways. IpaJ and VirA induce large-scale impairment of host protein secretion and block the recycling of surface receptors. Moreover, these two effectors decrease clathrin-dependent and -independent endocytosis. Therefore, S. flexneri infection induces a global blockage of host cell intracellular transport, affecting the exchange between cells and their external environment. The combined action of these effectors disorganizes the epithelial cell polarity, disturbs epithelial barrier integrity, promotes multiple invasion events, and enhances the pathogen capacity to penetrate into the colonic tissue in vivo.

Growth and host interaction of mouse segmented filamentous bacteria in vitro.

The gut microbiota plays a crucial role in the maturation of the intestinal mucosal immune system of its host. Within the thousand bacterial species present in the intestine, the symbiont segmented filamentous bacterium (SFB) is unique in its ability to potently stimulate the post-natal maturation of the B- and T-cell compartments and induce a striking increase in the small-intestinal Th17 responses. Unlike other commensals, SFB intimately attaches to absorptive epithelial cells in the ileum and cells overlying Peyer's patches. This colonization does not result in pathology; rather, it protects the host from pathogens. Yet, little is known about the SFB-host interaction that underlies the important immunostimulatory properties of SFB, because SFB have resisted in vitro culturing for more than 50 years. Here we grow mouse SFB outside their host in an SFB-host cell co-culturing system. Single-celled SFB isolated from monocolonized mice undergo filamentation, segmentation, and differentiation to release viable infectious particles, the intracellular offspring, which can colonize mice to induce signature immune responses. In vitro, intracellular offspring can attach to mouse and human host cells and recruit actin. In addition, SFB can potently stimulate the upregulation of host innate defence genes, inflammatory cytokines, and chemokines. In vitro culturing thereby mimics the in vivo niche, provides new insights into SFB growth requirements and their immunostimulatory potential, and makes possible the investigation of the complex developmental stages of SFB and the detailed dissection of the unique SFB-host interaction at the cellular and molecular levels.

ALPK1 controls TIFA/TRAF6-dependent innate immunity against heptose-1,7-bisphosphate of gram-negative bacteria.

During infection by invasive bacteria, epithelial cells contribute to innate immunity via the local secretion of inflammatory cytokines. These are directly produced by infected cells or by uninfected bystanders via connexin-dependent cell-cell communication. However, the cellular pathways underlying this process remain largely unknown. Here we perform a genome-wide RNA interference screen and identify TIFA and TRAF6 as central players of Shigella flexneri and Salmonella typhimurium-induced interleukin-8 expression. We show that threonine 9 and the forkhead-associated domain of TIFA are necessary for the oligomerization of TIFA in both infected and bystander cells. Subsequently, this process triggers TRAF6 oligomerization and NF-κB activation. We demonstrate that TIFA/TRAF6-dependent cytokine expression is induced by the bacterial metabolite heptose-1,7-bisphosphate (HBP). In addition, we identify alpha-kinase 1 (ALPK1) as the critical kinase responsible for TIFA oligomerization and IL-8 expression in response to infection with S. flexneri and S. typhimurium but also to Neisseria meningitidis. Altogether, these results clearly show that ALPK1 is a master regulator of innate immunity against both invasive and extracellular gram-negative bacteria.

Shigella flexneri modulates stress granule composition and inhibits stress granule aggregation.

Invasion and multiplication of the facultative, cytosolic, enteropathogen Shigella flexneri within the colonic epithelial lining leads to an acute inflammatory response, fever and diarrhea. During the inflammatory process, infected cells are subjected to numerous stresses including heat, oxidative stress and genotoxic stress. The evolutionarily conserved pathway of cellular stress management is the formation of stress granules that store translationally inactive cellular mRNAs and interfere with cellular signalling pathways by sequestering signalling components. In this study, we investigated the ability of S. flexneri-infected cells to form stress granules in response to exogenous stresses. We found that S. flexneri infection inhibits movement of the stress granule markers eIF3 and eIF4B into stress granules and prevents the aggregation of G3BP1 and eIF4G-containing stress granules. This inhibition occurred only with invasive, but not with non-invasive bacteria and occurred in response to stresses that induce translational arrest through the phosphorylation of eIF2α and by treating cells with pateamine A, a drug that induces stress granules by inhibiting the eIF4A helicase. The S. flexneri-mediated stress granule inhibition could be largely phenocopied by the microtubule-destabilizing drug nocodazole and while S. flexneri infection did not lead to microtubule depolymerization, infection greatly enhanced acetylation of alpha-tubulin. Our data suggest that qualitative differences in the microtubule network or subversion of the microtubule-transport machinery by S. flexneri may be involved in preventing the full execution of this cellular stress response.

