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P Guerry

Publications and source records attributed to P Guerry.

At least 19 recordsLinked to original sources
PubMed2001

Identification of the carbohydrate moieties and glycosylation motifs in Campylobacter jejuni flagellin.

Flagellins from three strains of Campylobacter jejuni and one strain of Campylobacter coli were shown to be extensively modified by glycosyl residues, imparting an approximate 6000-Da shift from the molecular mass of the protein predicted from the DNA sequence. Tryptic peptides from C. jejuni 81-176 flagellin were subjected to capillary liquid chromatography-electrospray mass spectrometry with a high/low orifice stepping to identify peptide segments of aberrant masses together with their corresponding glycosyl appendages. These modified peptides were further characterized by tandem mass spectrometry and preparative high performance liquid chromatography followed by nano-NMR spectroscopy to identify the nature and precise site of glycosylation. These analyses have shown that there are 19 modified Ser/Thr residues in C. jejuni 81-176 flagellin. The predominant modification found on C. jejuni flagellin was O-linked 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic acid (pseudaminic acid, Pse5Ac7Ac) with additional heterogeneity conferred by substitution of the acetamido groups with acetamidino and hydroxyproprionyl groups. In C. jejuni 81-176, the gene Cj1316c, encoding a protein of unknown function, was shown to be involved in the biosynthesis and/or the addition of the acetamidino group on Pse5Ac7Ac. Glycosylation is not random, since 19 of the total 107 Ser/Thr residues are modified, and all but one of these are restricted to the central, surface-exposed domain of flagellin when folded in the filament. The mechanism of attachment appears unrelated to a consensus peptide sequence but is rather based on surface accessibility of Ser/Thr residues in the folded protein.

Amino Acid Sequence↗
PubMed2001

A phase-variable capsule is involved in virulence of Campylobacter jejuni 81-176.

Campylobacter jejuni strain 81-176 (HS36, 23) synthesizes two distinct glycan structures, as visualized by immunoblotting of proteinase K-digested whole-cell preparations. A site-specific insertional mutant in the kpsM gene results in loss of expression of a high-molecular-weight (HMW) glycan (apparent Mr 26 kDa to > 85 kDa) and increased resolution of a second ladder-like glycan (apparent Mr 26-50 kDa). The kpsM mutant of 81-176 is no longer typeable in either HS23 or HS36 antisera, indicating that the HMW glycan structure is the serodeterminant of HS23 and HS36. Both the kpsM-dependent HMW glycan and the kpsM-independent ladder-like structure appear to be capsular in nature, as both are attached to phospholipid rather than lipid A. Additionally, the 81-176 kpsM gene can complement a deletion in Escherichia coli kpsM, allowing the expression of an alpha2,8 polysialic acid capsule in E. coli. Loss of the HMW glycan in 81-176 kpsM also increases the surface hydrophobicity and serum sensitivity of the bacterium. The kpsM mutant is also significantly reduced in invasion of INT407 cells and reduced in virulence in a ferret diarrhoeal disease model. The expression of the kpsM-dependent capsule undergoes phase variation at a high frequency.

ATP-Binding Cassette Transporters↗
PubMed2000

Campylobacter jejuni cytolethal distending toxin mediates release of interleukin-8 from intestinal epithelial cells.

Live cells of Campylobacter jejuni and Campylobacter coli can induce release of interleukin-8 (IL-8) from INT407 cells. Additionally, membrane fractions of C. jejuni 81-176, but not membrane fractions of C. coli strains, can also induce release of IL-8. Membrane preparations from 81-176 mutants defective in any of the three membrane-associated protein subunits of cytolethal distending toxin (CDT) were unable to induce IL-8. The presence of the three cdt genes on a shuttle plasmid in trans restored both CDT activity and the ability to release IL-8 to membrane fractions. However, CDT mutations did not affect the ability of 81-176 to induce IL-8 during adherence to or invasion of INT407 cells. When C. jejuni cdt genes were transferred on a shuttle plasmid into a C. coli strain lacking CDT, membrane preparations became positive in both CDT and IL-8 assays. Growth of C. jejuni in physiological levels of sodium deoxycholate released all three CDT proteins, as well as CDT activity and IL-8 activity, from membranes into supernatants. Antibodies against recombinant forms of each of the three CDT subunit proteins neutralized both CDT activity and the activity responsible for IL-8 release. The data suggest that C. jejuni can induce IL-8 release from INT407 cells by two independent mechanisms, one of which requires adherence and/or invasion and the second of which requires CDT.

