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Vinculin is a cytoskeletal linker strengthening cell adhesion. The Shigella IpaA invasion effector binds to vinculin to promote vinculin supra-activation associated with head-domain-mediated oligomerization. Our study investigates the impact of mutations of vinculin D1D2 subdomains' residues predicted to interact with IpaA VBS3. These mutations affected the rate of D1D2 trimer formation with distinct effects on monomer disappearance, consistent with structural modeling of a closed and open D1D2 conformer induced by IpaA. Notably, mutations targeting the closed D1D2 conformer significantly reduced Shigella invasion of host cells as opposed to mutations targeting the open D1D2 conformer and later stages of vinculin head-domain oligomerization. In contrast, all mutations affected the formation of focal adhesions (FAs), supporting the involvement of vinculin supra-activation in this process. Our findings suggest that IpaA-induced vinculin supra-activation primarily reinforces matrix adhesion in infected cells, rather than promoting bacterial invasion. Consistently, shear stress studies pointed to a key role for IpaA-induced vinculin supra-activation in accelerating and strengthening cell-matrix adhesion., (© 2024 Cocom-Chan et al.)

Shigella flexneri is a Gram-negative bacterium causing severe bloody dysentery. Its pathogenesis is largely dictated by a plasmid-encoded type III secretion system (T3SS) and its associated effectors. Among these, the effector OspG has been shown to bind to the ubiquitin conjugation machinery (E2~Ub) to activate its kinase activity. However, the cellular targets of OspG remain elusive despite years of extensive efforts. Here we show by unbiased phosphoproteomics that a major target of OspG is CAND1, a regulatory protein controlling the assembly of cullin-RING ubiquitin ligases (CRLs). CAND1 phosphorylation weakens its interaction with cullins, which is expected to impact a large panel of CRL E3s. Indeed, global ubiquitome profiling reveals marked changes in the ubiquitination landscape when OspG is introduced. Notably, OspG promotes ubiquitination of a class of cytoskeletal proteins called septins, thereby inhibiting formation of cage-like structures encircling cytosolic bacteria. Overall, we demonstrate that pathogens have evolved an elaborate strategy to modulate host ubiquitin signaling to evade septin-cage entrapment., (© 2024. The Author(s).)

Shigella , the causative agent of bacillary dysentery, subvert cytoskeletal and trafficking processes to invade and replicate in epithelial cells using an arsenal of bacterial effectors translocated through a type III secretion system. Here, we review the various roles of the type III effector IpgD, initially characterized as phosphatidylinositol 4,5 bisphosphate (PI4,5P 2 ) 4-phosphatase. By decreasing PI4,5P 2 levels, IpgD triggers the disassembly of cortical actin filaments required for bacterial invasion and cell migration. PI5P produced by IpgD further stimulates signaling pathways regulating cell survival, macropinosome formation, endosomal trafficking and dampening of immune responses. Recently, IpgD was also found to exhibit phosphotransferase activity leading to PI3,4P 2 synthesis adding a new flavor to this multipotent bacterial enzyme. The substrate of IpgD, PI4,5P 2 is also the main substrate hydrolyzed by endogenous phospholipases C to produce inositoltriphosphate (InsP 3 ), a major Ca 2+ second messenger. Hence, beyond the repertoire of effects associated with the direct diversion of phoshoinositides, IpgD indirectly down-regulates InsP 3 -mediated Ca 2+ release by limiting InsP 3 production. Furthermore, IpgD controls the intracellular lifestyle of Shigella promoting Rab8/11 -dependent recruitment of the exocyst at macropinosomes to remove damaged vacuolar membrane remnants and promote bacterial cytosolic escape. IpgD thus emerges as a key bacterial effector for the remodeling of host cell membranes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tran Van Nhieu, Latour-Lambert and Enninga.)

