Participation of CheR and CheB in the chemosensory response of Campylobacter jejuni

Microbiology ◽  
2011 ◽  
Vol 157 (5) ◽  
pp. 1279-1289 ◽  
Author(s):  
Doungjit Kanungpean ◽  
Tsutomu Kakuda ◽  
Shinji Takai
Keyword(s):  
Campylobacter Jejuni ◽  
Important Determinant ◽  
Commensal Bacterium ◽  
Gradient Sensing ◽  
Sds Page ◽  
Genes Encoding ◽  
Chemotaxis Proteins ◽  
Bacterial Gastroenteritis ◽  
Chemosensory Response

Campylobacter jejuni is a leading cause of bacterial gastroenteritis in humans and a commensal bacterium of the intestinal tracts of animals, especially poultry. Chemotaxis is an important determinant for chicken colonization of C. jejuni. Adaptation has a crucial role in the gradient-sensing mechanism that underlies chemotaxis. The genome sequence of C. jejuni reveals the presence of genes encoding putative adaptation proteins, CheB and CheR. In-frame deletions of cheB, cheR and cheBR were constructed and the chemosensory behaviour of the resultant mutants was examined on swarm plates. CheB and CheR proteins significantly influence chemotaxis but are not essential for this behaviour to occur. Increased mobility of two methyl-accepting chemotaxis proteins (MCPs), DocC and Tlp1, during SDS-PAGE was detected in the mutants lacking functional CheB in the presence of CheR, presumably resulting from stable methylation of receptors. In vitro studies using tissue culture revealed that deletion of cheR resulted in hyperadherent and hyperinvasive phenotypes, while deletion of cheB resulted in nonadherent, noninvasive phenotypes. Furthermore, the ΔcheBR mutant showed significantly reduced ability to colonize chick caeca. Our data suggest that modification of chemoreceptors by the CheBR system is involved in regulation of chemotaxis in C. jejuni although CheB is apparently not controlled by phosphorylation.

scholarly journals CapA, an Autotransporter Protein of Campylobacter jejuni, Mediates Association with Human Epithelial Cells and Colonization of the Chicken Gut

2006 ◽  
Vol 189 (5) ◽  
pp. 1856-1865 ◽  
Author(s):  
Sami S. A. Ashgar ◽  
Neil J. Oldfield ◽  
Karl G. Wooldridge ◽  
Michael A. Jones ◽  
Greg J. Irving ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Protein A ◽  
Phase Variation ◽  
Subcellular Fractionation ◽  
Immunogold Electron Microscopy ◽  
Insertion Mutant ◽  
Genes Encoding ◽  
Monospecific Antisera ◽  
Recombinant Polypeptides

ABSTRACT Two putative autotransporter proteins, CapA and CapB, were identified in silico from the genome sequence of Campylobacter jejuni NCTC11168. The genes encoding each protein contain homopolymeric tracts, suggestive of phase variation mediated by a slipped-strand mispairing mechanism; in each case the gene sequence contained frameshifts at these positions. The C-terminal two-thirds of the two genes, as well as a portion of the predicted signal peptides, were identical; the remaining N-terminal portions were gene specific. Both genes were cloned and expressed; recombinant polypeptides were purified and used to raise rabbit polyclonal monospecific antisera. Using immunoblotting, expression of the ca.116-kDa CapA protein was demonstrated for in vitro-grown cells of strain NCTC11168, for 4 out of 11 recent human fecal isolates, and for 2 out of 8 sequence-typed strains examined. Expression of CapB was not detected for any of the strains tested. Surface localization of CapA was demonstrated by subcellular fractionation and immunogold electron microscopy. Export of CapA was inhibited by globomycin, reinforcing the bioinformatic prediction that the protein is a lipoprotein. A capA insertion mutant had a significantly reduced capacity for association with and invasion of Caco-2 cells and failed to colonize and persist in chickens, indicating that CapA plays a role in host association and colonization by Campylobacter. In view of this demonstrated role, we propose that CapA stands for Campylobacter adhesion protein A.

scholarly journals Examination of Campylobacter jejuni Putative Adhesins Leads to the Identification of a New Protein, Designated FlpA, Required for Chicken Colonization