Bacterial Toxins↗
PubMed2000

Sialylation of lipooligosaccharide cores affects immunogenicity and serum resistance of Campylobacter jejuni.

Three genes involved in biosynthesis of the lipooligosaccharide (LOS) core of Campylobacter jejuni MSC57360, the type strain of the HS:1 serotype, whose structure mimics GM(2) ganglioside, have been cloned and characterized. Mutation of genes encoding proteins with homology to a sialyl transferase (cstII) and a putative N-acetylmannosamine synthetase (neuC1), part of the biosynthetic pathway of N-acetylneuraminic acid (NeuNAc), have identical phenotypes. The LOS cores of these mutants display identical changes in electrophoretic mobility, loss of reactivity with cholera toxin (CT), and enhanced immunoreactivity with a hyperimmune polyclonal antiserum generated against whole cells of C. jejuni MSC57360. Loss of sialic acid in the core of the neuC1 mutant was confirmed by fast atom bombardment mass spectrometry. Mutation of a gene encoding a putative beta-1,4-N-acetylgalactosaminyltransferase (Cgt) resulted in LOS cores intermediate in electrophoretic mobility between that of wild type and the mutants lacking NeuNAc, loss of reactivity with CT, and a reduced immunoreactivity with hyperimmune antiserum. Chemical analyses confirmed the loss of N-acetylgalactosamine (GalNAc) and the presence of NeuNAc in the cgt mutant. These data suggest that the Cgt enzyme is capable of transferring GalNAc to an acceptor with or without NeuNAc and that the Cst enzyme is capable of transferring NeuNAc to an acceptor with or without GalNAc. A mutant with a nonsialylated LOS core is more sensitive to the bactericidal effects of human sera than the wild type or the mutant lacking GalNAc.

Animals↗
PubMed2000

Involvement of a plasmid in virulence of Campylobacter jejuni 81-176.

Campylobacter jejuni strain 81-176 contains two, previously undescribed plasmids, each of which is approximately 35 kb in size. Although one of the plasmids, termed pTet, carries a tetO gene, conjugative transfer of tetracycline resistance to another strain of C. jejuni could not be demonstrated. Partial sequence analysis of the second plasmid, pVir, revealed the presence of four open reading frames which encode proteins with significant sequence similarity to Helicobacter pylori proteins, including one encoded by the cag pathogenicity island. All four of these plasmid-encoded proteins show some level of homology to components of type IV secretion systems. Mutation of one of these plasmid genes, comB3, reduced both adherence to and invasion of INT407 cells to approximately one-third that seen with wild-type strain 81-176. Mutation of comB3 also reduced the natural transformation frequency. A mutation in a second plasmid gene, a virB11 homolog, resulted in a 6-fold reduction in adherence and an 11-fold reduction in invasion compared to the wild type. The isogenic virB11 mutant of strain 81-176 also demonstrated significantly reduced virulence in the ferret diarrheal disease model. The virB11 homolog was detected on plasmids in 6 out of 58 fresh clinical isolates of C. jejuni, suggesting that plasmids are involved in the virulence of a subset of C. jejuni pathogens.

Animals↗
PubMed1999

Evidence for a system of general protein glycosylation in Campylobacter jejuni.

A genetic locus from Campylobacter jejuni 81-176 (O:23, 36) has been characterized that appears to be involved in glycosylation of multiple proteins, including flagellin. The lipopolysaccharide (LPS) core of Escherichia coli DH5alpha containing some of these genes is modified such that it becomes immunoreactive with O:23 and O:36 antisera and loses reactivity with the lectin wheat germ agglutinin (WGA). Site-specific mutation of one of these genes in the E. coli host causes loss of O:23 and O:36 antibody reactivity and restores reactivity with WGA. However, site-specific mutation of each of the seven genes in 81-176 failed to show any detectable changes in LPS. Multiple proteins from various cellular fractions of each mutant showed altered reactivity by Western blot analyses using O:23 and O:36 antisera. The changes in protein antigenicity could be restored in one of the mutants by the presence of the corresponding wild-type allele in trans on a shuttle vector. Flagellin, which is known to be a glycoprotein, was one of the proteins that showed altered reactivity with O:23 and O:36 antiserum in the mutants. Chemical deglycosylation of protein fractions from the 81-176 wild type suggests that the other proteins with altered antigenicity in the mutants are also glycosylated.

