scholarly journals The Virulence Regulatory Protein ToxR Mediates Enhanced Bile Resistance in Vibrio cholerae and Other PathogenicVibrio Species

2000 ◽  
Vol 68 (3) ◽  
pp. 1491-1497 ◽  
Author(s):  
Daniele Provenzano ◽  
Darren A. Schuhmacher ◽  
Justin L. Barker ◽  
Karl E. Klose
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Vibrio Cholerae ◽  
Outer Membrane Proteins ◽  
Regulatory Protein ◽  
Wild Type ◽  
Outer Membrane Porins ◽  
Mutant Strains ◽  
Bile Resistance ◽  
El Tor

ABSTRACT The transmembrane regulatory protein ToxR is required for expression of virulence factors in the human diarrheal pathogenVibrio cholerae, including cholera toxin (CT) and the toxin coregulated pilus (TCP). ToxR is necessary for transcription of the gene encoding a second regulatory protein, ToxT, which is the direct transcriptional activator of CT and TCP genes. However, ToxR, independent of ToxT, directly activates and represses transcription of the outer membrane porins OmpU and OmpT, respectively. The genes encoding TCP and CT (and including ToxT) lie on horizontally acquired genetic elements, while the toxR, ompU, andompT genes are apparently in the ancestralVibrio chromosome. The contribution of ToxR-dependent modulation of outer membrane porins to cholera pathogenesis has remained unknown. We demonstrate that ToxR mediates enhanced bile resistance in a ToxT-independent manner. In both classical and El Tor biotypes of V. cholerae, a toxR mutant strain has a reduced minimum bactericidal concentration (MBC) of bile, the bile component deoxycholate (DC), and the anionic detergent sodium dodecyl sulfate (SDS) compared to both wild-type and toxTmutant strains. Classical and El Tor toxR mutant strains also exhibit reduced growth rates at subinhibitory concentrations of DC and SDS. Growth of either V. cholerae biotype in subinhibitory concentrations of bile or DC induces increased ToxR-dependent production of a major 38-kDa outer membrane protein, which was confirmed to be OmpU by Western blot. Measurement of transcription of a ompUp-lacZ fusion in both biotypes reveals stimulation (about two- to threefold) of ToxR-dependent ompU transcription by the presence of bile or DC, suggesting that ToxR may respond to the presence of bile. ThetoxR mutant strains of three additional human intestinal pathogenic Vibrio species, V. mimicus, V. fluvialis, and V. parahaemolyticus, display lower MBCs of bile, DC, and SDS and have altered outer membrane protein profiles compared to the parental wild-type strains. Our results demonstrate a conserved role for ToxR in the modulation of outer membrane proteins and bile resistance of pathogenic Vibriospecies and suggest that these ToxR-dependent outer membrane proteins may mediate enhanced resistance to bile. We speculate that ToxR-mediated bile resistance was an early step in the evolution ofV. cholerae as an intestinal pathogen.

Inactivation of a fibronectin-binding TonB-dependent protein increases adhesion properties of Bacteroides fragilis

2013 ◽  
Vol 62 (10) ◽  
pp. 1524-1530 ◽  
Author(s):  
Heidi Pauer ◽  
Soraia N. V. Cavalcanti ◽  
Felipe L. Teixeira ◽  
Joaquim Santos-Filho ◽  
Rossiane C. Vommaro ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Bacteroides Fragilis ◽  
Strictly Anaerobic ◽  
Wild Type ◽  
Extracellular Matrix Components ◽  
Mutant Strains ◽  
Fibronectin Binding

