Post-translational targeting of Rab35 by the effector IcsB of Shigella determines intracellular bacterial niche formation.

Escape from the bacterial-containing vacuole (BCV) is a key step of Shigella host cell invasion. Rab GTPases subverted to in situ-formed macropinosomes in the vicinity of the BCV have been shown to promote its rupture. The involvement of the BCV itself has remained unclear. We demonstrate that Rab35 is non-canonically entrapped at the BCV. Stimulated emission depletion imaging localizes Rab35 directly on the BCV membranes before vacuolar rupture. The bacterial effector IcsB, a lysine Nε-fatty acylase, is a key regulator of Rab35-BCV recruitment, and we show post-translational acylation of Rab35 by IcsB in its polybasic region. While Rab35 and IcsB are dispensable for the first step of BCV breakage, they are needed for the unwrapping of damaged BCV remnants from Shigella. This provides a framework for understanding Shigella invasion implicating re-localization of a Rab GTPase via its bacteria-dependent post-translational modification to support the mechanical unpeeling of the BCV.

Cryo-EM structure of a Shigella podophage reveals a hybrid tail and novel decoration proteins.

There is a paucity of high-resolution structures of phages infecting Shigella, a human pathogen and a serious threat to global health. HRP29 is a Shigella podophage belonging to the Autographivirinae family, and has very low sequence identity to other known phages. Here, we resolved the structure of the entire HRP29 virion by cryo-EM. Phage HRP29 has a highly unusual tail that is a fusion of a T7-like tail tube and P22-like tailspikes mediated by interactions from a novel tailspike adaptor protein. Understanding phage tail structures is critical as they mediate hosts interactions. Furthermore, we show that the HRP29 capsid is stabilized by two novel, and essential decoration proteins, gp47 and gp48. Only one high resolution structure is currently available for Shigella podophages. The presence of a hybrid tail and an adapter protein suggests that it may be a product of horizontal gene transfer, and may be prevalent in other phages.

VirB, a key transcriptional regulator of Shigella virulence, requires a CTP ligand for its regulatory activities.

IMPORTANCE: Shigella species cause bacillary dysentery, the second leading cause of diarrheal deaths worldwide. There is a pressing need to identify novel molecular drug targets. Shigella virulence phenotypes are controlled by the transcriptional regulator, VirB. We show that VirB belongs to a fast-evolving, plasmid-borne clade of the ParB superfamily, which has diverged from versions with a distinct cellular role-DNA partitioning. We report that, like classic members of the ParB family, VirB binds a highly unusual ligand, CTP. Mutants predicted to be defective in CTP binding are compromised in a variety of virulence attributes controlled by VirB, likely because these mutants cannot engage DNA. This study (i) reveals that VirB binds CTP, (ii) provides a link between VirB-CTP interactions and Shigella virulence phenotypes, (iii) provides new insight into VirB-CTP-DNA interactions, and (iv) broadens our understanding of the ParB superfamily, a group of bacterial proteins that play critical roles in many bacteria.

Epithelial inflammasomes, gasdermins, and mucosal inflammation - Lessons from Salmonella and Shigella infected mice.

Besides its crucial function in nutrient absorbance and as barrier against the microbiota, the gut epithelium is essential for sensing pathogenic insults and mounting of an appropriate early immune response. In mice, the activation of the canonical NAIP/NLRC4 inflammasome is critical for the defense against enterobacterial infections. Activation of the NAIP/NLRC4 inflammasome triggers the extrusion of infected intestinal epithelial cells (IEC) into the gut lumen, concomitant with inflammasome-mediated lytic cell death. The membrane permeabilization, a hallmark of pyroptosis, is caused by the pore-forming proteins called gasdermins (GSDMs). Recent work has revealed that NAIP/NLRC4-dependent extrusion of infected IECs can, however, also be executed in the absence of GSDMD. In fact, several reports highlighted that various cell death pathways (e.g., pyroptosis or apoptosis) and unique mechanisms specific to particular infection models and stages of gut infection are in action during epithelial inflammasome defense against intestinal pathogens. Here, we summarize the current knowledge regarding the underlying mechanisms and speculate on the putative functions of the epithelial inflammasome activation and cell death, with a particular emphasis on mouse infection models for two prominent enterobacterial pathogens, Salmonella Typhimurium and Shigella flexneri.

