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T J Trust

Publications and source records attributed to T J Trust.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1995

Helicobacter pylori induces an increase in inositol phosphates in cultured epithelial cells.

Helicobacter pylori is a bacterial pathogen of humans that infects the gastric mucosa. This infection has been associated with gastritis, peptic ulcers, and gastric carcinomas. Diverse in vitro studies have described efficient adherence of H. pylori to different types of epithelial cells. Because of its varied effects on host cells, we have analysed signal transduction events in H. pylori-infected epithelial cells. Our results show that H. pylori induces an increase in inositol phosphates in all cultured epithelial cells used, including HeLa, Henle 407, Hep-2, and the human gastric adenocarcinoma cell line AGS. Bacterial growth medium supernatants induce a similar response in the host cell. The increase in inositol phosphates is not related to redistribution of cytoskeletal proteins such as actin or alpha-actinin nor tyrosine-phosphorylation of host cell proteins. The inositol phosphate increase is also observed in cells infected with low or non-adherent H. pylori mutants or mutants defective in the vacuolating toxin or urease holoenzyme. These results indicate that inositol phosphate release in H. pylori-infected cells is not dependent on bacterial adherence, and that a soluble bacterial factor, but not the vacuolating toxin or urease holoenzyme, mediates such an effect.

Cell Line↗
PubMed1995

Endogenous mutagenesis by an insertion sequence element identifies Aeromonas salmonicida AbcA as an ATP-binding cassette transport protein required for biogenesis of smooth lipopolysaccharide.

Analysis of an Aeromonas salmonicida A layer-deficient/O polysaccharide-deficient mutant carrying a Tn5 insertion in the structural gene for A protein (vapA) showed that the abcA gene immediately downstream of vapA had been interrupted by the endogenous insertion sequence element ISAS1. Immunoelectron microscopy showed that O polysaccharides did not accumulate at the inner membrane-cytoplasm interface of this mutant. abcA encodes an unusual protein; it carries both an amino-terminal ATP-binding cassette (ABC) domain showing high sequence similarity to ABC proteins implicated in the transport of certain capsular and O polysaccharides and a carboxyl-terminal potential DNA-binding domain, which distinguishes AbcA from other polysaccharide transport proteins in structural and evolutionary terms. The smooth lipopolysaccharide phenotype was restored by complementation with abcA but not by abcA carrying site-directed mutations in the sequence encoding the ATP-binding site of the protein. The genetic organization of the A. salmonicida ABC polysaccharide system differs from other bacteria. abcA also differs in apparently being required for both O-polysaccharide synthesis and in energizing the transport of O polysaccharides to the cell surface.

ATP-Binding Cassette Transporters↗
PubMed1995

Molecular analysis of an A-protein secretion mutant of Aeromonas salmonicida reveals a surface layer-specific protein secretion pathway.

The Aeromonas salmonicida Tn5 mutant, A449-TM1, is unable to secrete the surface layer protein (A-protein) through the outer membrane. Immunogold labeling of thin sections of A449-TM1, with polyclonal antisera against the A-protein, showed the accumulation of large quantities of A-protein in an enlarged periplasm. The majority of the labeled A-protein could be seen at the poles of the cells. The ability of A449-TM1 to secrete other extracellular proteins such as hemolysin and protease was not impaired by the Tn5 insertion, which indicates that the mutation in A449-TM1 interferes with a secretion pathway specifically for the translocation of the A-protein through the outer membrane. The mutant, A449-TM1, was shown to be avirulent for fish. A cosmid clone from a gene library of A449-TM1, which contains the Tn5 insertion from the chromosome, was used to identify a 1.4 kb SaII/ClaI fragment from immediately adjacent to the Tn5 insertion. This fragment was used to identify and clone a 4 kb HindIII fragment from a chromosomal DNA digest from the wild-type strain, A449. DNA sequence analysis of this clone identified an open reading frame (ORF) of 1656 bp. The deduced product of this ORF showed sequence similarity to a family of ATP-binding secretion proteins, but appeared to be phylogenetically distinct from these proteins, consistent with its participation in a secretory pathway specific for surface layer protein.