Host interactions with Segmented Filamentous Bacteria: an unusual trade-off that drives the post-natal maturation of the gut immune system.

Segmented Filamentous Bacteria (SFB) are present in the gut microbiota of a large number of vertebrate species where they are found intimately attached to the intestinal epithelium. SFB has recently attracted considerable attention due to its outstanding capacity to stimulate innate and adaptive host immune responses without causing pathology. Recent genomic analysis placed SFB between obligate and facultative symbionts, unraveled its highly auxotrophic needs, and provided a rationale for the complex SFB life-style in close contact with the epithelium. Herein, we examine how the SFB life-style may underlie its potent immunostimulatory properties and discuss how the trade-off set up between SFB and its hosts can simultaneously help to establish and maintain the ecological niche of SFB in the intestine and drive the post-natal maturation of the host gut immune barrier.

Diet prevents the expansion of segmented filamentous bacteria and ileo-colonic inflammation in a model of Crohn's disease.

BACKGROUND: Crohn's disease (CD) is associated with changes in the microbiota, and murine models of CD-like ileo-colonic inflammation depend on the presence of microbial triggers. Increased abundance of unknown Clostridiales and the microscopic detection of filamentous structures close to the epithelium of Tnf ΔARE mice, a mouse model of CD-like ileitis pointed towards segmented filamentous bacteria (SFB), a commensal mucosal adherent bacterium involved in ileal inflammation. RESULTS: We show that the abundance of SFB strongly correlates with the severity of CD-like ileal inflammation in two mouse models of ileal inflammation, including Tnf ΔARE and SAMP/Yit mice. SFB mono-colonization of germ-free Tnf ΔARE mice confirmed the causal link and resulted in severe ileo-colonic inflammation, characterized by elevated tissue levels of Tnf and Il-17A, neutrophil infiltration and loss of Paneth and goblet cell function. Co-colonization of SFB in human-microbiota associated Tnf ΔARE mice confirmed that SFB presence is indispensable for disease development. Screening of 468 ileal and colonic mucosal biopsies from adult and pediatric IBD patients, using previously published and newly designed human SFB-specific primer sets, showed no presence of SFB in human tissue samples, suggesting a species-specific functionality of the pathobiont. Simulating the human relevant therapeutic effect of exclusive enteral nutrition (EEN), EEN-like purified diet antagonized SFB colonization and prevented disease development in Tnf ΔARE mice, providing functional evidence for the protective mechanism of diet in modulating microbiota-dependent inflammation in IBD. CONCLUSIONS: We identified a novel pathogenic role of SFB in driving severe CD-like ileo-colonic inflammation characterized by loss of Paneth and goblet cell functions in Tnf ΔARE mice. A purified diet antagonized SFB colonization and prevented disease development in Tnf ΔARE mice in contrast to a fiber-containing chow diet, clearly demonstrating the important role of diet in modulating a novel IBD-relevant pathobiont and supporting a direct link between diet and microbial communities in mediating protective functions. Video Abstract.

Intracellular offspring released from SFB filaments are flagellated.

The gut commensal segmented filamentous bacterium (SFB) attaches to the ileal epithelium and potently stimulates the host immune system. Using transmission electron microscopy (TEM), we show that mouse and rat SFB are flagellated above the concave tip at the unicellular intracellular offspring (IO) stage and that flagellation occurs prior to full IO differentiation and release of IOs from SFB filaments. This finding adds a missing link to the SFB life cycle.

