Relationship between bacterial virulence and nucleotide metabolism: a mutation in the adenylate kinase gene renders Yersinia pestis avirulent.
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ABSTRACT:
SUBMITTER: Munier-Lehmann H
PROVIDER: S-EPMC1223521 | biostudies-other | 2003 Jul
REPOSITORIES: biostudies-other
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Project description:Plague caused by Yersinia pestis is one of the deadliest diseases. However, many molecular mechanisms of bacterial virulence remain unclear. This study engaged in the discovery of small open reading frame (sORF)-encoded peptides (SEPs) in Y. pestis. An integrated proteogenomic pipeline was established, and an atlas containing 76 SEPs was described. Bioinformatic analysis indicated that 20% of these SEPs were secreted or localized to the transmembrane and that 33% contained functional domains. Two SEPs, named SEPs-yp1 and -yp2 and encoded in noncoding regions, were selected by comparative peptidomics analysis under host-specific environments and high-salinity stress. They displayed important roles in the regulation of antiphagocytic capability in a thorough functional assay. Remarkable attenuation of virulence in mice was observed in the SEP-deleted mutants. Further global proteomic analysis indicated that SEPs-yp1 and -yp2 affected the bacterial metabolic pathways, and SEP-yp1 was associated with the bacterial virulence by modulating the expression of key virulence factors of the Yersinia type III secretion system. Our study provides a rich resource for research on Y. pestis and plague, and the findings on SEP-yp1 and SEP-yp2 shed light on the molecular mechanism of bacterial virulence.
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Project description:The complete nucleotide sequence and gene organization of the three virulence plasmids from Yersinia pestis KIM5 were determined. Plasmid pPCP1 (9,610 bp) has a GC content of 45.3% and encodes two previously known virulence factors, an associated protein, and a single copy of IS100. Plasmid pCD1 (70,504 bp) has a GC content of 44.8%. It is known to encode a number of essential virulence determinants, regulatory functions, and a multiprotein secretory system comprising the low-calcium response stimulation that is shared with the other two Yersinia species pathogenic for humans (Y. pseudotuberculosis and Y. enterocolitica). A new pseudogene, which occurs as an intact gene in the Y. enterocolitica and Y. pseudotuberculosis-derived analogues, was found in pCD1. It corresponds to that encoding the lipoprotein YlpA. Several intact and partial insertion sequences and/or transposons were also found in pCD1, as well as six putative structural genes with high homology to proteins of unknown function in other yersiniae. The sequences of the genes involved in the replication of pCD1 are highly homologous to those of the cognate plasmids in Y. pseudotuberculosis and Y. enterocolitica, but their localization within the plasmid differs markedly from those of the latter. Plasmid pMT1 (100,984 bp) has a GC content of 50.2%. It possesses two copies of IS100, which are located 25 kb apart and in opposite orientations. Adjacent to one of these IS100 inserts is a partial copy of IS285. A single copy of an IS200-like element (recently named IS1541) was also located in pMT1. In addition to 5 previously described genes, such as murine toxin, capsule antigen, capsule anchoring protein, etc., 30 homologues to genes of several bacterial species were found in this plasmid, and another 44 open reading frames without homology to any known or hypothetical protein in the databases were predicted.
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Project description:We have identified an approximately 22-kb region of the pgm locus of Yersinia pestis KIM6+ which encodes a number of iron-regulated proteins involved in the biosynthesis of the Y. pestis cognate siderophore, yersiniabactin (Ybt), and which is located immediately upstream of the pesticin/yersiniabactin receptor gene (psn). Sequence analysis and the construction of insertion and deletion mutants allowed us to determine the putative location of the irp1 gene and the positions of irp2, ybtT, and ybtE within the ybt operon. Mutations in the irp1, irp2, or ybtE gene yielded strains defective in siderophore production. Mutant strains were unable to grow on iron-deficient media at 37 degrees C but could be cross-fed by culture supernatants from yersiniabactin-producing strains of Y. pestis grown under iron-limiting conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of whole-cell extracts from Ybt+ and Ybt- strains grown in iron-deficient media revealed that expression of ybt-encoded proteins is not only iron regulated but also influenced by the presence of the siderophore itself. Finally, Y. pestis strains with mutations in either the psn or irp2 gene were avirulent in mice when inoculated subcutaneously.