Aeromonas↗
PubMed1995

Tyrosine phosphorylation of the tetragonal paracrystalline array of Aeromonas hydrophila: molecular cloning and high-level expression of the S-layer protein gene.

High virulence strains of the fish pathogenic bacterium Aeromonas hydrophila produce a tetragonally arranged paracrystalline surface protein array (S-layer). The gene (ahsA) encoding the S-protein subunit of A. hydrophila TF7 was cloned into lambda EMBL 3, and sub-cloned into pUC 18. Transformation into Escherichia coli led to stable high-level expression of full-size S-protein under the control of its native promoter. The DNA sequence revealed a 1406 base-pair open reading frame encoding a protein consisting of a 19 amino acid residue signal peptide, and a 448 residue 45,400 Da molecular mass mature protein with a predicted isoelectric point (pI) of 6.72 compared with the measured M(r) of 52,000 and pI of 4.6. This suggested that the S-protein was post-translationally modified and in vivo cell labelling with [32P]orthophosphate, acid phosphatase digestion of S-protein, ascending thin-layer chromatography of partially acid hydrolysed S-protein and Western blot analysis with monoclonal anti-phosphotyrosine antibody showed that the S-protein of strain TF7 contained phosphotyrosine. S-proteins produced by the other strains of motile aeromonads tested also reacted with this anti-phosphotyrosine antibody. Cell fractionation studies employing plasmid-encoded ahsA showed that in A. hydrophila the S-protein subunits were secreted across the outer membrane by the native S-protein secretion pathway, while in E. coli and A. salmonicida the cloned A. hydrophila S-protein inserted into the outer membrane of the foreign host. These findings indicate that the process employed to translocate Aeromonas S-proteins across the outer membrane is highly specific.

Acid Phosphatase↗
PubMed1995

The leucine zipper of Aeromonas salmonicida AbcA is required for the transcriptional activation of the P2 promoter of the surface-layer structural gene, vapA, in Escherichia coli.

The Aeromonas salmonicida AbcA protein is involved in the synthesis of the O-polysaccharide side-chains on the lipopolysaccharide and is also capable of enhancing the expression of the structural gene for the A-layer, vapA, when cloned into Escherichia coli. The P2 promoter of the vapA gene of A. salmonicida was cloned into a promoter probe vector and expression in E. coli was monitored. The expression of P2::lacZ was shown to be increased when abcA was provided in trans. AbcA contains an N-terminal ATP-binding domain as well as a C-terminal leucine zipper domain. Site-directed mutagenesis has been used to show that the ATP-binding domain is required for the synthesis of the O-polysaccharide side-chains, but not for the enhancement of vapA expression. Conversely, the leucine zipper is needed for the increase in vapA expression, but not for O-polysaccharide side-chain synthesis. This indicates that AbcA is a bifunctional protein that can influence the synthesis of the two principle antigenic components of the A. salmonicida cell surface.

ATP-Binding Cassette Transporters↗
PubMed1995

Molecular characterization of an Aeromonas salmonicida mutant with altered surface morphology and increased systemic virulence.

The asoA gene of Aeromonas salmonicida is located approximately 7 kb downstream of the A-layer structural gene, vapA. A 6 kb BamHI fragment containing asoA was cloned and marker-exchange mutagenesis using a kanamycin-resistance cassette was performed to generate an asoA mutation in the low-virulence strain A449L. When analysed by electron microscopy, the mutant A449L-MB exhibited an altered surface morphology. Strands and blebs of membranous material were observed protruding from the disorganized cell surface. This material was shown to contain lipopolysaccharide and A-layer subunit protein. The disorganization of the surface of A449L-MB had no apparent effect on virulence when the bacteria were administered to rainbow trout (Oncorhynchus mykiss) by bath immersion. However, when administered by intraperitoneal injection, the mutant A449L-MB was found to exhibit significantly increased virulence. The predicted amino acid sequence of AsoA shows homology to a number of polytopic membrane proteins involved in translocation across the cytoplasmic membrane.

Aeromonas↗
PubMed1995

Recombinant infectious hematopoietic necrosis virus and viral hemorrhagic septicemia virus glycoprotein epitopes expressed in Aeromonas salmonicida induce protective immunity in rainbow trout (Oncorhynchus mykiss).