Antigens, Bacterial↗
PubMed1999

Campylobacter jejuni-stimulated secretion of interleukin-8 by INT407 cells.

Incubation of INT407 cells with various clinical isolates of Campylobacter jejuni resulted in secretion of interleukin-8 (IL-8) at levels ranging from 96 to 554 pg/ml at 24 h. The strains which produced the highest levels of IL-8 secretion were 81-176 and BT44. Induction of IL-8 secretion required live cells of 81-176 and was dependent on de novo protein synthesis. Site-specific mutants of 81-176, which were previously shown to be defective in adherence and invasion, resulted in reduced levels of secretion of IL-8, and cheY mutants of strains 81-176 and 749, which are hyperadherent and hyperinvasive, resulted in higher levels of IL-8 secretion. Another mutant of 81-176, which adheres at about 43% of the wild-type levels but is noninvasive, also showed marked reduction in IL-8 levels, suggesting that invasion is necessary for high levels of IL-8 secretion. When gentamicin was added to INT407 cells at 2 h after infection with 81-176, IL-8 secretion 22 h later was equivalent to that of controls without gentamicin, suggesting that the events which trigger induction and release of IL-8 occur early in the interactions of bacteria and eukaryotic cells.

Bacterial Adhesion↗
PubMed1999

Evaluation of a truncated recombinant flagellin subunit vaccine against Campylobacter jejuni.

A recombinant protein comprising the maltose-binding protein (MBP) of Escherichia coli fused to amino acids 5 to 337 of the FlaA flagellin of Campylobacter coli VC167 was evaluated for immunogenicity and protective efficacy against challenge by a heterologous strain of campylobacter, Campylobacter jejuni 81-176, in two murine models. The sequence of the flaA gene of strain 81-176 revealed a predicted protein which was 98.1% similar to that of VC167 FlaA over the region expressed in the fusion protein. Mice were immunized intranasally with two doses of 3 to 50 microgram of MBP-FlaA, given 8 days apart, with or without 5 microgram of the mutant E. coli heat-labile enterotoxin (LT(R192G)) as a mucosal adjuvant. The full range of MBP-FlaA doses were effective in eliciting antigen-specific serum immunoglobulin G (IgG) responses, and these responses were enhanced by adjuvant use, except in the highest dosing group. Stimulation of FlaA-specific intestinal secretory IgA (sIgA) responses required immunization with higher doses of MBP-FlaA (>/=25 microgram) or coadministration of lower doses with the adjuvant. When vaccinated mice were challenged intranasally 26 days after immunization, the best protection was seen in animals given 50 microgram of MBP-FlaA plus LT(R192G). The protective efficacies of this dose against disease symptoms and intestinal colonization were 81.1 and 84%, respectively. When mice which had been immunized with 50 microgram of MBP-FlaA plus LT(R192G) intranasally were challenged orally with 8 x 10(10), 8 x 10(9), or 8 x 10(8) cells of strain 81-176, the protective efficacies against intestinal colonization at 7 days postinfection were 71.4, 71.4, and 100%, respectively.

Amino Acid Sequence↗
PubMed1997

CheY-mediated modulation of Campylobacter jejuni virulence.