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Project description:A Yersinia pestis-human protein interaction network is reported here to improve our understanding of its pathogenesis. Up to 204 interactions between 66 Y. pestis bait proteins and 109 human proteins were identified by yeast two-hybrid assay and then combined with 23 previously published interactions to construct a protein-protein interaction network. Topological analysis of the interaction network revealed that human proteins targeted by Y. pestis were significantly enriched in the proteins that are central in the human protein-protein interaction network. Analysis of this network showed that signaling pathways important for host immune responses were preferentially targeted by Y. pestis, including the pathways involved in focal adhesion, regulation of cytoskeleton, leukocyte transendoepithelial migration, and Toll-like receptor (TLR) and mitogen-activated protein kinase (MAPK) signaling. Cellular pathways targeted by Y. pestis are highly relevant to its pathogenesis. Interactions with host proteins involved in focal adhesion and cytoskeketon regulation pathways could account for resistance of Y. pestis to phagocytosis. Interference with TLR and MAPK signaling pathways by Y. pestis reflects common characteristics of pathogen-host interaction that bacterial pathogens have evolved to evade host innate immune response by interacting with proteins in those signaling pathways. Interestingly, a large portion of human proteins interacting with Y. pestis (16/109) also interacted with viral proteins (Epstein-Barr virus [EBV] and hepatitis C virus [HCV]), suggesting that viral and bacterial pathogens attack common cellular functions to facilitate infections. In addition, we identified vasodilator-stimulated phosphoprotein (VASP) as a novel interaction partner of YpkA and showed that YpkA could inhibit in vitro actin assembly mediated by VASP.
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Project description:Earlier, we reported the identification of new virulence factors/mechanisms of Yersinia pestis using an in vivo signature-tagged mutagenesis (STM) screening approach. From this screen, the role of rbsA, which encodes an ATP-binding protein of ribose transport system, and vasK, an essential component of the type VI secretion system (T6SS), were evaluated in mouse models of plague and confirmed to be important during Y. pestis infection. However, many of the identified genes from the screen remained uncharacterized. In this study, in-frame deletion mutants of ypo0815, ypo2884, ypo3614-3168 (cyoABCDE), and ypo1119-1120, identified from the STM screen, were generated. While ypo0815 codes for a general secretion pathway protein E (GspE) of the T2SS, the ypo2884-encoded protein has homology to the βγ crystallin superfamily, cyoABCDE codes for the cytochrome o oxidase operon, and the ypo1119-1120 genes are within the Tol-Pal system which has multiple functions. Additionally, as our STM screen identified three T6SS-associated genes, and, based on in silico analysis, six T6SS clusters and multiple homologs of the T6SS effector hemolysin-coregulated protein (Hcp) exist in Y. pestis CO92, we also targeted these T6SS clusters and effectors for generating deletion mutants. These deletion mutant strains exhibited varying levels of attenuation (up to 100%), in bubonic or pneumonic murine infection models. The attenuation could be further augmented by generation of combinatorial deletion mutants, namely ΔlppΔypo0815, ΔlppΔypo2884, ΔlppΔcyoABCDE, ΔvasKΔhcp6, and Δypo2720-2733Δhcp3. We earlier showed that deletion of the lpp gene, which encodes Braun lipoprotein (Lpp) and activates Toll-like receptor-2, reduced virulence of Y. pestis CO92 in murine models of bubonic and pneumonic plague. The surviving mice infected with ΔlppΔcyoABCDE, ΔvasKΔhcp6, and Δypo2720-2733Δhcp3 mutant strains were 55-100% protected upon subsequent re-challenge with wild-type CO92 in a pneumonic model. Further, evaluation of the attenuated T6SS mutant strains in vitro revealed significant alterations in phagocytosis, intracellular survival in murine macrophages, and their ability to induce cytotoxic effects on macrophages. The results reported here provide further evidence of the utility of the STM screening approach for the identification of novel virulence factors and to possibly target such genes for the development of novel live-attenuated vaccine candidates for plague.