Shigella Pathogenesis: New Insights through Advanced Methodologies.

Shigella is a genus of Gram-negative enteropathogens that have long been, and continue to be, an important public health concern worldwide. Over the past several decades, Shigella spp. have also served as model pathogens in the study of bacterial pathogenesis, and Shigella flexneri has become one of the best-studied pathogens on a molecular, cellular, and tissue level. In the arms race between Shigella and the host immune system, Shigella has developed highly sophisticated mechanisms to subvert host cell processes in order to promote infection, escape immune detection, and prevent bacterial clearance. Here, we give an overview of Shigella pathogenesis while highlighting innovative techniques and methods whose application has significantly advanced our understanding of Shigella pathogenesis in recent years.

Modulation of the gut microbiota to improve innate resistance.

One major benefit from the association of hosts with the complex microbial communities that establish at body surfaces is the resistance to pathogen infection. This protective role of symbiotic microbes is becoming ever more relevant, given the alarming rise of multidrug-resistant pathogens and severe infections in patients following extensive antibiotic treatment. Herein, we highlight some recent mechanistic studies that have provided insights into how the highly dynamic dialogue amongst intestinal bacteria and between intestinal bacteria and their host can contribute to protect the host against pathogens in and outside the gut. We then discuss how delineating the rules of this dialogue can help design strategies to modulate the microbiota and improve host resistance to infections.

Listeriolysin O: a phagosome-specific lysin.

Listeriolysin O (LLO) is a pore-forming toxin of the cholesterol-dependent cytolysin family and a primary virulence factor of the gram-positive, facultative intracellular pathogen Listeria monocytogenes. During the intracellular life cycle of L. monocytogenes, LLO is largely responsible for mediating rupture of the phagosomal membrane, thereby allowing the bacterium access to the host cytosol, its replicative niche. In the host cytosol, LLO activity is controlled at numerous levels to prevent perforation of the plasma membrane and loss of the intracellular environment. In this review, we focus primarily on the role of LLO in phagosomal escape and the multiple regulatory mechanisms that control LLO activity in the host cytosol.

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells.

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of infected cells, including organelle ultrastructure, cytoskeletal network organization, as well as cellular processes such as Stress Granule (SG) formation. The characterization of how pathogens subvert host processes is an important and integral part of the field of pathogenesis. While variable phenotypes may be readily visible, the precise analysis of the qualitative and quantitative differences in the cellular structures induced by pathogen challenge is essential for defining statistically significant differences between experimental and control samples. SG formation is an evolutionarily conserved stress response that leads to antiviral responses and has long been investigated using viral infections1. SG formation also affects signaling cascades and may have other still unknown consequences2. The characterization of this stress response to pathogens other than viruses, such as bacterial pathogens, is currently an emerging area of research3. For now, quantitative and qualitative analysis of SG formation is not yet routinely used, even in the viral systems. Here we describe a simple method for inducing and characterizing SG formation in uninfected cells and in cells infected with a cytosolic bacterial pathogen, which affects the formation of SGs in response to various exogenous stresses. Analysis of SG formation and composition is achieved by using a number of different SG markers and the spot detector plug-in of ICY, an open source image analysis tool.

Segmented filamentous bacteria, Th17 inducers and helpers in a hostile world.

The Th17 cell composition in the murine gut is strikingly dependent on the presence of the commensal segmented filamentous bacteria (SFB). SFB potently stimulates innate and adaptive immune responses and protects the host from pathogens both in and outside of the gut, partly due to its unique ability to promote a Th17-fostering environment. Recent work has highlighted the role of the tight adherence of SFB to the intestinal surface in mediating the potent immunostimulatory potential of SFB. Progress has also been made in our understanding of how SFB fosters this protective immune environment on the cellular and molecular level. This review focuses on the ability of SFB to specifically stimulate Th17 immunity.

A fluorescent reporter reveals on/off regulation of the Shigella type III secretion apparatus during entry and cell-to-cell spread.