Bacteroides fragilis is the Gram-negative strictly anaerobic bacterium most frequently isolated from clinical infections, including intra-abdominal abscess and bacteraemia. A number of factors can contribute to its virulence, including the expression of adhesins. Some of them are already characterized and can recognize and bind to extracellular matrix components, such as fibronectin. One of the molecules responsible for fibronectin-binding is an outer-membrane protein previously described by our group, which belongs to the TonB-dependent family. The aim of the present work was to characterize this protein. Initially, it was confirmed by fluorescence and electron microscopy that the fibronectin-binding molecules were located in the bacterial surface, but the distribution of these molecules on the surface was not uniform. To further evaluate the role of this protein, the gene bf1991, responsible for encoding this protein, was inactivated by a suicide vector and the mutant strains generated were used in several experiments to verify possible phenotypical alterations. In adherence assays with fibronectin immobilized on latex beads an increased adhesion was observed with the mutant strains compared with the wild-type strain. Western blot analysis in the mutant strain revealed the absence of the 120 kDa TonB-dependent outer-membrane protein and an alteration in the expression of an unknown 30 kDa protein. Killing assays using peritoneal macrophages were performed to evaluate the role of this protein as a virulence attribute and it was observed that the mutant strains were more efficiently internalized than the wild-type strains, with more internalization in the samples covered with fibronectin than in the samples not covered with it.

scholarly journals Outer Membrane Protein OmpW Is the Receptor for Typing Phage VP5 in the Vibrio cholerae O1 El Tor Biotype

2014 ◽  
Vol 88 (12) ◽  
pp. 7109-7111 ◽  
Author(s):  
D. Xu ◽  
J. Zhang ◽  
J. Liu ◽  
J. Xu ◽  
H. Zhou ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Vibrio Cholerae ◽  
Outer Membrane Protein ◽  
Typing Phage ◽  
Vibrio Cholerae O1 ◽  
El Tor

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 Unravelling potential virulence factor candidates inXanthomonas citri. subsp.citriby secretome analysis

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e1734 ◽  
Author(s):  
Rafael M. Ferreira ◽  
Leandro M. Moreira ◽  
Jesus A. Ferro ◽  
Marcia R.R. Soares ◽  
Marcelo L. Laia ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Type Strain ◽  
Wild Type Strain ◽  
Outer Membrane Proteins ◽  
Citrus Canker ◽  
Trna Synthetase ◽  
Hypothetical Proteins ◽  
Wild Type ◽  
In The Wild

Citrus canker is a major disease affecting citrus production in Brazil. It’s mainly caused byXanthomonas citrisubsp.citristrain 306 pathotype A (Xac). We analysed the differential expression of proteins secreted by wild typeXacand an asymptomatic mutant forhrpB4(ΔhrpB4) grown in Nutrient Broth (NB) and a medium mimicking growth conditions in the plant (XAM1). This allowed the identification of 55 secreted proteins, of which 37 were secreted by both strains when cultured in XAM1. In this secreted protein repertoire, the following stand out: Virk, Polyphosphate-selective porin, Cellulase, Endoglucanase, Histone-like protein, Ribosomal proteins, five hypothetical proteins expressed only in the wild type strain, Lytic murein transglycosylase, Lipoprotein, Leucyl-tRNA synthetase, Co-chaperonin, Toluene tolerance, C-type cytochrome biogenesis membrane protein, Aminopeptidase and two hypothetical proteins expressed only in the ΔhrpB4mutant. Furthermore, Peptidoglycan-associated outer membrane protein, Regulator of pathogenicity factor, Outer membrane proteins, Endopolygalacturonase, Chorismate mutase, Peptidyl-prolyl cis-trans isomerase and seven hypothetical proteins were detected in both strains, suggesting that there was no relationship with the secretion mediated by the type III secretory system, which is not functional in the mutant strain. Also worth mentioning is the Elongation factor Tu (EF-Tu), expressed only the wild type strain, and Type IV pilus assembly protein, Flagellin (FliC) and Flagellar hook-associated protein, identified in the wild-type strain secretome when grown only in NB. Noteworthy, that FliC, EF-Tu are classically characterized as PAMPs (Pathogen-associated molecular patterns), responsible for a PAMP-triggered immunity response. Therefore, our results highlight proteins potentially involved with the virulence. Overall, we conclude that the use of secretome data is a valuable approach that may bring more knowledge of the biology of this important plant pathogen, which ultimately can lead to the establishment of new strategies to combat citrus canker.