Fragments of the glycoprotein genes of viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) were cloned into a bacterial broad-host-range expression vector under the control of the plac promoter. Western blot (immunoblot) analysis with monoclonal antibodies specific to the glycoproteins demonstrated the inducible expression of the fusion proteins in Escherichia coli. Aeromonas salmonicida is the causative agent of furunculosis in salmonid fish. It was confirmed that an avirulent strain of A. salmonicida, A440, which contains a deletion in the structural gene for the paracrystalline surface protein array, will provide protective immunity against furunculosis when used as a live attenuated vaccine. The plasmid-encoded viral epitopes were then mobilized into A440 for use as a shuttle system for the expression of fragments of the glycoprotein genes of IHNV and VHSV. Vaccination of rainbow trout with A440 containing the viral epitopes resulted in the development of protective immunity against both VHSV and IHNV. This indicates that the use of cloned fragments of the glycoproteins and the use of A. salmonicida as a shuttle system constitute a feasible approach to fish vaccine development.

Aeromonas↗
PubMed1995

Isolation and characterization of a family of porin proteins from Helicobacter pylori.

Two-dimensional gel electrophoresis was used to identify heat-modifiable outer membrane proteins, which were candidates for porins, from Helicobacter pylori membrane preparations. Four such proteins with apparent molecular masses of 48, 49, 50, and 67 kDa were isolated. The four proteins copurified together after selective detergent solubilizations followed by anion-exchange chromatography, and each protein was ultimately purified to homogeneity by gel purification. These proteins were then tested for pore-forming ability with a planar lipid bilayer model membrane system. All four proteins appeared to be present as monomers, and they formed pores with low single-channel conductances in 1.0 M KCl of 0.36, 0.36, 0.30, and 0.25 nS, respectively, for the 48-, 49-, 50-, and 67-kDa proteins which we propose to designate HopA, HopB, HopC, and HopD. N-terminal amino acid sequence analyses showed a high degree of homology among all four proteins, and it appears that these proteins constitute a family of related porins in H. pylori.

Amino Acid Sequence↗
PubMed1995

Stimulation of interleukin-8 production in epithelial cell lines by Helicobacter pylori.

Following exposure to Helicobacter pylori cells, epithelial cell lines secreted interleukin-6 (IL-6) and IL-8 but not tumor necrosis factor alpha. Purified IL-6 alone did not stimulate IL-8 production from the cell lines tested, indicating that IL-6 was not an intermediary in IL-8 induction. Enhanced IL-8 secretion occurred in a time- and dose-dependent manner. None of 12 antibiotics tested exhibited a significant effect on IL-8-inducing activity, suggesting that preformed antigens were responsible for stimulating IL-8 secretion in vitro. Live bacterial cells caused the highest level of stimulation. Proteinase-digested and heated (56 or 100 degrees C) cells had significantly reduced stimulatory activities. Purified H. pylori lipopolysaccharide, but not exopolysaccharide, stimulated low-level secretion of IL-8, but only at high concentrations, while a water-extracted H. pylori antigen preparation was strongly stimulatory for HEp-2 cells. No reduction in IL-8-stimulatory activity was observed for H. pylori mutants negative for urease activity, production of a major lipoprotein, and motility. The noncytotoxic strain CCUG 915 stimulated lower IL-8 levels than other isolates. However, the otherwise isogenic cytotoxin-negative mutant 17874 delta vacA (S. H. Phadnis, D. Ilver, L. Janzon, S. Normark, and T. U. Westblom, Infect. Immun. 62:1557-1565, 1994) had the same IL-8-stimulatory ability as the parent strain, suggesting that surface proteins other than the vacuolating cytotoxin are involved in IL-8 stimulation.

Bacterial Proteins↗
PubMed1995

A conserved Aeromonas salmonicida porin provides protective immunity to rainbow trout.