2009 ◽  
Vol 77 (6) ◽  
pp. 2399-2407 ◽  
Author(s):  
Rebecca C. Flanagan ◽  
Jason M. Neal-McKinney ◽  
A. Singh Dhillon ◽  
William G. Miller ◽  
Michael E. Konkel
Keyword(s):  
Epithelial Cells ◽  
Campylobacter Jejuni ◽  
Blot Hybridization ◽  
Broiler Chicks ◽  
Cell Adherence ◽  
Dot Blot ◽  
Dot Blot Hybridization ◽  
Genes Encoding

ABSTRACT Campylobacter jejuni colonization of chickens is presumably dependent upon multiple surface-exposed proteins termed adhesins. Putative C. jejuni adhesins include CadF, CapA, JlpA, major outer membrane protein, PEB1, Cj1279c, and Cj1349c. We examined the genetic relatedness of 97 C. jejuni isolates recovered from human, poultry, bovine, porcine, ovine, and canine sources by multilocus sequence typing (MLST) and examined their profile of putative adhesin-encoding genes by dot blot hybridization. To assess the individual contribution of each protein in bacterium-host cell adherence, the C. jejuni genes encoding the putative adhesins were disrupted by insertional mutagenesis. The phenotype of each mutant was judged by performing in vitro cell adherence assays with chicken LMH hepatocellular carcinoma epithelial cells and in vivo colonization assays with broiler chicks. MLST analysis indicated that the C. jejuni isolates utilized in this study were genetically diverse. Dot blot hybridization revealed that the C. jejuni genes encoding the putative adhesins, with the exception of capA, were conserved among the isolates. The C. jejuni CadF, CapA, Cj1279c, and Cj1349c proteins were found to play a significant role in the bacterium's in vitro adherence to chicken epithelial cells, while CadF, PEB1, and Cj1279c were determined to play a significant role in the bacterium's in vivo colonization of broiler chicks. Collectively, the data indicate that Cj1279c is a novel adhesin. Because Cj1279c harbors fibronectin type III domains, we designated the protein FlpA, for fibronectin-like protein A.

scholarly journals A Campylobacter jejuni znuA Orthologue Is Essential for Growth in Low-Zinc Environments and Chick Colonization

2008 ◽  
Vol 191 (5) ◽  
pp. 1631-1640 ◽  
Author(s):  
Lindsay M. Davis ◽  
Tsutomu Kakuda ◽  
Victor J. DiRita
Keyword(s):  
Campylobacter Jejuni ◽  
Transport System ◽  
The United States ◽  
Gram Negative Bacteria ◽  
Limited Growth ◽  
E Coli ◽  
Poultry Products ◽  
Abc Transport ◽  
Bacterial Gastroenteritis

ABSTRACT Campylobacter jejuni infection is a leading cause of bacterial gastroenteritis in the United States and is acquired primarily through the ingestion of contaminated poultry products. Here, we describe the C. jejuni orthologue of ZnuA in other gram-negative bacteria. ZnuA (Cj0143c) is the periplasmic component of a putative zinc ABC transport system and is encoded on a zinc-dependent operon with Cj0142c and Cj0141c, which encode the other two likely components of the transport system of C. jejuni. Transcription of these genes is zinc dependent. A mutant lacking Cj0143c is growth deficient in zinc-limiting media, as well as in the chick gastrointestinal tract. The protein is glycosylated at asparagine 28, but this modification is dispensable for zinc-limited growth and chick colonization. Affinity-purified FLAG-tagged Cj0143c binds zinc in vitro. Based on our findings and on its homology to E. coli ZnuA, we conclude that Cj0143c encodes the C. jejuni orthologue of ZnuA.

scholarly journals Characterization and antigenicity of recombinant Campylobacter jejuni flagellar capping protein FliD

2014 ◽  
Vol 63 (4) ◽  
pp. 602-609 ◽  
Author(s):  
Hung-Yueh Yeh ◽  
Kelli L. Hiett ◽  
John E. Line ◽  
Bruce S. Seal
Keyword(s):  
Campylobacter Jejuni ◽  
Broiler Chickens ◽  
Nucleotide Sequencing ◽  
Calculated Molecular Mass ◽  
Ionization Time ◽  
Bacterial Flagella ◽  
Capping Protein ◽  
Flight Mass Spectrometry ◽  
Sds Page ◽  
Bacterial Gastroenteritis