scholarly journals A ToxR Homolog from Vibrio anguillarum Serotype O1 Regulates Its Own Production, Bile Resistance, and Biofilm Formation

2002 ◽  
Vol 184 (6) ◽  
pp. 1630-1639 ◽  
Author(s):  
Su-Yan Wang ◽  
Johan Lauritz ◽  
Jana Jass ◽  
Debra L. Milton
Keyword(s):  
Outer Membrane ◽  
Regulatory Protein ◽  
Vibrio Anguillarum ◽  
Wild Type ◽  
Environmental Signals ◽  
Environmental Stimuli ◽  
Outer Membrane Porin ◽  
Bile Resistance ◽  
Functional Analyses ◽  
Mutant Forms

ABSTRACT ToxR, a transmembrane regulatory protein, has been shown to respond to environmental stimuli. To better understand how the aquatic bacterium Vibrio anguillarum, a fish pathogen, responds to environmental signals that may be necessary for survival in the aquatic and fish environment, toxR and toxS from V. anguillarum serotype O1 were cloned. The deduced protein sequences were 59 and 67% identical to the Vibrio cholerae ToxR and ToxS proteins, respectively. Deletion mutations were made in each gene and functional analyses were done. Virulence analyses using a rainbow trout model showed that only the toxR mutant was slightly decreased in virulence, indicating that ToxR is not a major regulator of virulence factors. The toxR mutant but not the toxS mutant was 20% less motile than the wild type. Like many regulatory proteins, ToxR was shown to negatively regulate its own expression. Outer membrane protein (OMP) preparations from both mutants indicated that ToxR and ToxS positively regulate a 38-kDa OMP. The 38-kDa OMP was shown to be a major OMP, which cross-reacted with an antiserum to OmpU, an outer membrane porin from V. cholerae, and which has an amino terminus 75% identical to that of OmpU. ToxR and to a lesser extent ToxS enhanced resistance to bile. Bile in the growth medium increased expression of the 38-kDa OMP but did not affect expression of ToxR. Interestingly, a toxR mutant forms a better biofilm on a glass surface than the wild type, suggesting a new role for ToxR in the response to environmental stimuli.

scholarly journals Vibrio cholerae tolC Is Required for Bile Resistance and Colonization

2001 ◽  
Vol 69 (7) ◽  
pp. 4681-4685 ◽  
Author(s):  
James E. Bina ◽  
John J. Mekalanos
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Vibrio Cholerae ◽  
Antimicrobial Agents ◽  
Cell Envelope ◽  
Outer Membrane Proteins ◽  
Intestinal Colonization ◽  
Gene Encoding ◽  
Bile Resistance

ABSTRACT TolC and its homologues are outer membrane proteins that are essential for the transport of many molecules across the cell envelope. In this study we characterized the gene encoding Vibrio cholerae TolC. V. cholerae tolC mutants failed to secrete the RTX cytotoxin, were hypersensitive to antimicrobial agents, and were deficient in intestinal colonization.

scholarly journals The outer membrane protein OprQ and adherence of Pseudomonas aeruginosa to human fibronectin

Microbiology ◽  
2010 ◽  
Vol 156 (5) ◽  
pp. 1415-1423 ◽  
Author(s):  
Abraham Arhin ◽  
Cliff Boucher
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Membrane Protein ◽  
Nutrient Uptake ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Outer Membrane Proteins ◽  
Wild Type ◽  
Insertional Inactivation ◽  
Human Fibronectin