Enzymes from pathogens often modulate host protein post-translational modifications (PTMs), facilitating survival and proliferation of pathogens. Shigella virulence factors IpaJ and IcsB induce proteolytic cleavage and lysine fatty acylation on host proteins, which cause Golgi stress and suppress innate immunity, respectively. However, it is unknown whether host enzymes could reverse such modifications introduced by pathogens' virulence factors to suppress pathogenesis. Herein, we report that SIRT2, a potent lysine defatty-acylase, is upregulated by the transcription factor CREB3 under Golgi stress induced by Shigella infection. SIRT2 in turn removes the lysine fatty acylation introduced by Shigella virulence factor IcsB to enhance host innate immunity. SIRT2 knockout mice are more susceptible to Shigella infection than wildtype mice, demonstrating the importance of SIRT2 to counteract Shigella infection., (© 2022. The Author(s).)

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.

Shigellosis is characterized as diarrheal disease that causes a high mortality rate especially in children, elderly and immunocompromised patients. More recently, the World Health Organization advised safe vaccine designing against shigellosis due to the emergence of Shigella dysenteriae resistant strains. Therefore, the aim of this study is to identify novel drug targets as well as the design of the potential vaccine candidates and chimeric vaccine models against Shigella dysenteriae. A computational based Reverse Vaccinology along with subtractive genomics analysis is one of the robust approaches used for the prioritization of drug targets and vaccine candidates through direct screening of genome sequence assemblies. Herein, a successfully designed peptide-based novel highly antigenic chimeric vaccine candidate against Shigella dysenteriae sd197 strain is proposed. The study resulted in six epitopes from outer membrane WP_000188255.1 (Fe (3+) dicitrate transport protein FecA) that ultimately leads to the construction of twelve vaccine models. Moreover, V9 construct was found to be highly immunogenic, non-toxic, non-allergenic, highly antigenic, and most stable in terms of molecular docking and simulation studies against six HLAs and TLRS/MD complex. So far, this protein and multiepitope have never been characterized as vaccine targets against Shigella dysenteriae. The current study proposed that V9 could be a significant vaccine candidate against shigellosis and to ascertain that further experiments may be applied by the scientific community focused on shigellosis., (© 2022. The Author(s).)

Intracellular bacterial pathogens have evolved a plethora of strategies to invade eukaryotic cells. By manipulating host signaling pathways, in particular vesicular trafficking, these microbes subvert host functions to promote their internalization and to establish an intracellular niche. During these events, host endomembrane compartments are dynamically reorganized. Shigella flexneri, the causative agent of bacillary dysentery, recruits components of the host recycling pathway and the exocyst of non-phagocytic enterocytes in the vicinity of its entry site to facilitate its access to the host cytosol. These factors are either dynamically tethered to in situ formed macropinosomes or to the bacteria-containing vacuole itself. The underlying interactions cannot readily be monitored as individual bacterial infection events take place without synchronicity using cellular infection models. Therefore, time-resolved screens by fluorescence microscopy represent a powerful tool for the study of host subversion. Such screens can be performed with libraries of fluorescently tagged host factors. Using the cytosolic pathogenic agent Shigella flexneri as a model, we provide detailed protocols for such medium-to-high throughput multidimensional imaging screening of the dynamic host-pathogen cross talk. Our workflow is designed to be easily adapted for the study of different host factor libraries and different pathogen models., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Shigella spp. are highly adapted pathogens that cause bacillary dysentery in human and nonhuman primates. An unusual feature of Shigella pathogenesis is that this organism invades the colonic epithelia from the basolateral pole. Therefore, it has evolved the ability to disrupt the intestinal epithelial barrier to reach the basolateral surface. We have shown previously that the secreted serine protease A (SepA), which belongs to the family of serine protease autotransporters of Enterobacteriaceae , is responsible for the initial destabilization of the intestinal epithelial barrier that facilitates Shigella invasion. However, the mechanisms used by SepA to regulate this process remain unknown. To investigate the protein targets cleaved by SepA in the intestinal epithelium, we incubated a sample of homogenized human colon with purified SepA or with a catalytically inactive mutant of this protease. We discovered that SepA targets an array of 18 different proteins, including alpha-1 antitrypsin (AAT), a major circulating serine proteinase inhibitor in humans. In contrast to other serine proteases, SepA cleaved AAT without forming an inhibiting complex, which resulted in the generation of a neutrophil chemoattractant. We demonstrated that the products of the AAT-SepA reaction induce a mild but significant increase in neutrophil transepithelial migration in vitro . Moreover, the presence of AAT during Shigella infection stimulated neutrophil migration and dramatically enhanced the number of bacteria invading the intestinal epithelium in a SepA-dependent manner. We conclude that by cleaving AAT, SepA releases a chemoattractant that promotes neutrophil migration, which in turn disrupts the intestinal epithelial barrier to enable Shigella invasion. IMPORTANCE Shigella is the second leading cause of diarrheal death globally. In this study, we identified the host protein targets of SepA, Shigella 's major protein secreted in culture. We demonstrated that by cleaving AAT, a serine protease inhibitor important to protect surrounding tissue at inflammatory sites, SepA releases a neutrophil chemoattractant that enhances Shigella invasion. Moreover, SepA degraded AAT without becoming inhibited by the cleaved product, and SepA catalytic activity was enhanced at higher concentrations of AAT. Activation of SepA by an excess of AAT may be physiologically relevant at the early stages of Shigella infection, when the amount of synthesized SepA is very low compared to the concentration of AAT in the intestinal lumen. This observation may also help to explain the adeptness of Shigella infectivity at low dose, despite the requirement of reaching the basolateral side to invade and colonize the colonic epithelium.