Campylobacter jejuni, a flagellated, spiral-rod, Gram-negative bacterium, is the leading pathogen of human acute bacterial gastroenteritis worldwide, and chickens are regarded as a major reservoir of this micro-organism. Bacterial flagella, composed of more than 35 proteins, play important roles in colonization and adhesion to the mucosal surface of chicken caeca. In this study, the flagellar capping protein, FliD, encoded by the fliD gene, from the Campylobacter jenuni D1-39 isolate was expressed and characterized, and its antigenicity determined. The fliD gene comprised 1929 nt, potentially encoding a 642 aa peptide with a calculated molecular mass of 69.6 kDa. This gene was PCR amplified and overexpressed in Escherichia coli. The recombinant FliD protein was purified by cobalt-chelating affinity chromatography and confirmed by nucleotide sequencing of the expression plasmid, SDS-PAGE analysis, His tag detection and matrix-assisted laser desorption/ionization time of flight mass spectrometry. The immunoblot data showed that the purified recombinant FliD protein reacted strongly to sera from broiler chickens older than 4 weeks, indicating that anti-FliD antibody may be prevalent in the poultry population. These results provide a rationale for further evaluation of the FliD protein as a vaccine candidate for broiler chickens to improve food safety for poultry.

Reduction of Campylobacter jejuni and Campylobacter coli in Poultry Skin by Fruit Extracts

2010 ◽  
Vol 73 (3) ◽  
pp. 477-482 ◽  
Author(s):  
DIANA VALTIERRA-RODRÍGUEZ ◽  
NORMA L. HEREDIA ◽  
SANTOS GARCÍA ◽  
EDUARDO SÁNCHEZ
Keyword(s):  
Antimicrobial Activity ◽  
Campylobacter Jejuni ◽  
Antimicrobial Agents ◽  
Orange Peel ◽  
Campylobacter Coli ◽  
Sour Orange ◽  
Bacterial Gastroenteritis ◽  
Natural Foods ◽  
Promising Source

Campylobacter spp. are a major cause of foodborne bacterial gastroenteritis in humans, and current methods to control Campylobacter contamination in foods are not completely successful. Plants are a promising source of antimicrobial agents, particularly given the growing interest in “all natural” foods. In this study, the antimicrobial activity of extracts from 28 edible plants against Campylobacter jejuni and Campylobacter coli was evaluated in vitro and in a poultry skin model. Nine of 28 extracts exhibited antimicrobial activity in a diffusion assay, and MBCs were determined for the three most active extracts, i.e., lime, plum, and sour orange peel (MBCs of 2 to 3 mg/ml). Mixtures of the lime, plum, and sour orange peel extracts were applied to chicken skin inoculated with 105 CFU of Campylobacter to test for synergistic or antagonist effects. After incubation (48 h at 4°C) with any extract mixture, no Campylobacter CFUs were detectable. A panel of tasters determined that the mixture of lime and plum gave the best flavor to chicken wings. These active extracts from edible fruits are simple to prepare and are alternatives to reduce or eliminate Campylobacter contamination of chicken products.

scholarly journals Genetics behind the biosynthesis of nonulosonic acid containing lipooligosaccharides inCampylobacter coli

2018 ◽  
Author(s):  
Alejandra Kolehmainen ◽  
Mirko Rossi ◽  
Jacek Stupak ◽  
Jianjun Li ◽  
Michel Gilbert ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Foodborne Pathogen ◽  
Campylobacter Coli ◽  
Strong Impact ◽  
Large Set ◽  
Donor And Acceptor ◽  
Nonulosonic Acid ◽  
Bacterial Gastroenteritis ◽  
Acute Polyneuropathy

