scholarly journals Identification of OmpU of Vibrio vulnificus as a Fibronectin-Binding Protein and Its Role in Bacterial Pathogenesis

2006 ◽  
Vol 74 (10) ◽  
pp. 5586-5594 ◽  
Author(s):  
Sung Young Goo ◽  
Hyun-Ju Lee ◽  
Woo Hwang Kim ◽  
Kyu-Lee Han ◽  
Dae-Kyun Park ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Vibrio Vulnificus ◽  
Outer Membrane Proteins ◽  
Lethal Dose ◽  
Fold Increase ◽  
Host Cells ◽  
Wild Type ◽  
Binding Assays ◽  
Exogenous Addition ◽  
Main Components

ABSTRACT Vibrio vulnificus is a pathogenic bacterium that causes gastroenteritis and primary septicemia. To identify factors involved in microbial adherence to the host cells, we investigated bacterial proteins capable of binding to fibronectin, one of the main components comprised of the extracellular matrix of mammalian cells. A protein of ∼35 kDa was purified from the extracts of V. vulnificus by its property to bind to immobilized fibronectin. This protein was identified as OmpU, one of the major outer membrane proteins of V. vulnificus. In binding assays using immobilized fibronectin, the number of ompU mutant cells bound to fibronectin was only 4% of that of wild-type cells bound to fibronectin. In addition, the exogenous addition of antibodies against OmpU resulted in a decreased ability of wild-type V. vulnificus to adhere to fibronectin. The ompU mutant was also defective in its adherence to RGD tripeptide (5% of the adherence of the wild type to RGD), cytoadherence to HEp-2 cells (7% of the adherence of the wild type to HEp-2), cytotoxicity to cell cultures (39% of the cytotoxicity of the wild type), and mortality in mice (10-fold increase in the 50% lethal dose). The ompU mutant complemented with the intact ompU gene restored its abilities for adherence to fibronectin, RGD tripeptide, and HEp-2 cells; cytotoxicity to HEp-2 cells; and mouse lethality. This study indicates that OmpU is an important virulence factor involved in the adherence of V. vulnificus to the host cells.

scholarly journals Vibrio vulnificus rtxE Is Important for Virulence, and Its Expression Is Induced by Exposure to Host Cells

2008 ◽  
Vol 76 (4) ◽  
pp. 1509-1517 ◽  
Author(s):  
Byung Cheol Lee ◽  
Jeong Hyun Lee ◽  
Myung Won Kim ◽  
Byoung Sik Kim ◽  
Man Hwan Oh ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Vibrio Vulnificus ◽  
Lethal Dose ◽  
Host Cells ◽  
Transcriptional Unit ◽  
Intestinal Epithelial ◽  
Wild Type ◽  
Reading Frame ◽  
Human Intestinal Epithelial Cells

ABSTRACT Numerous secreted virulence factors have been proposed to account for the fulminating and destructive nature of Vibrio vulnificus infections. A mutant of V. vulnificus that exhibited less cytotoxicity to INT-407 human intestinal epithelial cells was screened from a library of mutants constructed by random transposon mutagenesis. A transposon-tagging method was used to identify and clone an open reading frame encoding an RTX toxin secretion ATP binding protein, RtxE, from V. vulnificus. The deduced amino acid sequence of RtxE from V. vulnificus was 91% identical to that reported from Vibrio cholerae. Functions of the rtxE gene in virulence were assessed by constructing an isogenic mutant whose rtxE gene was inactivated by allelic exchanges and by evaluating the differences between its virulence phenotype and that of the wild type in vitro and in mice. The disruption of rtxE blocked secretion of RtxA to the cell exterior and resulted in a significant reduction in cytotoxic activity against epithelial cells in vitro. Also, the intraperitoneal 50% lethal dose of the rtxE mutant was 104 to 105 times higher than that of the parental wild type, indicating that RtxE is essential for the virulence of V. vulnificus. Furthermore, the present study demonstrated that the rtxBDE genes are transcribed as one transcriptional unit under the control of a single promoter, P rtxBDE . The activity of V. vulnificus P rtxBDE is induced by exposure to INT-407 cells, and the induction requires direct contact of the bacteria with the host cells.

scholarly journals Salmonella Exploits Caspase-1 to Colonize Peyer's Patches in a Murine Typhoid Model