Outer membrane proteins of the Gram-negative organism Pseudomonas aeruginosa play a significant role in membrane permeability, antibiotic resistance, nutrient uptake, and virulence in the infection site. In this study, we show that the P. aeruginosa outer membrane protein OprQ, a member of the OprD superfamily, is involved in the binding of human fibronectin (Fn). Some members of the OprD subfamily have been reported to be important in the uptake of nutrients from the environment. Comparison of wild-type and mutant strains of P. aeruginosa revealed that inactivation of the oprQ gene does not reduce the growth rate. Although it does not appear to be involved in nutrient uptake, an increased doubling time was reproducibly observed with the loss of OprQ in P. aeruginosa. Utilizing an oprQ–xylE transcriptional fusion, we determined that the PA2760 gene, encoding OprQ, was upregulated under conditions of decreased iron and magnesium. This upregulation appears to occur in early exponential phase. Insertional inactivation of PA2760 in the P. aeruginosa wild-type background did not produce a significant increase in resistance to any antibiotic tested, a phenotype that is typical of OprD family members. Interestingly, the in trans expression of OprQ in the ΔoprQ PAO1 mutant resulted in increased sensitivity to certain antibiotics. These findings suggest that OprQ is under dual regulation with other P. aeruginosa genes. Intact P. aeruginosa cells are capable of binding human Fn. We found that loss of OprQ resulted in a reduction of binding to plasmatic Fn in vitro. Finally, we present a discussion of the possible role of the P. aeruginosa outer membrane protein OprQ in adhesion to epithelial cells, thereby increasing colonization and subsequently enhancing lung destruction by P. aeruginosa.

scholarly journals Characterization of Native Outer Membrane Vesicles from lpxL Mutant Strains of Neisseria meningitidis for Use in Parenteral Vaccination

2005 ◽  
Vol 73 (7) ◽  
pp. 4070-4080 ◽  
Author(s):  
Makda Fisseha ◽  
Ping Chen ◽  
Brenda Brandt ◽  
Todd Kijek ◽  
Elizabeth Moran ◽  
...  
Keyword(s):  
Neisseria Meningitidis ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Wild Type ◽  
Necrosis Factor Alpha ◽  
Mutant Strains ◽  
Reduced Toxicity

ABSTRACT Native outer membrane vesicles (NOMV) of Neisseria meningitidis consist of intact outer membrane and contain outer membrane proteins (OMP) and lipooligosaccharides (LOS) in their natural conformation and membrane environment. NOMV have been safely used intranasally in P1 studies with encouraging results, but they are too toxic for parenteral vaccination. We now report the preparation and characterization of lpxL mutants that express LOS with reduced toxicity, and the evaluation of the potential of NOMV from these strains for use as a parenteral vaccine. A series of deletion mutants were prepared with knockouts of one or more of the lpxL1, lpxL2, or synX genes. The ΔlpxL2 mutants had a reduced growth rate, reduced level of LOS expression, and increased sensitivity to surfactants. In addition, ΔsynX ΔlpxL2 double mutants had reduced viability in stationary phase. The ΔlpxL1 ΔlpxL2 double mutant behaved essentially the same as the ΔlpxL2 single mutant. LOS from both lpxL mutant strains exhibited altered migration on polyacrylamide gels. The LOS of ΔlpxL2 mutants of L3,7 strains were fully sialylated. NOMV prepared from lpxL2 mutants was about 200-fold less active than wild-type NOMV in rabbit pyrogen tests and in tumor necrosis factor alpha release assays. Bactericidal titers induced in animals by ΔlpxL2 mutant NOMV were lower than those induced by ΔlpxL1 or wild-type NOMV. However, immunogenicity could be largely restored by use of an adjuvant. These results provide evidence that NOMV from ΔlpxL2 mutant strains will be safe and immunogenic in humans when given parenterally.

scholarly journals The Helicobacter pylori Autotransporter ImaA (HP0289) Modulates the Immune Response and Contributes to Host Colonization

2012 ◽  
Vol 80 (7) ◽  
pp. 2286-2296 ◽  
Author(s):  
William E. Sause ◽  
Andrea R. Castillo ◽  
Karen M. Ottemann
Keyword(s):  
Immune Response ◽  
Helicobacter Pylori ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Dependent Manner ◽  
Wild Type ◽  
Content Type ◽  
H Pylori ◽  
Murine Infections