Diffusible signal factors (DSFs) bind and repress VirF, the leading virulence activator of Shigella flexneri.

Shigella, the aetiological agent of human bacillary dysentery, controls the expression of its virulence determinants through an environmentally stimulated cascade of transcriptional activators. VirF is the leading activator and is essential for proper virulence expression. In this work, we report on in vitro and in vivo experiments showing that two autoinducers of the DSF family, XcDSF and BDSF interact with the jelly roll module of VirF causing its inhibition and affecting the expression of the entire virulence system of Shigella, including its ability to invade epithelial cells. We propose a molecular model explaining how the binding of XcDSF and BDSF causes inhibition of VirF by preventing its dimerization. Overall, our experimental results suggest that XcDSF and BDSF may contribute to "colonisation resistance" in the human gut or, alternatively, may be exploited for the fine-tuning of Shigella virulence expression as the bacterium migrates from the lumen to approach the intestinal mucosa. Our findings also stress how a detailed understanding of the interaction of DSF ligands with VirF may contribute to the rational development of innovative antivirulence drugs to treat shigellosis.

Shigella IpaA mediates actin bundling through diffusible vinculin oligomers with activation imprint.

Upon activation, vinculin reinforces cytoskeletal anchorage during cell adhesion. Activating ligands classically disrupt intramolecular interactions between the vinculin head and tail domains that bind to actin filaments. Here, we show that Shigella IpaA triggers major allosteric changes in the head domain, leading to vinculin homo-oligomerization. Through the cooperative binding of its three vinculin-binding sites (VBSs), IpaA induces a striking reorientation of the D1 and D2 head subdomains associated with vinculin oligomerization. IpaA thus acts as a catalyst producing vinculin clusters that bundle actin at a distance from the activation site and trigger the formation of highly stable adhesions resisting the action of actin relaxing drugs. Unlike canonical activation, vinculin homo-oligomers induced by IpaA appear to keep a persistent imprint of the activated state in addition to their bundling activity, accounting for stable cell adhesion independent of force transduction and relevant to bacterial invasion.

Shigella IpaH9.8 limits GBP1-dependent LPS release from intracytosolic bacteria to suppress caspase-4 activation.

Pyroptosis is an inflammatory form of cell death induced upon recognition of invading microbes. During an infection, pyroptosis is enhanced in interferon-gamma-exposed cells via the actions of members of the guanylate-binding protein (GBP) family. GBPs promote caspase-4 (CASP4) activation by enhancing its interactions with lipopolysaccharide (LPS), a component of the outer envelope of Gram-negative bacteria. Once activated, CASP4 promotes the formation of noncanonical inflammasomes, signaling platforms that mediate pyroptosis. To establish an infection, intracellular bacterial pathogens, like Shigella species, inhibit pyroptosis. The pathogenesis of Shigella is dependent on its type III secretion system, which injects ~30 effector proteins into host cells. Upon entry into host cells, Shigella are encapsulated by GBP1, followed by GBP2, GBP3, GBP4, and in some cases, CASP4. It has been proposed that the recruitment of CASP4 to bacteria leads to its activation. Here, we demonstrate that two Shigella effectors, OspC3 and IpaH9.8, cooperate to inhibit CASP4-mediated pyroptosis. We show that in the absence of OspC3, an inhibitor of CASP4, IpaH9.8 inhibits pyroptosis via its known degradation of GBPs. We find that, while some LPS is present within the host cell cytosol of epithelial cells infected with wild-type Shigella, in the absence of IpaH9.8, increased amounts are shed in a GBP1-dependent manner. Furthermore, we find that additional IpaH9.8 targets, likely GBPs, promote CASP4 activation, even in the absence of GBP1. These observations suggest that by boosting LPS release, GBP1 provides CASP4-enhanced access to cytosolic LPS, thus promoting host cell death via pyroptosis.

Septins and K63 ubiquitin chains are present in separate bacterial microdomains during autophagy of entrapped Shigella.