2000 ◽  
Vol 192 (2) ◽  
pp. 249-258 ◽  
Author(s):  
Denise M. Monack ◽  
David Hersh ◽  
Nafisa Ghori ◽  
Donna Bouley ◽  
Arturo Zychlinsky ◽  
...  
Keyword(s):  
Proinflammatory Cytokines ◽  
Mammalian Cells ◽  
Yersinia Pseudotuberculosis ◽  
Lethal Dose ◽  
Cysteine Proteases ◽  
Peyer's Patches ◽  
Peyer’S Patches ◽  
Wild Type ◽  
M Cell ◽  
Caspase 1

Salmonella typhimurium invades host macrophages and induces apoptosis and the release of mature proinflammatory cytokines. SipB, a protein translocated by Salmonella into the cytoplasm of macrophages, is required for activation of Caspase-1 (Casp-1, an interleukin [IL]-1β–converting enzyme), which is a member of a family of cysteine proteases that induce apoptosis in mammalian cells. Casp-1 is unique among caspases because it also directly cleaves the proinflammatory cytokines IL-1β and IL-18 to produce bioactive cytokines. We show here that mice lacking Casp-1 (casp-1−/− mice) had an oral S. typhimurium 50% lethal dose (LD50) that was 1,000-fold higher than that of wild-type mice. Salmonella breached the M cell barrier of casp-1−/− mice efficiently; however, there was a decrease in the number of apoptotic cells, intracellular bacteria, and the recruitment of polymorphonuclear lymphocytes in the Peyer's patches (PP) as compared with wild-type mice. Furthermore, Salmonella did not disseminate systemically in the majority of casp-1−/− mice, as demonstrated by significantly less colonization in the PP, mesenteric lymph nodes, and spleens of casp-1−/− mice after an oral dose of S. typhimurium that was 100-fold higher than the LD50. The increased resistance in casp-1−/− animals appears specific for Salmonella infection since these mice were susceptible to colonization by another enteric pathogen, Yersinia pseudotuberculosis, which normally invades the PP. These results show that Casp-1, which is both proapoptotic and proinflammatory, is essential for S. typhimurium to efficiently colonize the cecum and PP and subsequently cause systemic typhoid-like disease in mice.

scholarly journals Avoidance of Autophagy Mediated by PlcA or ActA Is Required for Listeria monocytogenes Growth in Macrophages

2015 ◽  
Vol 83 (5) ◽  
pp. 2175-2184 ◽  
Author(s):  
Gabriel Mitchell ◽  
Liang Ge ◽  
Qiongying Huang ◽  
Chen Chen ◽  
Sara Kianian ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Wild Type Strain ◽  
Fold Increase ◽  
Host Cells ◽  
Listeriolysin O ◽  
Wild Type ◽  
Conjugation System ◽  
Content Type ◽  
A Cell ◽  
Gain Access

Listeria monocytogenesis a facultative intracellular pathogen that escapes from phagosomes and grows in the cytosol of infected host cells. Most of the determinants that govern its intracellular life cycle are controlled by the transcription factor PrfA, including the pore-forming cytolysin listeriolysin O (LLO), two phospholipases C (PlcA and PlcB), and ActA. We constructed a strain that lacked PrfA but expressed LLO from a PrfA-independent promoter, thereby allowing the bacteria to gain access to the host cytosol. This strain did not grow efficiently in wild-type macrophages but grew normally in macrophages that lacked ATG5, a component of the autophagy LC3 conjugation system. This strain colocalized more with the autophagy marker LC3 (42% ± 7%) at 2 h postinfection, which constituted a 5-fold increase over the colocalization exhibited by the wild-type strain (8% ± 6%). While mutants lacking the PrfA-dependent virulence factor PlcA, PlcB, or ActA grew normally, a double mutant lacking both PlcA and ActA failed to grow in wild-type macrophages and colocalized more with LC3 (38% ± 5%). Coexpression of LLO and PlcA in a PrfA-negative strain was sufficient to restore intracellular growth and decrease the colocalization of the bacteria with LC3. In a cell-free assay, purified PlcA protein blocked LC3 lipidation, a key step in early autophagosome biogenesis, presumably by preventing the formation of phosphatidylinositol 3-phosphate (PI3P). The results of this study showed that avoidance of autophagy byL. monocytogenesprimarily involves PlcA and ActA and that either one of these factors must be present forL. monocytogenesgrowth in macrophages.

scholarly journals Enteropathogenic EscherichiacoliVirulence Factor Bundle-Forming Pilus Has a Binding Specificity for Phosphatidylethanolamine

