Membrane chaperone SecDF plays a role in the secretion of Listeria monocytogenes major virulence factors.
Ontology highlight
ABSTRACT:
SUBMITTER: Burg-Golani T
PROVIDER: S-EPMC3837963 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:As an organism that has evolved to live in environments ranging from soil to the cytosol of mammalian cells, Listeria monocytogenes must regulate the secretion and activity of protein products that promote survival within these habitats. The post-translocation chaperone PrsA2 has been adapted to assist in the folding and activity of L. monocytogenes secreted proteins required for bacterial replication within host cells. Here we present the first structure/function investigation of the contributions of PrsA2 to protein secretion and activity as well as to bacterial virulence. Domain swap experiments with the closely related L. monocytogenes PrsA1 protein combined with targeted mutagenesis indicate distinct functional roles for the PrsA2 peptidyl-prolyl isomerase (PPIase) and the N- and C-terminal domains in pathogenesis. In contrast to other PrsA-like proteins described thus far in the literature, an absolute in vivo requirement for PrsA2 PPIase activity is evident in mouse infection models. This work illustrates the diversity of function associated with L. monocytogenes PrsA2 that serves to promote bacterial life within the infected host.
| S-EPMC3115453 | biostudies-literature
Project description:The Gram-positive bacterium Listeria monocytogenes is a facultative intracellular pathogen that relies on the regulated secretion and activity of a variety of proteins that sustain life within diverse environments. PrsA2 has recently been identified as a secreted peptidyl-prolyl cis/trans isomerase and chaperone that is dispensable for bacterial growth in broth culture but essential for L. monocytogenes virulence. Following host infection, PrsA2 contributes to the proper folding and activity of secreted proteins that are required for bacterial replication within the host cytosol and for bacterial spread to adjacent cells. PrsA2 is one member of a family of Gram-positive secretion chaperones that appear to play important roles in bacterial physiology; however, it is not known how these proteins recognize their substrate proteins or the degree to which their function is conserved across diverse Gram-positive species. We therefore examined PrsA proteins encoded by a variety of Gram-positive bacteria for functional complementation of L. monocytogenes mutants lacking prsA2. PrsA homologues encoded by Bacillus subtilis, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus mutans, Staphylococcus aureus, and Lactococcus lactis were examined for functional complementation of a variety of L. monocytogenes PrsA2-associated phenotypes central to L. monocytogenes pathogenesis and bacterial cell physiology. Our results indicate that while selected aspects of PrsA2 function are broadly conserved among diverse Gram-positive bacteria, PrsA2 exhibits unique specificity for L. monocytogenes target proteins required for pathogenesis. The L. monocytogenes PrsA2 chaperone thus appears evolutionarily optimized for virulence factor secretion within the host cell cytosol while still maintaining aspects of activity relevant to more general features of Gram-positive protein translocation.
| S-EPMC4567654 | biostudies-literature
Project description:Listeria monocytogenes is an intracellular facultative pathogen that causes listeriosis, a foodborne zoonotic infection. There are differences in the pathogenic potential of L. monocytogenes subtypes and strains. Comparison of the genome sequences among L. monocytogenes pathogenic strains EGD-e and F2365 with nonpathogenic L. innocua CLIP1182 and L. monocytogenes strain HCC23 revealed a set of proteins that were present in pathogenic strains and had no orthologs among the nonpathogenic strains. Among the candidate virulence factors are five proteins: putrescine carbamoyltransferase; InlH/InlC2 family class 1 internalin; phosphotransferase system (PTS) fructose transporter subunit EIIC; putative transketolase; and transcription antiterminator BglG family. To determine if these proteins have a role in adherence and invasion of intestinal epithelial Caco-2 cells and/or contribute to virulence, five mutant strains were constructed. F2365?inlC2, F2365?eiic, and F2365?tkt exhibited a significant (p < 0.05) reduction in adhesion to Caco-2 cells compared to parent F2365 strain. The invasion of F2365?aguB, F2365?inlC2, and F2365?bglG decreased significantly (p < 0.05) compared with the parent strain. Bacterial loads in mouse liver and spleen infected by F2365 was significantly (p < 0.05) higher than it was for F2365?aguB, F2365?inlC2, F2365?eiic, F2365?tkt, and F2365?bglG strains. This study demonstrates that aguB, inlC2, eiic, tkt, and bglG play a role in L. monocytogenes pathogenicity.
| S-EPMC6783856 | biostudies-literature
Project description:Bacterial pathogens often employ RNA regulatory elements located in the 5' untranslated regions (UTRs) to control gene expression. Using a comparative structural analysis, we examine the structure of 5' UTRs at a global scale in the pathogenic bacterium Listeria monocytogenes under different conditions. In addition to discovering an RNA thermoswitch and detecting simultaneous interaction of ribosomes and small RNAs with mRNA, we identify structural changes in the 5' UTR of an mRNA encoding the post-translocation chaperone PrsA2 during infection conditions. We demonstrate that the 5' UTR of the prsA2 mRNA base pairs with the 3' UTR of the full-length hly mRNA encoding listeriolysin O, thus preventing RNase J1-mediated degradation of the prsA2 transcript. Mutants lacking the hly-prsA2 interaction exhibit reduced virulence properties. This work highlights an additional level of RNA regulation, where the mRNA encoding a chaperone is stabilized by the mRNA encoding its substrate.