ABSTRACTCampylobacter jejuniandCampylobacter coliare the most common cause of bacterial gastroenteritis in the world. Ganglioside mimicry byC. jejunilipooligosaccharide (LOS) is the triggering factor of Guillain-Barré syndrome (GBS), an acute polyneuropathy. Sialyltransferases from the glycosyltransferase (GT) family 42 are essential for the expression of ganglioside mimics inC. jejuni. Recently, two novel GT-42 genes,cstIVandcstV, have been identified inC. coli.Despite being present in ∼11% of currently availableC. coligenomes, the biological role ofcstIVandcstVis unknown. In the present study, mutation studies in two strains expressing eithercstIVorcstVwere performed and mass spectrometry was used to investigate differences in the chemical composition of LOS. Attempts were made to identify donor and acceptor molecules usingin vitroactivity tests with recombinant GT-42 enzymes. Here, we show that CstIV and CstV are involved inC. coliLOS biosynthesis. In particular,cstVis associated with LOS sialylation, whilecstIVis linked to the addition of a diacetylated nonulosonic acid residue.IMPORTANCEDespite being a major foodborne pathogen,Campylobacter coliglycobiology has been largely neglected. The genetic makeup of theC. colilipooligosaccharide biosynthesis locus was largely unknown until recently.C. coliharbour a large set of genes associated to lipooligosaccharide biosynthesis, including several putative glycosyltransferases involved in the synthesis of sialylated lipooligosaccharide inCampylobacter jejuni. In the present study,C. coliwas found to express lipooligosaccharide structures containing sialic acid and other nonulosonate acids. These findings have a strong impact in understandingC. coliecology, host-pathogen interaction, and pathogenesis.

scholarly journals Molecular Characterization of Carbapenem-Nonsusceptible Enterobacterial Isolates Collected during a Prospective Interregional Survey in France and Susceptibility to the Novel Ceftazidime-Avibactam and Aztreonam-Avibactam Combinations

2015 ◽  
Vol 60 (1) ◽  
pp. 215-221 ◽  
Author(s):  
Hervé Dupont ◽  
Olivier Gaillot ◽  
Anne-Sophie Goetgheluck ◽  
Claire Plassart ◽  
Jean-Philippe Emond ◽  
...  
Keyword(s):  
Outer Membrane Proteins ◽  
Surveillance Program ◽  
Northwestern Part ◽  
The Novel ◽  
Content Type ◽  
Active Efflux ◽  
Fixed Concentration ◽  
Sds Page ◽  
Genes Encoding

ABSTRACTAn interregional surveillance program was conducted in the northwestern part of France to determine the prevalence of carbapenem-nonsusceptibleEnterobacteriaceae(CNSE) isolates and their susceptibility to ceftazidime-avibactam and aztreonam-avibactam combinations. Nonduplicate CNSE clinical isolates were prospectively collected from six hospitals between June 2012 and November 2013. MICs of ceftazidime and aztreonam, alone or combined with a fixed concentration of avibactam (4 μg/ml), and those of carbapenems (comparator agents) were determined. MICs of ertapenem in combination with phenylalanine arginine-naphthylamide dihydrochloride (PAβN) were also determined to assess active efflux. Genes encoding carbapenemases, plasmid-mediated AmpC enzymes, extended-spectrum β-lactamases (ESBLs), and major outer membrane proteins (OMPs) were amplified and sequenced. OMPs were also extracted for SDS-PAGE analysis. Among the 139 CNSE isolates, mainlyEnterobacterspp. andKlebsiella pneumoniae, 123 (88.4%) were ertapenem nonsusceptible, 12 (8.6%) exhibited reduced susceptibility to all carbapenems, and 4Proteeaeisolates (2.9%) were resistant to imipenem. Carbapenemase production was detected in only two isolates (producing OXA-48 and IMI-3). In contrast, OMP deficiency, in association with AmpCs and/or ESBLs (mainly CTX-M-9, SHV-12, and CTX-M-15), was largely identified among CNSE isolates. The ceftazidime-avibactam and aztreonam-avibactam combinations exhibited potent activity against CNSE isolates (MIC50/MIC90, 1/1 μg/ml and 0.5/0.5 μg/ml, respectively) compared to that of ceftazidime and aztreonam alone (MIC50/MIC90, 512/512 μg/ml and 128/512 μg/ml, respectively). This study reveals thein vitroactivity of ceftazidime-avibactam and aztreonam-avibactam combinations against a large collection of porin-deficient enterobacterial isolates that are representative of the CNSE recovered in the northern part of France.

