Actin Assembly around the Shigella-Containing Vacuole Promotes Successful Infection.
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ABSTRACT:
SUBMITTER: Kuhn S
PROVIDER: S-EPMC7225751 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Many cytosolic bacterial pathogens hijack the host actin polymerization machinery to form actin tails that promote direct cell-to-cell spread, enabling these pathogens to avoid extracellular immune defenses. However, these pathogens are still susceptible to intracellular cell-autonomous immune responses that restrict bacterial actin-based motility. Two classes of cytosolic antimotility factors, septins and guanylate-binding proteins (GBPs), have recently been established to block actin tail formation by the human-adapted bacterial pathogen Shigella flexneri. Both septin cages and GBP1 microcapsules restrict S. flexneri cell-to-cell spread by blocking S. flexneri actin-based motility. While septins assemble into cage-like structures around immobile S. flexneri, GBP1 forms microcapsules around both motile and immobile bacteria. The interplay between these two defense programs remains elusive. Here, we demonstrate that GBP1 microcapsules block septin cage assembly, likely by interfering with the function of S. flexneri IcsA, the outer membrane protein that promotes actin-based motility, as this protein is required for septin cage formation. However, S. flexneri that escape from GBP1 microcapsules via the activity of IpaH9.8, a type III secreted effector that promotes the degradation of GBPs, are often captured within septin cages. Thus, our studies reveal how septin cages and GBP1 microcapsules represent complementary host cell antimotility strategies.
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Project description:Pathogenic Shigella bacteria are a paradigm to address key issues of cell and infection biology. Polar localisation of the Shigella autotransporter protein IcsA is essential for actin tail formation, which is necessary for the bacterium to travel from cell-to-cell; yet how proteins are targeted to the bacterial cell pole is poorly understood. The bacterial actin homologue MreB has been extensively studied in broth culture using model organisms including Escherichia coli, Bacillus subtilis and Caulobacter crescentus, but has never been visualised in rod-shaped pathogenic bacteria during infection of host cells. Here, using single-cell analysis of intracellular Shigella, we discover that MreB accumulates at the cell pole of bacteria forming actin tails, where it colocalises with IcsA. Pharmacological inhibition of host cell actin polymerisation and genetic deletion of IcsA is used to show, respectively, that localisation of MreB to the cell poles precedes actin tail formation and polar localisation of IcsA. Finally, by exploiting the MreB inhibitors A22 and MP265, we demonstrate that MreB polymerisation can support actin tail formation. We conclude that Shigella MreB promotes polar IcsA positioning for actin tail formation, and suggest that understanding the bacterial cytoskeleton during host-pathogen interactions can inspire development of new therapeutic regimes for infection control.This article has an associated First Person interview with the first author of the paper.
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Project description:Intracellular pathogens such as Shigella flexneri and Listeria monocytogenes achieve dissemination in the intestinal epithelium by displaying actin-based motility in the cytosol of infected cells. As they reach the cell periphery, motile bacteria form plasma membrane protrusions that resolve into vacuoles in adjacent cells, through a poorly understood mechanism. Here, we report on the role of the class II phosphatidylinositol 3-phosphate kinase PIK3C2A in S. flexneri dissemination. Time-lapse microscopy revealed that PIK3C2A was required for the resolution of protrusions into vacuoles through the formation of an intermediate membrane-bound compartment that we refer to as a vacuole-like protrusion (VLP). Genetic rescue of PIK3C2A depletion with RNA interference (RNAi)-resistant cDNA constructs demonstrated that VLP formation required the activity of PIK3C2A in primary infected cells. PIK3C2A expression was required for production of phosphatidylinositol 3-phosphate [PtdIns(3)P] at the plasma membrane surrounding protrusions. PtdIns(3)P production was not observed in the protrusions formed by L. monocytogenes, whose dissemination did not rely on PIK3C2A. PIK3C2A-mediated PtdIns(3)P production in S. flexneri protrusions was regulated by host cell tyrosine kinase signaling and relied on the integrity of the S. flexneri type 3 secretion system (T3SS). We suggest a model of S. flexneri dissemination in which the formation of VLPs is mediated by the PIK3C2A-dependent production of the signaling lipid PtdIns(3)P in the protrusion membrane, which relies on the T3SS-dependent activation of tyrosine kinase signaling in protrusions.