| S-EPMC8722363 | biostudies-literature
Project description:The Gram-positive bacterium Listeria monocytogenes transitions from an environmental organism to an intracellular pathogen following its ingestion by susceptible mammalian hosts. Bacterial replication within the cytosol of infected cells requires activation of the central virulence regulator PrfA followed by a PrfA-dependent induction of secreted virulence factors. The PrfA-induced secreted chaperone PrsA2 and the chaperone/protease HtrA contribute to the folding and stability of select proteins translocated across the bacterial membrane. L. monocytogenes strains that lack both prsA2 and htrA exhibit near-normal patterns of growth in broth culture but are severely attenuated in vivo We hypothesized that, in the absence of PrsA2 and HtrA, the increase in PrfA-dependent protein secretion that occurs following bacterial entry into the cytosol results in misfolded proteins accumulating at the bacterial membrane with a subsequent reduction in intracellular bacterial viability. Consistent with this hypothesis, the introduction of a constitutively activated allele of prfA (prfA*) into ?prsA2 ?htrA strains was found to essentially inhibit bacterial growth at 37°C in broth culture. ?prsA2 ?htrA strains were additionally found to be defective for cell invasion and vacuole escape in selected cell types, steps that precede full PrfA activation. These data establish the essential requirement for PrsA2 and HtrA in maintaining bacterial growth under conditions of PrfA activation. In addition, chaperone function is required for efficient bacterial invasion and rapid vacuole lysis within select host cell types, indicating roles for PrsA2/HtrA prior to cytosolic PrfA activation and the subsequent induction of virulence factor secretion.
| S-EPMC5038075 | biostudies-literature
Project description:The bulk of bacterial protein secretion occurs through the conserved SecY translocation channel that is powered by SecA-dependent ATP hydrolysis. Many Gram-positive bacteria, including the human pathogen Listeria monocytogenes, possess an additional nonessential specialized ATPase, SecA2. SecA2-dependent secretion is required for normal cell morphology and virulence in L. monocytogenes; however, the mechanism of export via this pathway is poorly understood. L. monocytogenes secA2 mutants form rough colonies, have septation defects, are impaired for swarming motility, and form small plaques in tissue culture cells. In this study, 70 spontaneous mutants were isolated that restored swarming motility to L. monocytogenes secA2 mutants. Most of the mutants had smooth colony morphology and septated normally, but all were lysozyme sensitive. Five representative mutants were subjected to whole-genome sequencing. Four of the five had mutations in proteins encoded by the lmo2769 operon that conferred lysozyme sensitivity and increased swarming but did not rescue virulence defects. A point mutation in secY was identified that conferred smooth colony morphology to secA2 mutants, restored wild-type plaque formation, and increased virulence in mice. This secY mutation resembled a prl suppressor known to expand the repertoire of proteins secreted through the SecY translocation complex. Accordingly, the ΔsecA2prlA1 mutant showed wild-type secretion levels of P60, an established SecA2-dependent secreted autolysin. Although the prl mutation largely suppressed almost all of the measurable SecA2-dependent traits, the ΔsecA2prlA1 mutant was still less virulent in vivo than the wild-type strain, suggesting that SecA2 function was still required for pathogenesis.
| S-EPMC4325107 | biostudies-literature
Project description:Upon bacterial entry into the cytosol of infected mammalian host cells, the central virulence regulator PrfA of Listeria monocytogenes becomes activated and induces the expression of numerous factors which contribute to bacterial pathogenesis. The mechanism or signal by which PrfA becomes activated during the course of infection has not yet been determined; however, several amino acid substitutions within PrfA (known as PrfA* mutations) that appear to lock the protein into a constitutively activated state have been identified. In this study, the PrfA activation statuses of several L. monocytogenes mutant strains were subjected to direct isogenic comparison and the mutant with the highest activity, the prfA(L140F) mutant, was identified. The prfA(L140F) strain was subsequently used as a tool to identify gene products secreted as a result of PrfA activation. By use of two-dimensional gel electrophoresis followed by liquid chromatography-electrospray ionization-tandem mass spectroscopy analyses, 15 proteins were identified as up-regulated in the prfA(L140F) secretome, while the secretion of two proteins was found to be reduced. Although some of the proteins identified were known to be subject to direct regulation by PrfA, the majority have not previously been associated with PrfA regulation and their expression or secretion may be influenced indirectly by a PrfA-dependent regulatory pathway. Plasmid insertion inactivation of the genes encoding four novel secreted products indicated that three of the four have significant roles in L. monocytogenes virulence. The use of mutationally activated prfA alleles therefore provides a useful approach towards identifying gene products that contribute to L. monocytogenes pathogenesis.