2001 ◽  
Vol 69 (11) ◽  
pp. 6573-6579 ◽  
Author(s):  
C. Khursigara ◽  
M. Abul-Milh ◽  
B. Lau ◽  
J. A. Girón ◽  
C. A. Lingwood ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Solid Phase ◽  
Specific Interaction ◽  
Laboratory Strain ◽  
Host Cells ◽  
Wild Type ◽  
Binding Assays ◽  
Thin Layer Chromatogram ◽  
Cultured Epithelial Cells ◽  
Overlay Assay

ABSTRACT The bundle-forming pilus (BFP) of enteropathogenicEscherichia coli (EPEC), an established virulence factor encoded on the EPEC adherence factor (EAF) plasmid, has been implicated in the formation of bacterial autoaggregates and in the localized adherence of EPEC to cultured epithelial cells. While understanding of the pathogenic mechanism of this organism is rapidly improving, a receptor ligand for BFP has not yet been identified. We now report, using both solid-phase and liposome binding assays, that BFP expression correlates with phosphatidylethanolamine (PE) binding. In a thin-layer chromatogram overlay assay, specific recognition of PE was documented for BFP-expressing strains, including E2348/69, a wild-type EPEC clinical isolate, as well as a laboratory strain, HB101, transformed with a bfp-carrying plasmid. Strains which did not express BFP did not bind PE, including a bfpAdisruptional mutant of E2348/69, EAF plasmid-cured E2348/69, and HB101. E2348/69 also aggregated PE-containing liposomes but not phosphatidylcholine- or phosphatidylserine-containing liposomes, while BFP-negative strains did not produce aggregates with any tested liposomes. Purified BFP preparations bound commercial PE standards as well as a PE-containing band within lipid extracts from human epithelial cells and from E2348/69. Our results therefore indicate a specific interaction between BFP and PE and suggest that PE may serve as a BFP receptor for bacterial autoaggregation and may promote localized adherence to host cells, both of which contribute to bacterial pathogenesis.

scholarly journals Oral Immunization with an rfaH Mutant Elicits Protection against Salmonellosis in Mice

2004 ◽  
Vol 72 (7) ◽  
pp. 4297-4301 ◽  
Author(s):  
Gábor Nagy ◽  
Ulrich Dobrindt ◽  
Jörg Hacker ◽  
Levente Emödy
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Lethal Dose ◽  
Fold Increase ◽  
Oral Immunization ◽  
Oral Infection ◽  
Wild Type ◽  
Virulence Attenuation ◽  
Serovar Typhimurium

ABSTRACT Loss of the transcriptional antiterminator RfaH results in virulence attenuation (>104-fold increase in 50% lethal dose) of the archetypal Salmonella enterica serovar Typhimurium strain SL1344 by both orogastric and intraperitoneal routes of infection in BALB/c mice. Oral immunization with the mutant efficiently protects mice against a subsequent oral infection with the wild-type strain. Interestingly, in vitro immunoreactivity is not confined to strain SL1344; rather, it is directed also towards other serovars of S. enterica and even Salmonella bongori strains.

scholarly journals A Synthetic E7 Gene of Human Papillomavirus Type 16 That Yields Enhanced Expression of the Protein in Mammalian Cells and Is Useful for DNA Immunization Studies

2003 ◽  
Vol 77 (8) ◽  
pp. 4928-4937 ◽  
Author(s):  
Angel Cid-Arregui ◽  
Victoria Juárez ◽  
Harald zur Hausen
Keyword(s):  
Human Papillomavirus ◽  
Protein Expression ◽  
Mammalian Cells ◽  
Host Cells ◽  
Dna Immunization ◽  
Wild Type ◽  
Animal Cells ◽  
E7 Gene ◽  
Early Protein ◽  
Enhanced Expression

ABSTRACT A synthetic E7 gene of human papillomavirus (HPV) type 16 was generated that consists entirely of preferred human codons. Expression analysis of the synthetic E7 gene in human and animal cells showed levels of E7 protein 20- to 100-fold higher than those obtained with wild-type E7. Enhanced expression of E7 protein resulted from highly efficient translation, as well as increased stability of the E7 mRNA due to its codon optimization. Higher levels of E7 protein in cells transfected with synthetic E7 correlated with significant loss of cell viability in various human cell lines. In contrast, lower E7 protein expression driven by the wild-type gene resulted in a slight induction of cell proliferation. Furthermore, mice inoculated with plasmids expressing the synthetic E7 gene produced significantly higher levels of E7 antibodies than littermates injected with wild-type E7, suggesting that synthetic E7 may be useful for DNA immunization studies and the development of genetic vaccines against HPV-16. In view of these results, we hypothesize that HPVs may have retained a pattern of G + C content and codon usage distinct from that of their host cells in response to selective pressure. Thus, the nonhuman codon bias may have been conserved by HPVs to prevent compromising viability of the host cells by excessive viral early protein expression, as well as to evade the immune system.