A protein with an apparent M(r) of 28,000 was isolated from outer membrane preparations of Aeromonas salmonicida A440. The protein was tested for the ability to form pores, using a planar lipid bilayer model membrane system. The protein appeared to be a monomer with a single-channel conductance in 1.0 M KCl of 1.96 nS and a cation/anion permeability ratio of 2.91 +/- 0.68. These data show that the porin channel is comparable in size to OmpC and OmpF of Escherichia coli and is relatively nonselective, having some preference for cations over anions. The porin was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and a polyclonal antibody was raised. Immunoblot analysis showed that an immunologically cross-reactive protein was present in other Aeromonas strains but not in strains of Vibrio or Yersinia. The N-terminal amino acid sequence of the porin was determined and was found to show some homology to an Aeromonas hydrophila outer membrane protein. This is the second porin species of A. salmonicida to be described, and it differs from the other in subunit molecular weight, aggregation properties, peptidoglycan association, pore size, and antigenicity. Rainbow trout (Oncorhynchus mykiss) immunized intraperitoneally with the purified porin protein were significantly protected from experimental A. salmonicida challenge. This is the first report of successful vaccination against A. salmonicida with a purified outer membrane component.

Aeromonas↗
PubMed1995

A specific PulD homolog is required for the secretion of paracrystalline surface array subunits in Aeromonas hydrophila.

Aeromonas hydrophila is an important pathogen of fish, and its high-virulence strains display a two-dimensional paracrystalline layer (S-layer) on their outermost surfaces. The nucleotide sequence of a 4.1-kb region located 700 bp upstream of the A. hydrophila TF7 S-layer protein gene (ahsA) has been determined. A sequence analysis of the region revealed the presence of three complete open reading frames ending in a gene encoding a 79.8-kDa polypeptide that shows high homology to the PulD family of secretion proteins. The sequenced region displays both organizational and sequence homology to the Xanthomonas campestris pv. campestris Xps secretory system. Insertional inactivation of the spsD (S-protein secretion D) gene showed that the loss of expression of the PulD homolog coincided with the localization of the S-protein in the periplasm and the loss of the S-layer from the surface of the bacterium. However, the secretion of the enzymes hemolysin, amylase, and protease was unaffected in the mutant with the nonfunctional spsD gene, as was the export of flagella and fimbrial proteins. Southern blot analysis showed that the spsD gene was not conserved among all strains of S-protein-producing A. hydrophila or Aeromonas veronii biotype sobria. Use of the promoterless chloramphenicol acetyltransferase gene showed that unlike pulD and its homologs, spsD contains its own promoter. A. hydrophila has been shown to contain the exe operon, which is responsible for the secretion of a number of extracellular enzymes in this bacterium. A fragment of DNA was generated from the exeD gene of A. hydrophilia Ah65 by PCR and was subsequently used in hybridization studies to probe the chromosome of A. hydrophila TF7. The presence of an exeD homolog in A. hydrophila TF7 was found; therefore, the spsD gene encodes a second pulD homolog that displays a high specificity for the secretion of the S-protein. This gene appears to be part of a second terminal branch of the general secretory pathway in A. hydrophila.

Aeromonas hydrophila↗
PubMed1995

Isolation and characterization of a conserved porin protein from Helicobacter pylori.

Helicobacter pylori is a causative agent of gastritis in humans and is correlated with gastric ulcer formation. Infections with this bacterium have proven difficult to treat with antimicrobial agents. To better understand how this bacterium transports compounds such as antimicrobial agents across its outer membrane, identification of porin proteins is important. We have recently identified a family of H. pylori porins (HopA to HopD) (M. M. Exner, P. Doig, T. J. Trust, and R. E. W. Hancock, Infect. Immun. 63:1567-1572, 1995). Here, we report on an unrelated porin species (HopE) from this bacterium. This protein had a apparent molecular mass of 31 kDa and was seen to form 50- and 90-kDa aggregates that were designated putative dimeric and trimeric forms, respectively. The protein was purified to homogeneity and, with a model planar lipid membrane system, was shown to act as a nonselective pore with a single channel conductance in 1.0 M KCl of 1.5 nS, similarly to other bacterial nonspecific porins. An internal peptide sequence of HopE shared homology with the P2 porin of Haemophilus influenzae. HopE was also shown to be antigenic in vivo as assessed by sera taken from H. pylori-infected individuals and was immunologically conserved with both patient sera and specific monoclonal antibodies. From these data, it appears that HopE is a major nonselective porin of H. pylori. The implications of these findings are discussed.