Numerous pathogenic Gram-negative bacteria use a type three secretion apparatus (T3SA) to translocate effector proteins into host cells. Detecting and monitoring the T3SA of intracellular bacteria within intact host cells has been challenging. Taking advantage of the tight coupling between T3S effector-gene transcription and T3SA activity in Shigella flexneri, together with a fast-maturing green fluorescent protein, we developed reporters to monitor T3SA activity in real time. These reporters reveal a dynamic temporal regulation of the T3SA during the course of infection. T3SA is activated initially during bacterial entry and downregulated subsequently when bacteria gain access to the host cell cytoplasm, allowing replenishment of the bacterial stores of T3S substrates necessary for invading neighboring cells. Reactivation of the T3SA was strictly dependent on actin-based motility and formation of plasma membrane protrusions during cell-to-cell spread. Thus, the T3SA is subject to a tight on/off regulation within the bacterial intracellular niche.

Genome Sequence of "Candidatus Arthromitus" sp. Strain SFB-Mouse-NL, a Commensal Bacterium with a Key Role in Postnatal Maturation of Gut Immune Functions.

"Candidatus Arthromitus" sp. strain SFB-mouse-NL (SFB, segmented filamentous bacteria) is a commensal bacterium necessary for inducing the postnatal maturation of homeostatic innate and adaptive immune responses in the mouse gut. Here, we report the genome sequence of this bacterium, which sets it apart from earlier sequenced mouse SFB isolates.

Quantitative RT-PCR profiling of the rabbit immune response: assessment of acute Shigella flexneri infection.

Quantitative reverse transcription PCR analysis is an important tool to monitor changes in gene expression in animal models. The rabbit is a widely accepted and commonly used animal model in the study of human diseases and infections by viral, fungal, bacterial and protozoan pathogens. Only a limited number of rabbit genes have, however, been analyzed by this method as the rabbit genome sequence remains unfinished. Recently, increasing coverage of the genome has permitted the prediction of a growing number of genes that are relevant in the context of the immune response. We hereby report the design of twenty-four quantitative PCR primer pairs covering common cytokines, chemoattractants, antimicrobials and enzymes for a rapid, sensitive and quantitative analysis of the rabbit immune response. Importantly, all primer pairs were designed to be used under identical experimental conditions, thereby enabling the simultaneous analysis of all genes in a high-throughput format. This tool was used to analyze the rabbit innate immune response to infection with the human gastrointestinal pathogen Shigella flexneri. Beyond the known inflammatory mediators, we identified IL-22, IL-17A and IL-17F as highly upregulated cytokines and as first responders to infection during the innate phase of the host immune response. This set of qPCR primers also provides a convenient tool for monitoring the rabbit immune response during infection with other pathogens and other inflammatory conditions.

Preventing acute gut wall damage in infectious diarrhoeas with glycosylated dendrimers.

Intestinal pathogens use the host's excessive inflammatory cytokine response, designed to eliminate dangerous bacteria, to disrupt epithelial gut wall integrity and promote their tissue invasion. We sought to develop a non-antibiotic-based approach to prevent this injury. Molecular docking studies suggested that glycosylated dendrimers block the TLR4-MD-2-LPS complex, and a 13.6 kDa polyamidoamine (PAMAM) dendrimer glucosamine (DG) reduced the induction of human monocyte interleukin (IL)-6 by Gram-negative bacteria. In a rabbit model of shigellosis, PAMAM-DG prevented epithelial gut wall damage and intestinal villous destruction, reduced local IL-6 and IL-8 expression, and minimized bacterial invasion. Computational modelling studies identified a 3.3 kDa polypropyletherimine (PETIM)-DG as the smallest likely bioactive molecule. In human monocytes, high purity PETIM-DG potently inhibited Shigella Lipid A-induced IL-6 expression. In rabbits, PETIM-DG prevented Shigella-induced epithelial gut wall damage, reduced local IL-6 and IL-8 expression, and minimized bacterial invasion. There was no change in ß-defensin, IL-10, interferon-ß, transforming growth factor-ß, CD3 or FoxP3 expression. Small and orally delivered DG could be useful for preventing gut wall tissue damage in a wide spectrum of infectious diarrhoeal diseases.