The pore-forming protein gasdermin D (GSDMD) executes lytic cell death called pyroptosis to eliminate the replicative niche of intracellular pathogens. Evolution favors pathogens that circumvent this host defense mechanism. Here, we show that the Shigella ubiquitin ligase IpaH7.8 functions as an inhibitor of GSDMD. Shigella is an enteroinvasive bacterium that causes hemorrhagic gastroenteritis in primates, but not rodents. IpaH7.8 contributes to species specificity by ubiquitinating human, but not mouse, GSDMD and targeting it for proteasomal degradation. Accordingly, infection of human epithelial cells with IpaH7.8-deficient Shigella flexneri results in increased GSDMD-dependent cell death compared with wild type. Consistent with pyroptosis contributing to murine disease resistance, eliminating GSDMD from NLRC4-deficient mice, which are already sensitized to oral infection with Shigella flexneri, leads to further enhanced bacterial replication and increased disease severity. This work highlights a species-specific pathogen arms race focused on maintenance of host cell viability., Competing Interests: Declaration of interests All authors except J.R., R.A.C., and R.E.V. are employees of Genentech. R.E.V. is an investigator of the Howard Hughes Medical Institute and a consultant for Ventus Therapeutics and Tempest Therapeutics., (Copyright © 2021 Genentech. Published by Elsevier Inc. All rights reserved.)

Many bacterial pathogens require a type 3 secretion system (T3SS) to establish a niche. Host contact activates bacterial T3SS assembly of a translocon pore in the host plasma membrane. Following pore formation, the T3SS docks onto the translocon pore. Docking establishes a continuous passage that enables the translocation of virulence proteins, effectors, into the host cytosol. Here we investigate the contribution of actin polymerization to T3SS-mediated translocation. Using the T3SS model organism Shigella flexneri, we show that actin polymerization is required for assembling the translocon pore in an open conformation, thereby enabling effector translocation. Opening of the pore channel is associated with a conformational change to the pore, which is dependent upon actin polymerization and a coiled-coil domain in the pore protein IpaC. Analysis of an IpaC mutant that is defective in ruffle formation shows that actin polymerization-dependent pore opening is distinct from the previously described actin polymerization-dependent ruffles that are required for bacterial internalization. Moreover, actin polymerization is not required for other pore functions, including docking or pore protein insertion into the plasma membrane. Thus, activation of the T3SS is a multilayered process in which host signals are sensed by the translocon pore leading to the activation of effector translocation., Competing Interests: The authors have declared that no competing interests exist.