Four motile, non-adherent and non-invasive mutants of Campylobacter jejuni 81-176 generated by a site-specific insertional mutagenesis scheme were characterized at the molecular level and all contained a duplication of the same region of the chromosome. When this region was cloned from wild-type 81-176 and transferred into 81-176 on a shuttle plasmid, the same non-invasive phenotype as the original mutants was observed, suggesting that the region contained a repressor of adherence and invasion. The smallest piece of DNA identified which was capable of repressing adherence and invasion was a 0.8 kb fragment encoding the cheY gene of C.jejuni. To confirm further that CheY was responsible for the observed non-adherent and non-invasive phenotypes, the cheY gene was inserted into the arylsulfatase gene of 81-176 to generate a strain with two chromosomal copies of cheY. This diploid strain displayed the same non-adherent and non-invasive phenotype as the original mutants. Insertional inactivation of the cheY gene in 81-176 resulted in an approx. threefold increase in adherence and invasion in vitro, but this strain was unable to colonize or cause disease in animals. The diploid cheY strain, although able to colonize mice, was attenuated in a ferret disease model.

Amino Acid Sequence↗
PubMed1997

Nonlipopolysaccharide surface antigens of Campylobacter species.

Among the protein antigens of Campylobacter species, flagellin, the subunit of the flagellar filament, is the best characterized. The motility imparted by this locomotory organelle is absolutely essential for Campylobacter organisms to colonize the gastrointestinal tract and to cause diarrheal disease. Flagellin is the immunodominant protein recognized during infection and has been suggested to be involved in the protective immune response. Campylobacter flagellins are glycosylated, which is an unusual posttranslational modification for prokaryotic proteins. Although the chemical structure of the glycosylated moiety is undetermined, the posttranslational modification includes sialic acid. The association of glycosylated flagellin with development of Guillain-Barré syndrome remains speculative, but the possibility of molecular mimicry between glycosylated flagellin and eukaryotic glycoproteins exists.

Antigens, Bacterial↗
PubMed1997

A genetic locus involved in iron utilization unique to some Campylobacter strains.

Two genes involved in iron utilization in Campylobacter coli VC167 T1 have been characterized. The cfrA gene encodes a protein with a predicted Mr of 77,653 which, after processing of the leader sequence, has a predicted Mr of 75,635. This protein has significant sequence identity to siderophore receptors of several bacteria, and site-specific mutants defective in cfrA do not synthesize one of two major iron-repressible outer membrane proteins. An adjacent gene encodes a TonB-like protein; a mutant in this gene lost the ability to utilize hemin, ferrichrome, and enterochelin as iron sources. The cfrA and tonB genes of VC167 T1 hybridized to all strains of C. coli and most strains of C. jejuni examined but did not hybridize to several other strains of C. jejuni, suggesting that the thermophilic campylobacters can be separated into two categories based on the presence of these two iron utilization genes.

Amino Acid Sequence↗
PubMed1997

The flgE gene of Campylobacter coli is under the control of the alternative sigma factor sigma54.

The flgE gene encoding the flagellar hook protein of Campylobacter coli VC167-T1 was cloned by immunoscreening of a genomic library constructed in lambdaZAP Express. The flgE DNA sequence was 2,553 bp in length and encoded a protein with a deduced molecular mass of 90,639 Da. The sequence had significant homology to the 5' and 3' sequences of the flgE genes of Helicobacter pylori, Treponema phagedenis, and Salmonella typhimurium. Primer extension analysis indicated that the VC167 flgE gene is controlled by a sigma54 promoter. PCR analysis showed that the flgE gene size and the 5' and 3' DNA sequences were conserved among C. coli and C. jejuni strains. Southern hybridization analyses confirmed that there is considerable sequence identity among the hook genes of C. coli and C. jejuni but that there are also regions within the genes which differ. Mutants of C. coli defective in hook production were generated by allele replacement. These mutants were nonmotile and lacked flagellar filaments. Analyses of flgE mutants indicated that the carboxy terminus of FlgE is necessary for assembly of the hook structure but not for secretion of FlgE and that, unlike salmonellae, the lack of flgE expression does not result in repression of flagellin expression.

Amino Acid Sequence↗
PubMed1996

Identification and characterization of genes required for post-translational modification of Campylobacter coli VC167 flagellin.