During host cell invasion, Shigella escapes to the cytosol and polymerizes actin for cell-to-cell spread. To restrict cell-to-cell spread, host cells employ cell-autonomous immune responses including antibacterial autophagy and septin cage entrapment. How septins interact with the autophagy process to target Shigella for destruction is poorly understood. Here, we employed a correlative light and cryo-soft X-ray tomography (cryo-SXT) pipeline to study Shigella septin cage entrapment in its near-native state. Quantitative cryo-SXT showed that Shigella fragments mitochondria and enabled visualization of X-ray-dense structures (∼30 nm resolution) surrounding Shigella entrapped in septin cages. Using Airyscan confocal microscopy, we observed lysine 63 (K63)-linked ubiquitin chains decorating septin-cage-entrapped Shigella. Remarkably, septins and K63 chains are present in separate bacterial microdomains, indicating they are recruited separately during antibacterial autophagy. Cryo-SXT and live-cell imaging revealed an interaction between septins and LC3B-positive membranes during autophagy of Shigella. Together, these findings demonstrate how septin-caged Shigella are targeted for autophagy and provide fundamental insights into autophagy-cytoskeleton interactions.

Localized modulation of DNA supercoiling, triggered by the Shigella anti-silencer VirB, is sufficient to relieve H-NS-mediated silencing.

In Bacteria, nucleoid structuring proteins govern nucleoid dynamics and regulate transcription. In Shigella spp., at ≤30°C, the histone-like nucleoid structuring protein (H-NS) transcriptionally silences many genes on the large virulence plasmid. Upon a switch to 37°C, VirB, a DNA binding protein and key transcriptional regulator of Shigella virulence, is produced. VirB functions to counter H-NS-mediated silencing in a process called transcriptional anti-silencing. Here, we show that VirB mediates a loss of negative DNA supercoils from our plasmid-borne, VirB-regulated PicsP-lacZ reporter in vivo. The changes are not caused by a VirB-dependent increase in transcription, nor do they require the presence of H-NS. Instead, the VirB-dependent change in DNA supercoiling requires the interaction of VirB with its DNA binding site, a critical first step in VirB-dependent gene regulation. Using two complementary approaches, we show that VirB:DNA interactions in vitro introduce positive supercoils in plasmid DNA. Subsequently, by exploiting transcription-coupled DNA supercoiling, we reveal that a localized loss of negative supercoils is sufficient to alleviate H-NS-mediated transcriptional silencing independently of VirB. Together, our findings provide novel insight into VirB, a central regulator of Shigella virulence and, more broadly, a molecular mechanism that offsets H-NS-dependent silencing of transcription in bacteria.

Structural insight into the recognition of the linear ubiquitin assembly complex by Shigella E3 ligase IpaH1.4/2.5.

Pathogenic bacteria deliver virulence factors called effectors into host cells in order to facilitate infection. The Shigella effector proteins IpaH1.4 and IpaH2.5 are members of the 'novel E3 ligase' (NEL)-type bacterial E3 ligase family. These proteins ubiquitinate the linear ubiquitin assembly complex (LUBAC) to inhibit nuclear factor (NF)-κB activation and, concomitantly, the inflammatory response. However, the molecular mechanisms underlying the interaction and recognition between IpaH1.4 and IpaH2.5 and LUBAC are unclear. Here we present the crystal structures of the substrate-recognition domains of IpaH1.4 and IpaH2.5 at resolutions of 1.4 and 3.4 Å, respectively. The LUBAC-binding site on IpaH1.4 was predicted based on structural comparisons with the structures of other NEL-type E3s. Structural and biochemical data were collected and analysed to determine the specific residues of IpaH1.4 that are involved in interactions with LUBAC and influence NF-κB signaling. The new structural insight presented here demonstrates how bacterial pathogens target innate immune signaling pathways.

Structural mechanisms of calmodulin activation of Shigella effector OspC3 to ADP-riboxanate caspase-4/11 and block pyroptosis.

The caspase-4/11-GSDMD pyroptosis axis recognizes cytosolic lipopolysaccharide for antibacterial defenses. Shigella flexneri employs an OspC3 effector to block pyroptosis by catalyzing NAD+-dependent arginine ADP-riboxanation of caspase-4/11. Here, we identify Ca2+-free calmodulin (CaM) that binds and stimulates OspC3 ADP-riboxanase activity. Crystal structures of OspC3-CaM and OspC3-caspase-4 binary complexes reveal unique CaM binding to an OspC3 N-terminal domain featuring an ADP-ribosyltransferase-like fold and specific recognition of caspase-4 by an OspC3 ankryin repeat domain, respectively. CaM-OspC3-caspase-4 ternary complex structures show that NAD+ binding reorganizes the catalytic pocket, in which D231 and D177 activate the substrate arginine for initial ADP-ribosylation and ribosyl 2'-OH in the ADP-ribosylated arginine, respectively, for subsequent deamination. We also determine structures of unmodified and OspC3-ADP-riboxanated caspase-4. Mechanisms derived from this series of structures covering the entire process of OspC3 action are supported by biochemical analyses in vitro and functional validation in S. flexneri-infected mice.