The Anticoagulant Properties of a Modified Form of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 298-304 ◽  
Author(s):  
C A Mitchell ◽  
S M Kelemen ◽  
H H Salem
Keyword(s):  
Monoclonal Antibody ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Human Neutrophil Elastase ◽  
Factor X ◽  
Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

In Vitro Stability of a Tissue-Type Plasminogen Activator Mutant, BM 06.022, in Human Plasma

1994 ◽  
Vol 72 (06) ◽  
pp. 906-911 ◽  
Author(s):  
D C Rijken ◽  
E Groeneveld ◽  
M M Barrett-Bergshoeff
Keyword(s):  
Plasminogen Activator ◽  
Half Life ◽  
Polyacrylamide Gel Electrophoresis ◽  
Tissue Type ◽  
Single Chain ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Sds Page ◽  
Chain Form

SummaryBM 06.022 is a non-glycosylated mutant of human tissue-type plasminogen activator (t-PA) comprising only the kringle-2 and proteinase domains. The in vivo half-life of BM 06.022 antigen is 4- to 5-fold longer than that of t-PA antigen. The in vitro half-life of the activity of BM 06.022 at therapeutic concentrations in plasma is shorter than that of t-PA. In this study the inactivation of BM 06.022 in plasma was further investigated.Varying concentrations of BM 06.022 were incubated in plasma for 0-150 min. Activity assays on serial samples showed a dose-dependent decline of BM 06.022 activity with a half-life from 72 min at 0.3 μg/ml to 38 min at 10 μg/ml. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fibrin autography showed the generation of several BM 06.022-complexes. These complexes could be completely precipitated with antibodies against Cl-inactivator, α2-antiplasmin and α1-antitrypsin.During the incubation of BM 06.022 in plasma, plasmin was generated dose-dependently as revealed by varying degrees of a2-anti-plasmin consumption and fibrinogen degradation. SDS-PAGE and immunoblotting showed that single-chain BM 06.022 was rapidly (i. e. within 45 min) converted into its two-chain form at concentrations of 5 μg/ml BM 06.022 and higher.In conclusion, BM 06.022 at therapeutic concentrations in plasma was inactivated by Cl-inactivator, a2-antiplasmin and a j-antitrypsin. The half-life of the activity decreased at increasing BM 06.022 concentrations, probably as a result of the generation of two-chain BM 06.022 which may be inactivated faster than the single-chain form.

scholarly journals Role of Satb1 and Satb2 Transcription Factors in the Glutamate Receptors Expression and Ca2+ Signaling in the Cortical Neurons In Vitro

2021 ◽  
Vol 22 (11) ◽  
pp. 5968
Author(s):  
Egor A. Turovsky ◽  
Maria V. Turovskaya ◽  
Evgeniya I. Fedotova ◽  
Alexey A. Babaev ◽  
Viktor S. Tarabykin ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Nmda Receptor ◽  
Cortical Neurons ◽  
Target Genes ◽  
Cortical Cell ◽  
Inhibitory System ◽  
Selective Agonist ◽  
Genes Encoding ◽  
Deficient Mice

Transcription factors Satb1 and Satb2 are involved in the processes of cortex development and maturation of neurons. Alterations in the expression of their target genes can lead to neurodegenerative processes. Molecular and cellular mechanisms of regulation of neurotransmission by these transcription factors remain poorly understood. In this study, we have shown that transcription factors Satb1 and Satb2 participate in the regulation of genes encoding the NMDA-, AMPA-, and KA- receptor subunits and the inhibitory GABA(A) receptor. Deletion of gene for either Satb1 or Satb2 homologous factors induces the expression of genes encoding the NMDA receptor subunits, thereby leading to higher amplitudes of Ca2+-signals in neurons derived from the Satb1-deficient (Satb1fl/+ * NexCre/+) and Satb1-null mice (Satb1fl/fl * NexCre/+) in response to the selective agonist reducing the EC50 for the NMDA receptor. Simultaneously, there is an increase in the expression of the Gria2 gene, encoding the AMPA receptor subunit, thus decreasing the Ca2+-signals of neurons in response to the treatment with a selective agonist (5-Fluorowillardiine (FW)). The Satb1 deletion increases the sensitivity of the KA receptor to the agonist (domoic acid), in the cortical neurons of the Satb1-deficient mice but decreases it in the Satb1-null mice. At the same time, the Satb2 deletion decreases Ca2+-signals and the sensitivity of the KA receptor to the agonist in neurons from the Satb1-null and the Satb1-deficient mice. The Satb1 deletion affects the development of the inhibitory system of neurotransmission resulting in the suppression of the neuron maturation process and switching the GABAergic responses from excitatory to inhibitory, while the Satb2 deletion has a similar effect only in the Satb1-null mice. We show that the Satb1 and Satb2 transcription factors are involved in the regulation of the transmission of excitatory signals and inhibition of the neuronal network in the cortical cell culture.

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