ABSTRACTThe human pathogenHelicobacter pyloriemploys a diverse collection of outer membrane proteins to colonize, persist, and drive disease within the acidic gastric environment. In this study, we sought to elucidate the function of the host-induced geneHP0289, which encodes an uncharacterized outer membrane protein. We first generated an isogenicH. pylorimutant that lacksHP0289and found that the mutant has a colonization defect in single-strain infections and is greatly outcompeted in mouse coinfection experiments with wild-typeH. pylori. Furthermore, we used protease assays and biochemical fractionation coupled with an HP0289-targeted peptide antibody to verify that the HP0289 protein resides in the outer membrane. Our previous findings showed that theHP0289promoter is upregulated in the mouse stomach, and here we demonstrate thatHP0289expression is induced under acidic conditions in an ArsRS-dependent manner. Finally, we have shown that theHP0289mutant induces greater expression of the chemokine interleukin-8 (IL-8) and the cytokine tumor necrosis factor alpha (TNF-α) in gastric carcinoma cells (AGS). Similarly, transcription of the IL-8 homolog keratinocyte-derived chemokine (KC) is elevated in murine infections with the HP0289 mutant than in murine infections with wild-typeH. pylori. On the basis of this phenotype, we renamed HP0289 ImaA forimmunomodulatoryautotransporter protein. Our work has revealed that genes inducedin vivoplay an important role inH. pyloripathogenesis. Specifically, the outer membrane protein ImaA modulates a component of the host inflammatory response, and thus may allowH. pylorito fine tune the host immune response based on ImaA expression.

scholarly journals Regulated Delayed Expression of rfaH in an Attenuated Salmonellaenterica Serovar Typhimurium Vaccine Enhances Immunogenicity of Outer Membrane Proteins and a Heterologous Antigen

2009 ◽  
Vol 77 (12) ◽  
pp. 5572-5582 ◽  
Author(s):  
Qingke Kong ◽  
Qing Liu ◽  
Kenneth L. Roland ◽  
Roy Curtiss
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Lethal Dose ◽  
Enteric Bacteria ◽  
Lymphoid Tissues ◽  
Heterologous Antigen ◽  
O Antigen ◽  
Mutant Strains ◽  
Serovar Typhimurium

ABSTRACT RfaH is a transcriptional antiterminator that reduces the polarity of long operons encoding secreted and surface-associated cell components of Salmonella enterica serovar Typhimurium, including O antigen and lipopolysaccharide core sugars. A ΔrfaH mutant strain is attenuated in mice (50% lethal dose [LD50], >108 CFU). To examine the potential for using rfaH in conjunction with other attenuating mutations, we designed a series of strains in which we replaced the native rfaH promoter with the tightly regulated arabinose-dependent araC PBAD promoter so that rfaH expression was dependent on exogenously supplied arabinose provided during in vitro growth. Following colonization of host lymphoid tissues, where arabinose was not available, the PBAD promoter was no longer active and rfaH was not expressed. In the absence of RfaH, O antigen and core sugars were not synthesized. We constructed three mutant strains that expressed different levels of RfaH by altering the ribosome-binding sequence and start codon. One mutation, ΔPrfaH178, was introduced into the attenuated vaccine strain χ9241 (ΔpabA ΔpabB ΔasdA) expressing the pneumococcal surface protein PspA from an Asd+ balanced-lethal plasmid. Mice immunized with this strain and boosted 4 weeks later induced higher levels of serum immunoglobulin G specific for PspA and for outer membrane proteins from other enteric bacteria than either an isogenic ΔrfaH derivative or the isogenic RfaH+ parent. Eight weeks after primary oral immunization, mice were challenged with 200 LD50 of virulent S treptococcus pneumoniae WU2. Immunization with ΔPrfaH178 mutant strains led to increased levels of protection compared to that of the parent χ9241 and of a ΔrfaH derivative of χ9241.

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