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Project description:Human α-defensins are 3- to 5-kDa disulfide-bridged peptides with a multitude of antimicrobial activities and immunomodulatory functions. Recent studies show that human enteric α-defensin 5 (HD5), a host defense peptide important for intestinal homeostasis and innate immunity, aids the highly infectious enteropathogen Shigella in breaching the intestinal epithelium in vitro and in vivo Whether and how HD5 influences Shigella infection of resident macrophages following its invasion of the intestinal epithelium remain poorly understood. Here, we report that HD5 greatly promoted phagocytosis of Shigella by macrophages by targeting the bacteria to enhance bacterium-to-cell contacts in a structure- and sequence-dependent fashion. Subsequent intracellular multiplication of phagocytosed Shigella led to massive necrotic cell death and release of the bacteria. HD5-promoted phagocytosis of Shigella was independent of the status of the type 3 secretion system. Furthermore, HD5 neither inhibited nor enhanced phagosomal escape of Shigella Collectively, these findings confirm a potential pathogenic role of HD5 in Shigella infection of not only epithelial cells but also macrophages, illuminating how an enteropathogen exploits a host protective factor for virulence and infection.
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Project description:The enteric bacterial pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), utilizes two type III secretion systems (T3SSs) to invade host cells, survive and replicate intracellularly. T3SS1 and its dedicated effector proteins are required for bacterial entry into non-phagocytic cells and establishment and trafficking of the nascent Salmonella-containing vacuole (SCV). Here we identify the first T3SS1 effector required to maintain the integrity of the nascent SCV as SopF. SopF associates with host cell membranes, either when translocated by bacteria or ectopically expressed. Recombinant SopF binds to multiple phosphoinositides in protein-lipid overlays, suggesting that it targets eukaryotic cell membranes via phospholipid interactions. In yeast, the subcellular localization of SopF is dependent on the activity of Mss4, a phosphatidylinositol 4-phosphate 5-kinase that generates PI(4,5)P2 from PI(4)P, indicating that membrane recruitment of SopF requires specific phospholipids. Ectopically expressed SopF partially colocalizes with specific phosphoinositide pools present on the plasma membrane in mammalian cells and with cytoskeletal-associated markers at the leading edge of cells. Translocated SopF concentrates on plasma membrane ruffles and around intracellular bacteria, presumably on the SCV. SopF is not required for bacterial invasion of non-phagocytic cells but is required for maintenance of the internalization vacuole membrane as infection with a S. Typhimurium ΔsopF mutant led to increased lysis of the SCV compared to wild type bacteria. Our structure-function analysis shows that the carboxy-terminal seven amino acids of SopF are essential for its membrane association in host cells and to promote SCV membrane stability. We also describe that SopF and another T3SS1 effector, SopB, act antagonistically to modulate nascent SCV membrane dynamics. In summary, our study highlights that a delicate balance of type III effector activities regulates the stability of the Salmonella internalization vacuole.
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Project description:Coxiella burnetii is an intracellular bacterium that replicates within an expansive phagolysosome-like vacuole. Fusion between the Coxiella-containing vacuole (CCV) and late endosomes/multivesicular bodies requires Rab7, the HOPS tethering complex, and SNARE proteins, with actin also speculated to play a role. Here, we investigated the importance of actin in CCV fusion. Filamentous actin patches formed around the CCV membrane that were preferred sites of vesicular fusion. Accordingly, the mediators of endolysosomal fusion Rab7, VAMP7, and syntaxin 8 were concentrated in CCV actin patches. Generation of actin patches required C. burnetii type 4B secretion and host retromer function. Patches decorated with VPS29 and VPS35, components of the retromer, FAM21 and WASH, members of the WASH complex that engage the retromer, and Arp3, a component of the Arp2/3 complex that generates branched actin filaments. Depletion by siRNA of VPS35 or VPS29 reduced CCV actin patches and caused Rab7 to uniformly distribute in the CCV membrane. C. burnetii grew normally in VPS35 or VPS29 depleted cells, as well as WASH-knockout mouse embryo fibroblasts, where CCVs are devoid of actin patches. Endosome recycling to the plasma membrane and trans-Golgi of glucose transporter 1 (GLUT1) and cationic-independent mannose-6-phosphate receptor (CI-M6PR), respectively, was normal in infected cells. However, siRNA knockdown of retromer resulted in aberrant trafficking of GLUT1, but not CI-M6PR, suggesting canonical retrograde trafficking is unaffected by retromer disruption. Treatment with the specific Arp2/3 inhibitor CK-666 strongly inhibited CCV formation, an effect associated with altered endosomal trafficking of transferrin receptor. Collectively, our results show that CCV actin patches generated by retromer, WASH, and Arp2/3 are dispensable for CCV biogenesis and stability. However, Arp2/3-mediated production of actin filaments required for cargo transport within the endosomal system is required for CCV generation. These findings delineate which of the many actin related events that shape the endosomal compartment are important for CCV formation.