Roles of Two-Component Signal Transduction Systems in Shigella Virulence.

Two-component signal transduction systems (TCSs) are widespread types of protein machinery, typically consisting of a histidine kinase membrane sensor and a cytoplasmic transcriptional regulator that can sense and respond to environmental signals. TCSs are responsible for modulating genes involved in a multitude of bacterial functions, including cell division, motility, differentiation, biofilm formation, antibiotic resistance, and virulence. Pathogenic bacteria exploit the capabilities of TCSs to reprogram gene expression according to the different niches they encounter during host infection. This review focuses on the role of TCSs in regulating the virulence phenotype of Shigella, an intracellular pathogen responsible for severe human enteric syndrome. The pathogenicity of Shigella is the result of the complex action of a wide number of virulence determinants located on the chromosome and on a large virulence plasmid. In particular, we will discuss how five TCSs, EnvZ/OmpR, CpxA/CpxR, ArcB/ArcA, PhoQ/PhoP, and EvgS/EvgA, contribute to linking environmental stimuli to the expression of genes related to virulence and fitness within the host. Considering the relevance of TCSs in the expression of virulence in pathogenic bacteria, the identification of drugs that inhibit TCS function may represent a promising approach to combat bacterial infections.

In silico analysis and a comparative genomics approach to predict pathogenic trehalase genes in the complete genome of Antarctica Shigella sp. PAMC28760.

Although four Shigella species (S. flexneri, S. sonnei, S. dysenteriae, and S. boydii) have been reported, S. sp. PAMC 28760, an Antarctica isolate, is the only one with a complete genome deposited in NCBI database as an uncharacterized isolate. Because it is the world's driest, windiest, and coldest continent, Antarctica provides an unfavourable environment for microorganisms. Computational analysis of genomic sequences of four Shigella species and our uncategorized Antarctica isolates Shigella sp. PAMC28760 was performed using MP3 (offline version) program to predict trehalase encoding genes as a pathogenic or non-pathogenic form. Additionally, we employed RAST and Prokka (offline version) annotation programs to determine locations of periplasmic (treA) and cytoplasmic (treF) trehalase genes in studied genomes. Our results showed that only 56 out of 134 Shigella strains had two different trehalase genes (treF and treA). It was revealed that the treF gene tends to be prevalent in Shigella species. In addition, both treA and treF genes were present in our strain S. sp. PAMC28760. The main objective of this study was to predict the prevalence of two different trehalase genes (treF and treA) in the complete genome of Shigella sp. PAMC28760 and other complete genomes of Shigella species. Till date, it is the first study to show that two types of trehalase genes are involved in Shigella species, which could offer insight on how the bacteria use accessible carbohydrate like glucose produced from the trehalose degradation pathway, and importance of periplasmic trehalase involvement in bacterial virulence.

Epithelial and Neutrophil Interactions and Coordinated Response to Shigella in a Human Intestinal Enteroid-Neutrophil Coculture Model.