scholarly journals Three Yersinia pestis Adhesins Facilitate Yop Delivery to Eukaryotic Cells and Contribute to Plague Virulence

2010 ◽  
Vol 78 (10) ◽  
pp. 4134-4150 ◽  
Author(s):  
Suleyman Felek ◽  
Tiffany M. Tsang ◽  
Eric S. Krukonis
Keyword(s):  
Yersinia Pestis ◽  
Lethal Dose ◽  
Fold Increase ◽  
Growth Conditions ◽  
Host Cells ◽  
Eukaryotic Cells ◽  
Cell Binding ◽  
Cytokine Responses ◽  
Successful Infection ◽  
Mouse Tissues

ABSTRACT To establish a successful infection, Yersinia pestis requires the delivery of cytotoxic Yops to host cells. Yops inhibit phagocytosis, block cytokine responses, and induce apoptosis of macrophages. The Y. pestis adhesin Ail facilitates Yop translocation and is required for full virulence in mice. To determine the contributions of other adhesins to Yop delivery, we deleted five known adhesins of Y. pestis. In addition to Ail, plasminogen activator (Pla) and pH 6 antigen (Psa) could mediate Yop translocation to host cells. The contribution of each adhesin to binding and Yop delivery was dependent upon the growth conditions. When cells were pregrown at 28°C and pH 7, the order of importance for adhesins in cell binding and cytotoxicity was Ail > Pla > Psa. Y. pestis grown at 37°C and pH 7 had equal contributions from Ail and Pla but an undetectable role for Psa. At 37°C and pH 6, both Ail and Psa contributed to binding and Yop delivery, while Pla contributed minimally. Pla-mediated Yop translocation was independent of protease activity. Of the three single mutants, the Δail mutant was the most defective in mouse virulence. The expression level of ail was also the highest of the three adhesins in infected mouse tissues. Compared to an ail mutant, additional deletion of psaA (encoding Psa) led to a 130,000-fold increase in the 50% lethal dose for mice relative to that of the KIM5 parental strain. Our results indicate that in addition to Ail, Pla and Psa can serve as environmentally specific adhesins to facilitate Yop secretion, a critical virulence function of Y. pestis.

scholarly journals Lack of Outer Membrane Protein A Enhances the Release of Outer Membrane Vesicles and Survival ofVibrio choleraeand Suppresses Viability ofAcanthamoeba castellanii

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Soni Priya Valeru ◽  
Salah Shanan ◽  
Haifa Alossimi ◽  
Amir Saeed ◽  
Gunnar Sandström ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Outer Membrane Proteins ◽  
Protein A ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Host Cells ◽  
Wild Type ◽  
Outer Membrane Protein A

Vibrio cholerae, the causative agent of the diarrhoeal disease cholera, survives in aquatic environments. The bacterium has developed a survival strategy to grow and survive insideAcanthamoeba castellanii. It has been shown thatV. choleraeexpresses outer membrane proteins as virulence factors playing a role in the adherence to interacted host cells. This study examined the role of outer membrane protein A (OmpA) and outer membrane vesicles (OMVs) in survival ofV. choleraealone and during its interaction withA. castellanii. The results showed that anOmpAmutant ofV. choleraesurvived longer than wild-typeV. choleraewhen cultivated alone. Cocultivation withA. castellaniienhanced the survival of both bacterial strains andOmpAprotein exhibited no effect on attachment, engulfment, and survival inside the amoebae. However, cocultivation of theOmpAmutant ofV. choleraedecreased the viability ofA. castellaniiand this bacterial strain released more OMVs than wild-typeV. cholerae. Surprisingly, treatment of amoeba cells with OMVs isolated from theOmpAmutant significantly decreased viable counts of the amoeba cells. In conclusion, the results might highlight a regulating rule forOmpAin survival ofV. choleraeand OMVs as a potent virulence factor for this bacterium towards eukaryotes in the environment.

scholarly journals Impact of either Elevated or Decreased Levels of Cytochrome bd Expression on Shigella flexneri Virulence