Listeriolysin O secreted by Listeria monocytogenes into the host cell cytosol is degraded by the N-end rule pathway.

The intracellular pathogen Listeria monocytogenes escapes from a phagosomal compartment into the cytosol by secreting the pore-forming cytolysin listeriolysin O (LLO). During the proliferation of L. monocytogenes bacteria in the mammalian cell cytosol, the secreted LLO is targeted for degradation by the ubiquitin system. We report here that LLO is a substrate of the ubiquitin-dependent N-end rule pathway, which recognizes LLO through its N-terminal Lys residue. Specifically, we demonstrated by reverse-genetic and pharmacological methods that LLO was targeted for degradation by the N-end rule pathway in reticulocyte extracts and mouse NIH 3T3 cells and after its secretion by intracellular bacteria into the mouse cell cytosol. Replacing the N-terminal Lys of LLO with a stabilizing residue such as Val increased the in vivo half-life of LLO but did not strongly affect the intracellular growth or virulence of L. monocytogenes. Nevertheless, this replacement decreased the virulence of L. monocytogenes by nearly twofold, suggesting that a destabilizing N-terminal residue of LLO may stem from positive selection during the evolution of this and related bacteria. A double mutant strain of L. monocytogenes in which upregulated secretion of LLO was combined with a stabilizing N-terminal residue was severely toxic to infected mammalian cells, resulting in reduced intracellular growth of bacteria and an approximately 100-fold-lower level of virulence. In summary, we showed that LLO is degraded by the N-end rule pathway and that the degradation of LLO can reduce the toxicity of L. monocytogenes during infection, a property of LLO that may have been selected for its positive effects on fitness during the evolution of L. monocytogenes.

Phosphorylation, ubiquitination and degradation of listeriolysin O in mammalian cells: role of the PEST-like sequence.

Listeria monocytogenes is a bacterial pathogen that grows within the cytosol of infected host cells. Entry into the cytosol is largely mediated by a secreted bacterial cytolysin, listeriolysin O (LLO). In order to prevent host cell damage, the pore-forming activity of LLO is restricted to the phagosome. Compartmentalization of LLO requires a PEST-like sequence; PEST sequences can direct eukaryotic proteins for proteasomal degradation. Here we test the hypothesis that LLO's PEST-like sequence compartmentalizes pore-forming activity by targeting this bacterial protein for degradation in the host cytosol. We show that intracellular LLO was degraded in a proteasome-dependent manner, and that, prior to degradation, LLO was ubiquitinated and was phosphorylated within the PEST-like sequence. However, wild-type LLO and PEST region mutants had similarly short intracellular half-lives and both the wild-type and mutant proteins were stabilized by inhibitors of host proteasomes. Additionally, blocking host proteasomes did not cause toxicity in a wild-type infection, but enhanced the cytotoxicity of PEST region mutants. Together with the observation that PEST region mutants exhibit higher intracellular LLO levels than wild-type bacteria, these data suggest that LLO's PEST-like region does not mediate proteasomal degradation by the host, but controls LLO production in the cytosol.

Regulated translation of listeriolysin O controls virulence of Listeria monocytogenes.

Listeria monocytogenes is an intracytosolic bacterial pathogen that escapes from the phagosome using a secreted cytolysin, listeriolysin O (LLO). In the host cytosol, LLO activity is minimized to prevent pore formation in the host plasma membrane; premature lysis of the infected host cell exposes the bacteria to extracellular immune defences of the host and is detrimental to infection. Here we identified nucleotide substitutions in the coding sequence of the LLO gene (hly) that did not alter the protein sequence, yet caused over-production of LLO, cytotoxicity and loss of virulence. These phenotypes were independent of the promoter and, under conditions in which the mutants produced more LLO protein than wild type, levels of hly mRNA were similar. Finally, negative regulation of LLO was maintained even when bacteria were engineered to produce elevated levels of the wild-type hly transcript. Together, our data demonstrate that translational regulation of LLO is critical for L. monocytogenes pathogenesis.