Two genes have been identified in Campylobacter coli VC167 which are required for the biosynthesis of post-translational modifications on flagellin proteins. The ptmA gene encodes a protein of predicted M(r) 28,486 which shows significant homology to a family of alcohol dehydrogenases from a variety of bacteria. The ptmB gene encodes a protein of predicted M(r) 26,598 with significant homology to CMP-N-acetylneuraminic acid synthetase enzymes involved in sialic acid capsular biosynthesis in Neisseria meninigitidis and Escherichia coli K1. Site-specific mutation of either ptmA or ptmB caused loss of reactivity with antisera specific to the post-translational modifications and a change in the isoelectric focusing fingerprints relative to the parent strains. Mutation of ptmB, but not of ptmA, caused a change in apparent M(r) of the flagellin subunit in SDS-PAGE gels. The ptmA and ptmB genes are present in other strains of Campylobacter. In a rabbit model the ptmA mutant showed a reduced ability to elicit protection against subsequent challenge with heterologous strains of the same Lior serotype compared to the parental wild-type strain. This suggests that the surface-exposed post-translational modifications may play a significant role in the protective immune response.

Amino Acid Sequence↗
PubMed1996

Characterization of a post-translational modification of Campylobacter flagellin: identification of a sero-specific glycosyl moiety.

The flagellins of Campylobacter spp. differ antigenically. In variants of C. coli strain VC167, two antigenic flagellin types determined by sero-specific antibodies have been described (termed T1 and T2). Post-translational modification has been suggested to be responsible for T1 and T2 epitopes, and, using mild periodate treatment and biotin hydrazide labelling, flagellin from both VC167-T1 and T2 were shown to be glycosylated. Glycosylation was also shown to be present on other Campylobacter flagellins. The ability to label all Campylobacter flagellins examined with the lectin LFA demonstrated the presence of a terminal sialic acid moiety. Furthermore, mild periodate treatment of the flagellins of VC167 eliminated reactivity with T1 and T2 specific antibodies LAH1 and LAH2, respectively, and LFA could also compete with LAH1 and LAH2 antibodies for binding to their respective flagellins. These data implicate terminal sialic acid as part of the LAH strain-specific epitopes. However, using mutants in genes affecting LAH serorecognition of flagellin it was demonstrated that sialic acid alone is not the LAH epitope. Rather, the epitope(s) is complex, probably involving multiple glycosyl and/or amino acid residues.

Campylobacter coli↗
PubMed1996

An environmentally regulated pilus-like appendage involved in Campylobacter pathogenesis.

Examination of strains of Campylobacter jejuni, Campylobacter coli, and Campylobacter fetus by electron microscopy revealed that they produced peritrichous pilus-like appendages when the bacteria were grown in the presence of bile salts. Various bile-salt supplements were used and it was found that deoxycholate and chenodeoxycholic acid caused a significant enhancement of pilus production and resulted in a highly aggregative phenotype. Morphologically, the pili were between 4 and 7 nm in width and were greater than 1 micron in length. A gene, termed pspA, which encodes a predicted protein resembling protease IV of Escherichia coli, was identified in C. jejuni strain 81-176. A site-specific insertional mutation within this gene resulted in the loss of pilus synthesis as determined by electron microscopy. Insertions upstream and downstream of the gene had no effect on pilus production. The non-piliated mutant of strain 81-176 showed no reduction in adherence to or invasion of INT 407 cells in vitro. However, this mutant, while still possessing the ability to colonize ferrets, caused significantly reduced disease symptoms in this animal model.

Amino Acid Sequence↗
PubMed1996

Restriction fragment length polymorphism of flagellin genes of Campylobacter jejuni and/or C. coli isolates from Egypt.

The conservation of flagellin genes from thermophilic Campylobacter spp. strains isolated in Egypt was evaluated by a restriction fragment length polymorphism (RFLP) assay. The flaA and flaB genes were amplified from 59 independent clinical isolates and digested with EcoRI and PstI, and the resulting patterns were compared with each other and with previously described RFLP groups. The results indicate that the isolates fell into 14 groups for flaA and 11 groups for flaB, 9 of which have been described, and that considerable genetic variability exists among isolates belonging to the same LIO serogroup. In most cases, the flaB gene displayed the same RFLP pattern as that of the flaA gene of the same strain, although some variability was observed. The data suggest that more variability of flagellin genes exists within the LIO serogroups common to Campylobacter field isolates from Egypt than has previously been reported for North American isolates.

Base Sequence↗