Polymorphonuclear neutrophils (PMN) are recruited to the gastrointestinal mucosa in response to inflammation, injury, and infection. Here, we report the development and the characterization of an ex vivo tissue coculture model consisting of human primary intestinal enteroid monolayers and PMN, and a mechanistic interrogation of PMN-epithelial cell interaction and response to Shigella, a primary cause of childhood dysentery. Cellular adaptation and tissue integration, barrier function, PMN phenotypic and functional attributes, and innate immune responses were examined. PMN within the enteroid monolayers acquired a distinct activated/migratory phenotype that was influenced by direct epithelial cell contact as well as by molecular signals. Seeded on the basal side of the intestinal monolayer, PMN were intercalated within the epithelial cells and moved paracellularly toward the apical side. Cocultured PMN also increased basal secretion of interleukin 8 (IL-8). Shigella added to the apical surface of the monolayers evoked additional PMN phenotypic adaptations, including increased expression of cell surface markers associated with chemotaxis and cell degranulation (CD47, CD66b, and CD88). Apical Shigella infection triggered rapid transmigration of PMN to the luminal side, neutrophil extracellular trap (NET) formation, and bacterial phagocytosis and killing. Shigella infection modulated cytokine production in the coculture; apical monocyte chemoattractant protein (MCP-1), tumor necrosis factor alpha (TNF-α), and basolateral IL-8 production were downregulated, while basolateral IL-6 secretion was increased. We demonstrated, for the first time, PMN phenotypic adaptation and mobilization and coordinated epithelial cell-PMN innate response upon Shigella infection in the human intestinal environment. The enteroid monolayer-PMN coculture represents a technical innovation for mechanistic interrogation of gastrointestinal physiology, host-microbe interaction, innate immunity, and evaluation of preventive/therapeutic tools. IMPORTANCE Studies of mucosal immunity and microbial host cell interaction have traditionally relied on animal models and in vitro tissue culture using immortalized cancer cell lines, which yield nonphysiological and often unreliable results. Herein, we report the development and characterization of an ex vivo enteroid-PMN coculture consisting of normal human intestinal epithelium and a mechanistic interrogation of PMN and epithelial cell interaction and function in the context of Shigella infection. We demonstrated tissue-driven phenotypic and functional adaptation of PMN and a coordinated epithelial cell and PMN response to Shigella, a primary cause of dysentery in young children in the developing world.

Shigella Outer Membrane Vesicles as Promising Targets for Vaccination.

The clinical symptoms of shigellosis, a gastrointestinal infection caused by Shigella spp. range from watery diarrhea to fulminant dysentery. Endemic infections, particularly among children in developing countries, represent the majority of clinical cases. The situation is aggravated due to the high mortality rate of shigellosis, the rapid dissemination of multi-resistant Shigella strains and the induction of only serotype-specific immunity. Thus, infection prevention due to vaccination, encompassing as many of the circulating serotypes as possible, has become a topic of interest. However, vaccines have turned out to be ineffective so far. Outer membrane vesicles (OMVs) are promising novel targets for vaccination. OMVs are constitutively secreted by Gram-negative bacteria including Shigella during growth. They are composed of soluble luminal portions and an insoluble membrane and can contain toxins, bioactive periplasmic and cytoplasmic (lipo-) proteins, (phospho-) lipids, nucleic acids and/or lipopolysaccharides. Thus, OMVs play an important role in bacterial cell-cell communication, growth, survival and pathogenesis. Furthermore, they modulate the secretion and transport of biomolecules, the stress response, antibiotic resistance and immune responses of the host. Thus, OMVs serve as novel secretion machinery. Here, we discuss the current literature and highlight the properties of OMVs as potent vaccine candidates because of their immunomodulatory, antigenic and adjuvant properties.

Composition and Biophysical Properties of the Sorting Platform Pods in the Shigella Type III Secretion System.

Shigella flexneri, causative agent of bacillary dysentery (shigellosis), uses a type III secretion system (T3SS) as its primary virulence factor. The T3SS injectisome delivers effector proteins into host cells to promote entry and create an important intracellular niche. The injectisome's cytoplasmic sorting platform (SP) is a critical assembly that contributes to substrate selection and energizing secretion. The SP consists of oligomeric Spa33 "pods" that associate with the basal body via MxiK and connect to the Spa47 ATPase via MxiN. The pods contain heterotrimers of Spa33 with one full-length copy associated with two copies of a C-terminal domain (Spa33C). The structure of Spa33C is known, but the precise makeup and structure of the pods in situ remains elusive. We show here that recombinant wild-type Spa33 can be prepared as a heterotrimer that forms distinct stable complexes with MxiK and MxiN. In two-hybrid analyses, association of the Spa33 complex with these proteins occurs via the full-length Spa33 component. Furthermore, these complexes each have distinct biophysical properties. Based on these properties, new high-resolution cryo-electron tomography data and architectural similarities between the Spa33 and flagellar FliM-FliN complexes, we provide a preliminary model of the Spa33 heterotrimers within the SP pods. From these findings and evolving models of SP interfaces and dynamics in the Yersinia and Salmonella T3SS, we suggest a model for SP function in which two distinct complexes come together within the context of the SP to contribute to form the complete pod structures during the recruitment of T3SS secretion substrates.