| S-EPMC5650977 | biostudies-literature
Project description:BackgroundYersinia pestis is the causative agent of plague, which is transmitted primarily between fleas and mammals and is spread to humans through the bite of an infected flea or contact with afflicted animals. Hfq is proposed to be a global post-transcriptional regulator that acts by mediating interactions between many regulatory small RNAs (sRNAs) and their mRNA targets. Sequence comparisons revealed that Y. pestis appears to produce a functional homologue of E. coli Hfq.Methodology and principal findingsPhenotype comparisons using in vitro assays demonstrated that Y. pestis Hfq was involved in resistance to H(2)O(2), heat and polymyxin B and contributed to growth under nutrient-limiting conditions. The role of Hfq in Y. pestis virulence was also assessed using macrophage and mouse infection models, and the gene expression affected by Hfq was determined using microarray-based transcriptome and real time PCR analysis. The macrophage infection assay showed that the Y. pestis hfq deletion strain did not have any significant difference in its ability to associate with J774A.1 macrophage cells. However, hfq deletion appeared to significantly impair the ability of Y. pestis to resist phagocytosis and survive within macrophages at the initial stage of infection. Furthermore, the hfq deletion strain was highly attenuated in mice after subcutaneous or intravenous injection. Transcriptome analysis supported the results concerning the attenuated phenotype of the hfq mutant and showed that the deletion of the hfq gene resulted in significant alterations in mRNA abundance of 243 genes in more than 13 functional classes, about 23% of which are known or hypothesized to be involved in stress resistance and virulence.Conclusions and significanceOur results indicate that Hfq is a key regulator involved in Y. pestis stress resistance, intracellular survival and pathogenesis. It appears that Hfq acts by controlling the expression of many virulence- and stress-associated genes, probably in conjunction with small noncoding RNAs.
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Project description:In all Yersinia pestis strains examined, the adhesin/invasin yadA gene is a pseudogene, yet Y. pestis is invasive for epithelial cells. To identify potential surface proteins that are structurally and functionally similar to YadA, we searched the Y. pestis genome for open reading frames with homology to yadA and found three: the bicistronic operon yadBC (YPO1387 and YPO1388 of Y. pestis CO92; y2786 and y2785 of Y. pestis KIM5), which encodes two putative surface proteins, and YPO0902, which lacks a signal sequence and likely is nonfunctional. In this study we characterized yadBC regulation and tested the importance of this operon for Y. pestis adherence, invasion, and virulence. We found that loss of yadBC caused a modest loss of invasiveness for epithelioid cells and a large decrease in virulence for bubonic plague but not for pneumonic plague in mice.
| S-EPMC2223446 | biostudies-literature
Project description:Yersinia pestis, the causative agent of plague, uses a type III secretion system (T3SS) to inject cytotoxic Yop proteins directly into the cytosol of mammalian host cells. The T3SS can also be activated in vitro at 37°C in the absence of calcium. The chromosomal gene rfaL (waaL) was recently identified as a virulence factor required for proper function of the T3SS. RfaL functions as a ligase that adds the terminal N-acetylglucosamine to the lipooligosaccharide core of Y. pestis. We previously showed that deletion of rfaL prevents secretion of Yops in vitro. Here we show that the divalent cations calcium, strontium, and magnesium can partially or fully rescue Yop secretion in vitro, indicating that the secretion phenotype of the rfaL mutant may be due to structural changes in the outer membrane and the corresponding feedback inhibition on the T3SS. In support of this, we found that the defect can be overcome by deleting the regulatory gene lcrQ. Consistent with a defective T3SS, the rfaL mutant is less virulent than the wild type. We show here that the virulence defect of the mutant correlates with a decrease in both T3SS gene expression and ability to inject innate immune cells, combined with an increased sensitivity to cationic antimicrobial peptides.