1999 ◽  
Vol 181 (4) ◽  
pp. 1229-1237 ◽  
Author(s):  
Sing Sing Way ◽  
Sandra Sallustio ◽  
Richard S. Magliozzo ◽  
Marcia B. Goldberg
Keyword(s):  
Shigella Flexneri ◽  
Lethal Dose ◽  
Fold Increase ◽  
Intracellular Survival ◽  
Bacterial Invasion ◽  
Wild Type ◽  
Terminal Oxidases ◽  
Cytochrome Bd ◽  
Transport Chain ◽  
Pathogenic Process

ABSTRACT Shigella spp. are the major cause of bacillary dysentery worldwide. The pathogenic process involves bacterial invasion and lysis of the phagocytic vacuole, followed by replication and movement within the cell cytoplasm and, ultimately, spread directly into adjacent cells. This study demonstrates that S. flexneri cytochrome bd expression is necessary for normal intracellular survival and virulence. Cytochrome bdis one of two terminal oxidases in the bacterial respiratory chain that reduce molecular oxygen to water, utilizing intermediates shuttled through the electron transport chain. S. flexneri mutants that contain a disruption in the cydC locus, which leads to defective cytochrome bd expression, or in the riboflavin (ribE) or ubiquinol-8 (ubiH) biosynthetic pathway, which leads to elevated cytochrome bd expression, were evaluated in intracellular survival and virulence assays. ThecydC mutant formed significantly smaller plaques, had significantly decreased intracellular survival, and had a 100-fold increase in lethal dose for mice compared with the wild type. TheribE and ubiH mutants each formed significantly larger plaques and had a 10-fold decrease in lethal dose for mice compared with the wild type. The data indicate that expression of cytochrome bd is required for S. flexneriintracellular survival and virulence.

scholarly journals An ordered sequential mechanism for Factor IX and Factor IXa binding to platelet receptors in the assembly of the Factor X-activating complex

2005 ◽  
Vol 390 (1) ◽  
pp. 157-167 ◽  
Author(s):  
Xia Yang ◽  
Peter N. Walsh
Keyword(s):  
Fold Increase ◽  
Factor Ix ◽  
Phospholipid Vesicles ◽  
Phospholipid Membranes ◽  
Factor X ◽  
Wild Type ◽  
Binding Assays ◽  
Epidermal Growth Factor Domain ◽  
Factor Ixa ◽  
Activated Platelets

To define the contributions of the Ω-loop of the Gla (γ-carboxyglutamic acid) domain and the EGF2 (second epidermal growth factor) domain of FIXa (Factor IXa) in the assembly of the FX-activating complex on activated platelets and phospholipid membranes, three recombinant FIXa chimeras were prepared with corresponding residues from the homologous coagulation protein, FVII: (i) Gly4–Gln11 (FIXa7Ωloop), (ii) Cys88–Cys124 (FIXa7EGF2), and (iii) both Gly4–Gln11 and Cys88–Cys124 (FIXa7Ωloop7EGF2). All three chimeras were similar to wild-type FIXa, as assessed by SDS/PAGE, active-site titration, content of Gla residues, activation rates by FXIa and rates of FXa generation in solution. Titrations of FX or FVIIIa on SFLLRN peptide-activated platelets and on phospholipid vesicles in the presence of FVIIIa revealed normal substrate and cofactor binding to all chimeras. In kinetic assays in the presence of phospholipid vesicles and FVIIIa, compared with wild-type FIXa Kd, app∼4 nM, the FIX7Ωloop chimera showed a 1.6-fold increase in Kd, app, the FIX7EGF2 chimera had a 7.4-fold increase in Kd, app, and the FIX7Ωloop7EGF2 chimera showed a 21-fold increase in Kd, app. In kinetic assays and equilibrium platelet-binding assays with activated platelets and FVIIIa, compared with wild-type FIXa (Vmax∼5 nM min−1; Kd, app∼0.5 nM; Bmax∼550 sites/platelet; Kd∼0.5 nM), the FIX7Ωloop chimera displayed 2-fold decreases in Vmax and Bmax and 2-fold increases in Kd, app and Kd. The FIX7EGF2 chimera displayed 2-fold decreases in Vmax and Bmax and 10-fold increases in Kd, app and Kd. The FIX7Ωloop7EGF2 chimera showed non-saturable curves and severely impaired rates of FXa generation, and non-saturable, non-specific, low-level binding to activated platelets. Thus both the Gla domain Ω-loop (Gly4–Gln11) and the EGF2 domain (Cys88–Cys124) are required to mediate the normal assembly of the FX-activating complex on activated platelets and on phospholipid membranes.

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