| S-EPMC2168340 | biostudies-literature
Project description:Outer membrane vesicles produced by Gram-negative bacteria have been studied for half a century but the possibility that Gram-positive bacteria secrete extracellular vesicles (EVs) was not pursued until recently due to the assumption that the thick peptidoglycan cell wall would prevent their release to the environment. However, following their discovery in fungi, which also have cell walls, EVs have now been described for a variety of Gram-positive bacteria. EVs purified from Gram-positive bacteria are implicated in virulence, toxin release, and transference to host cells, eliciting immune responses, and spread of antibiotic resistance. Listeria monocytogenes is a Gram-positive bacterium that causes listeriosis. Here we report that L. monocytogenes produces EVs with diameters ranging from 20 to 200 nm, containing the pore-forming toxin listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC). Cell-free EV preparations were toxic to mammalian cells, the murine macrophage cell line J774.16, in a LLO-dependent manner, evidencing EV biological activity. The deletion of plcA increased EV toxicity, suggesting PI-PLC reduced LLO activity. Using simultaneous metabolite, protein, and lipid extraction (MPLEx) multiomics we characterized protein, lipid, and metabolite composition of bacterial cells and secreted EVs and found that EVs carry the majority of listerial virulence proteins. Using immunogold EM we detected LLO at several organelles within infected human epithelial cells and with high-resolution fluorescence imaging we show that dynamic lipid structures are released from L. monocytogenes during infection. Our findings demonstrate that L. monocytogenes uses EVs for toxin release and implicate these structures in mammalian cytotoxicity.
| S-EPMC6349127 | biostudies-literature
Project description:The interaction of Listeria monocytogenes with endothelial cells represents a crucial step in the pathogenesis of listeriosis. Incubation of human umbilical vein endothelial cells (HUVEC) with wild-type L. monocytogenes (EGD) provoked immediate strong NO synthesis, attributable to listerial presentation of listeriolysin O (LLO), as the NO release was missed upon employment of a deletion mutant for LLO (EGD hly mutant) and was reproduced by purified LLO. Studies of conditions lacking extracellular Ca(2+) suggested LLO-elicited Ca(2+) flux as the underlying mechanism. In addition, HUVEC incubation with EGD turned out to be a potent stimulus for sustained (>12-h) upregulation of proinflammatory cytokine generation (interleukin 6 [IL-6], IL-8, and granulocyte-macrophage colony-stimulating factor). Use of deletion mutants for LLO (EGD hly mutant), listerial phosphatidylinositol-specific phospholipase C (EGD plcA mutant), broad-spectrum phospholipase C (EGD plcB mutant) and internalin B (EGD inlB mutant), as well as purified LLO, identified LLO as largely responsible for the cytokine response. Endothelial cells responded with diacylglycerole and ceramide generation as well as nuclear translocation of NF-kappa B to the stimulation with the LLO-producing strains EGD and Listeria innocua. The endothelial PC-phospholipase C inhibitor tricyclodecan-9-yl-xanthogenate as well as two independent inhibitors of NF-kappa B activation, pyrolidine dithiocarbamate and caffeic acid phenethyl ester, suppressed both the NF-kappa B translocation and the upregulation of cytokine synthesis. We conclude that L. monocytogenes is a potent stimulus of NO release and sustained upregulation of proinflammatory cytokine synthesis in human endothelial cells, both events being largely attributable to LLO presentation. LLO-induced transmembrane Ca(2+) flux as well as a sequence of endothelial phospholipase activation and the appearance of diacylglycerole, ceramide, and NF-kappa B are suggested as underlying host signaling events. These endothelial responses to L. monocytogenes may well contribute to the pathogenic sequelae in severe listerial infection and sepsis.
| S-EPMC97967 | biostudies-literature
Project description:A multi-virulence-locus sequence typing (MVLST) scheme was developed for subtyping Listeria monocytogenes, and the results obtained using this scheme were compared to those of pulsed-field gel electrophoresis (PFGE) and the published results of other typing methods, including ribotyping (RT) and multilocus sequence typing (MLST). A set of 28 strains (eight different serotypes and three known genetic lineages) of L. monocytogenes was selected from a strain collection (n > 1,000 strains) to represent the genetic diversity of this species. Internal fragments (ca. 418 to 469 bp) of three virulence genes (prfA, inlB, and inlC) and three virulence-associated genes (dal, lisR, and clpP) were sequenced and analyzed. Multiple DNA sequence alignment identified 10 (prfA), 19 (inlB), 13 (dal), 10 (lisR), 17 (inlC), and 16 (clpP) allelic types and a total of 28 unique sequence types. Comparison of MVLST with automated EcoRI-RT and PFGE with ApaI enzymatic digestion showed that MVLST was able to differentiate strains that were indistinguishable by RT (13 ribotypes; discrimination index = 0.921) or PFGE (22 profiles; discrimination index = 0.970). Comparison of MVLST with housekeeping-gene-based MLST analysis showed that MVLST provided higher discriminatory power for serotype 1/2a and 4b strains than MLST. Cluster analysis based on the intragenic sequences of the selected virulence genes indicated a strain phylogeny closely related to serotypes and genetic lineages. In conclusion, MVLST may improve the discriminatory power of MLST and provide a convenient tool for studying the local epidemiology of L. monocytogenes.
| S-EPMC348834 | biostudies-literature