| S-EPMC3639585 | biostudies-literature
Project description:A comprehensive TnphoA mutant library was constructed in Yersinia pestis KIM6 to identify surface proteins involved in Y. pestis host cell invasion and bacterial virulence. Insertion site analysis of the library repeatedly identified a 9,042-bp chromosomal gene (YPO3944), intimin/invasin-like protein (Ilp), similar to the Gram-negative intimin/invasin family of surface proteins. Deletion mutants of ilp were generated in Y. pestis strains KIM5(pCD1(+)) Pgm(-) (pigmentation negative)/, KIM6(pCD1(-)) Pgm(+), and CO92. Comparative analyses were done with the deletions and the parental wild type for bacterial adhesion to and internalization by HEp-2 cells in vitro, infectivity and maintenance in the flea vector, and lethality in murine models of systemic and pneumonic plague. Deletion of ilp had no effect on bacterial blockage of flea blood feeding or colonization. The Y. pestis KIM5 ?ilp strain had reduced adhesion to and internalization by HEp-2 cells compared to the parental wild-type strain (P < 0.05). Following intravenous challenge with Y. pestis KIM5 ?ilp, mice had a delayed time to death and reduced dissemination to the lungs, livers, and kidneys as monitored by in vivo imaging using a lux reporter system (in vivo imaging system [IVIS]) and bacterial counts. Intranasal challenge in mice with Y. pestis CO92 ?ilp had a 55-fold increase in the 50% lethal dose ([LD(50)] 1.64 × 10(4) CFU) compared to the parental wild-type strain LD(50) (2.98 × 10(2) CFU). These findings identified Ilp as a novel virulence factor of Y. pestis.
| S-EPMC3457552 | biostudies-literature
Project description:Yersinia pestis, the causative agent of plague, encodes several essential virulence factors on a 70 kb plasmid, including the Yersinia outer proteins (Yops) and a multifunctional virulence antigen (V). V is uniquely able to inhibit the host immune response; aid in the expression, secretion, and injection of the cytotoxic Yops via a type III secretion system (T3SS)-dependent mechanism; be secreted extracellularly; and enter the host cell by a T3SS-independent mechanism, where its activity is unknown. To elucidate the intracellular trafficking and target(s) of V, time-course experiments were performed with macrophages (MPhis) infected with Y. pestis or Y. pseudotuberculosis at intervals from 5 min to 6 h. The trafficking pattern was discerned from results of parallel microscopy, immunoblotting, and flow cytometry experiments. The MPhis were incubated with fluorescent or gold conjugated primary or secondary anti-V (antibodies [Abs]) in conjunction with organelle-associated Abs or dyes. The samples were observed for co-localization by immuno-fluorescence and electron microscopy. For fractionation studies, uninfected and infected MPhis were lysed and subjected to density gradient centrifugation coupled with immunoblotting with Abs to V or to organelles. Samples were also analyzed by flow cytometry after lysis and dual-staining with anti-V and anti-organelle Abs. Our findings indicate a co-localization of V with (1) endosomal proteins between 10-45 min of infection, (2) lysosomal protein(s) between 1-2 h of infection, (3) mitochondrial proteins between 2.5-3 h infection, and (4) Golgi protein(s) between 4-6 h of infection. Further studies are being performed to determine the specific intracellular interactions and role in pathogenesis of intracellularly localized V.
| S-EPMC2707630 | biostudies-literature