The Pretreatment Gut Microbiome Is Associated With Lack of Response to Methotrexate in New-Onset Rheumatoid Arthritis.

OBJECTIVE: Although oral methotrexate (MTX) remains the anchor drug for rheumatoid arthritis (RA), up to 50% of patients do not achieve a clinically adequate outcome. In addition, there is a lack of prognostic tools for treatment response prior to drug initiation. This study was undertaken to investigate whether interindividual differences in the human gut microbiome can aid in the prediction of MTX efficacy in new-onset RA. METHODS: We performed 16S ribosomal RNA gene and shotgun metagenomic sequencing on the baseline gut microbiomes of drug-naive patients with new-onset RA (n = 26). Results were validated in an additional independent cohort (n = 21). To gain insight into potential microbial mechanisms, we conducted ex vivo experiments coupled with metabolomics analysis to evaluate the association between microbiome-driven MTX depletion and clinical response. RESULTS: Our analysis revealed significant associations of the abundance of gut bacterial taxa and their genes with future clinical response (q < 0.05), including orthologs related to purine and MTX metabolism. Machine learning techniques were applied to the metagenomic data, resulting in a microbiome-based model that predicted lack of response to MTX in an independent group of patients. Finally, MTX levels remaining after ex vivo incubation with distal gut samples from pretreatment RA patients significantly correlated with the magnitude of future clinical response, suggesting a possible direct effect of the gut microbiome on MTX metabolism and treatment outcomes. CONCLUSION: Taken together, these findings are the first step toward predicting lack of response to oral MTX in patients with new-onset RA and support the value of the gut microbiome as a possible prognostic tool and as a potential target in RA therapeutics.

Direct binding of polymeric GBP1 to LPS disrupts bacterial cell envelope functions.

In the outer membrane of gram-negative bacteria, O-antigen segments of lipopolysaccharide (LPS) form a chemomechanical barrier, whereas lipid A moieties anchor LPS molecules. Upon infection, human guanylate binding protein-1 (hGBP1) colocalizes with intracellular gram-negative bacterial pathogens, facilitates bacterial killing, promotes activation of the lipid A sensor caspase-4, and blocks actin-driven dissemination of the enteric pathogen Shigella. The underlying molecular mechanism for hGBP1's diverse antimicrobial functions is unknown. Here, we demonstrate that hGBP1 binds directly to LPS and induces "detergent-like" LPS clustering through protein polymerization. Binding of polymerizing hGBP1 to the bacterial surface disrupts the O-antigen barrier, thereby unmasking lipid A, eliciting caspase-4 recruitment, enhancing antibacterial activity of polymyxin B, and blocking the function of the Shigella outer membrane actin motility factor IcsA. These findings characterize hGBP1 as an LPS-binding surfactant that destabilizes the rigidity of the outer membrane to exert pleiotropic effects on the functionality of gram-negative bacterial cell envelopes.

Targeting the Early Endosome-to-Golgi Transport of Shiga Toxins as a Therapeutic Strategy.

Shiga toxin (STx) produced by Shigella and closely related Shiga toxin 1 and 2 (STx1 and STx2) synthesized by Shiga toxin-producing Escherichia coli (STEC) are bacterial AB5 toxins. All three toxins target kidney cells and may cause life-threatening renal disease. While Shigella infections can be treated with antibiotics, resistance is increasing. Moreover, antibiotic therapy is contraindicated for STEC, and there are no definitive treatments for STEC-induced disease. To exert cellular toxicity, STx, STx1, and STx2 must undergo retrograde trafficking to reach their cytosolic target, ribosomes. Direct transport from early endosomes to the Golgi apparatus is an essential step that allows the toxins to bypass degradative late endosomes and lysosomes. The essentiality of this transport step also makes it an ideal target for the development of small-molecule inhibitors of toxin trafficking as potential therapeutics. Here, we review the recent advances in understanding the molecular mechanisms of the early endosome-to-Golgi transport of STx, STx1, and STx2, as well as the development of small-molecule inhibitors of toxin trafficking that act at the endosome/Golgi interface.