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Project description:Two Shigella species, Shigella flexneri and Shigella sonnei, cause approximately 90% of bacterial dysentery worldwide. While S. flexneri is the dominant species in low-income countries, S. sonnei causes the majority of infections in middle- and high-income countries. S. flexneri is a prototypic cytosolic bacterium; once intracellular, it rapidly escapes the phagocytic vacuole and causes pyroptosis of macrophages, which is important for pathogenesis and bacterial spread. In contrast, little is known about the invasion, vacuole escape, and induction of pyroptosis during S. sonnei infection of macrophages. We demonstrate here that S. sonnei causes substantially less pyroptosis in human primary monocyte-derived macrophages and THP1 cells. This is due to reduced bacterial uptake and lower relative vacuole escape, which results in fewer cytosolic S. sonnei and hence reduced activation of caspase-1 inflammasomes. Mechanistically, the O-antigen (O-Ag), which in S. sonnei is contained in both the lipopolysaccharide and the capsule, was responsible for reduced uptake and the type 3 secretion system (T3SS) was required for vacuole escape. Our findings suggest that S. sonnei has adapted to an extracellular lifestyle by incorporating multiple layers of O-Ag onto its surface compared to other Shigella species.IMPORTANCE Diarrheal disease remains the second leading cause of death in children under five. Shigella remains a significant cause of diarrheal disease with two species, S. flexneri and S. sonnei, causing the majority of infections. S. flexneri are well known to cause cell death in macrophages, which contributes to the inflammatory nature of Shigella diarrhea. Here, we demonstrate that S. sonnei causes less cell death than S. flexneri due to a reduced number of bacteria present in the cell cytosol. We identify the O-Ag polysaccharide which, uniquely among Shigella spp., is present in two forms on the bacterial cell surface as the bacterial factor responsible. Our data indicate that S. sonnei differs from S. flexneri in key aspects of infection and that more attention should be given to characterization of S. sonnei infection.
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Project description:S. flexneri is an important human pathogen that causes bacillary dysentery. During infection, S. flexneri invades colonic epithelial cells, hijacks the host cell cytoskeleton to move in the cytosol of infected cells, and spreads from cell to cell through formation of membrane protrusions that project into adjacent cells and resolve into double membrane vacuoles (DMVs). S. flexneri cell-to-cell spread requires the integrity of the bacterial type three secretion system (T3SS). However, the exact role of the T3SS effector proteins in the dissemination process remains poorly understood. Here, we investigated the role of the T3SS effector protein IpgB1 in S. flexneri dissemination. IpgB1 was previously characterized as a guanine nucleotide exchange factor (GEF) that contributes to invasion. In addition to the invasion defect, we showed that the ipgB1 mutant formed smaller infection foci in HT-29 cells. Complementation of this phenotype required the GEF activity of IpgB1. Using live confocal microscopy, we showed that the ipgB1 mutant is specifically impaired in DMV escape. Depletion of Rac1, the host cell target of IpgB1 during invasion, as well as pharmacological inhibition of Rac1 signaling, reduced cell-to-cell spread and DMV escape. In a targeted siRNA screen, we uncovered that RhoA depletion restored ipgB1 cell-to-cell spread and DMV escape, revealing a critical role for the IpgB1-Rac1 axis in antagonizing RhoA-mediated restriction of DMV escape. Using an infant rabbit model of shigellosis, we showed that the ipgB1 mutant formed fewer and smaller infection foci in the colon of infected animals, which correlated with attenuated symptoms of disease, including epithelial fenestration and bloody diarrhea. Our results demonstrate that, in addition to its role during invasion, IpgB1 modulates Rho family small GTPase signaling to promote cell-to-cell spread, DMV escape, and S. flexneri pathogenesis.
| S-EPMC8903249 | biostudies-literature
Project description:Shigella flexneri is an intracellular bacterium that hijacks the host actin cytoskeleton to invade and disseminate within the colonic epithelium. Shigella's virulence factors induce actin polymerization, leading to bacterial uptake, actin tail formation, actin-mediated motility, and cell-to-cell spreading. Many host factors involved in the Shigella-prompted actin rearrangements remain elusive. Here, we studied the role of a host protein receptor for activated C kinase 1 (RACK1) in actin cytoskeleton dynamics and Shigella infection. We used time-lapse imaging to demonstrate that RACK1 facilitates Shigella-induced actin cytoskeleton remodeling at multiple levels during infection of epithelial cells. Silencing RACK1 expression impaired Shigella-induced rapid polymerizing structures, reducing host cell invasion, bacterial motility, and cell-to-cell spreading. In uninfected cells, RACK1 silencing reduced jasplakinolide-mediated filamentous actin aggregate formation and negatively affected actin turnover in fast polymerizing structures, such as membrane ruffles. Our findings provide a role of RACK1 in actin cytoskeleton dynamics and Shigella infection.
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