Amino Acid Sequence↗
PubMed1995

The putative neuraminyllactose-binding hemagglutinin HpaA of Helicobacter pylori CCUG 17874 is a lipoprotein.

The ability of certain strains of Helicobacter pylori to cause sialic acid-sensitive agglutination of erythrocytes has been attributed to the HpaA protein (D.G. Evans, T.K. Karjalainen, D. J. Evans, Jr., D. Y. Graham, and C.H. Lee, J. Bacteriol. 175:674-683, 1993), the gene for which has been cloned and sequenced. On the basis of the hydropathy plot of HpaA and the presence of a potential lipoprotein signal sequence and modification site, and because of the similarities of these features with those of the cell envelope lipoprotein Lpp20 of H. pylori, we examined the possibility that HpaA was also a lipoprotein. Posttranslational processing of the HpaA protein expressed by the cloned gene was sensitive to globomycin, an inhibitor of the lipoprotein-specific signal peptidase II. Antibodies raised to the putative sialic acid-binding region of HpaA failed to bind to the surface of H. pylori cells in immunoelectron microscopy but instead were observed to have labeled the cytoplasm when thin sections were examined. This antibody recognized a 29,000-M(r) protein in Western blots (immunoblots) of cell extracts of H. pylori and Escherichia coli cells expressing the cloned hpaA gene. Determination of the sequence of hpaA from strain CCUG 17874 indicated significant differences from that determined by Evans and coworkers in the above-mentioned study, including extension of the gene into the open reading frame 3 downstream of hpaA to produce a protein with an M(r) of 26,414. Localization of HpaA indicated that it was predominantly located in the cytoplasmic fraction of the cell in both E. coli and H. pylori. HpaA was not observed in the sarkosyl-insoluble outer membrane fraction. An isogenic mutant generated by insertional inactivation of hpaA was unaffected in its ability to bind four different human cell lines as well as fixed sections of gastric tissue and had hemagglutination properties identical to those of the wild type. The data collectively suggest that HpaA is a nonessential lipoprotein internal to the H. pylori cell and that it is not involved in adhesion.

Adhesins, Bacterial↗
PubMed1994

Mutagenesis of the paracrystalline surface protein array of Aeromonas salmonicida by endogenous insertion elements.

The tetragonal paracrystalline surface protein array (A-layer) of the fish pathogenic bacterium Aeromonas salmonicida is a virulence factor and bacteria which are unable to produce A-layer are attenuated in their ability to kill fish. Ten independent mutants of Aeromonas salmonicida which were unable to produce A-layer were isolated by growth at 30 degrees C. These mutants displayed either reduced synthesis of the A-layer subunit, synthesis of a truncated subunit, or complete loss of the ability to produce the subunit protein. Restriction mapping and analysis by polymerase chain reaction showed that the mutations had resulted from insertion of two different insertion sequence (IS) elements into different sites in the A-layer subunit gene (vapA) and its promoter. Sequence comparisons indicated that ISAS1 is unique among reported IS elements. It is 1223 bp long with imperfect terminal inverted repeats of 22 bp and insertion resulted in a duplicated 8 bp target sequence in vapA. ISAS1 expressed a 42,000 molecular weight (M(r)) protein in mini-cells. ISAS2 was 1084 bp long, expressed proteins of M(r) 38,000 and 39,000 in vitro, had imperfect 29 bp terminal inverted repeats and had duplicated a 3 bp target sequence. Sequence comparisons indicated that ISAS2 was also unique to A. salmonicida; however, the proteins encoded by ISAS2 showed strong homology to the putative transposases encoded by the IS30 family of IS elements. Southern analyses showed that both ISAS1 and ISAS2 were restricted to A. salmonicida strains A449 and A450 where they were present in low copy number. The ability of these two IS elements to mutate the ability of A. salmonicida to produce its paracrystalline surface array provides a novel method for the attenuation of virulence.

Aeromonas↗
PubMed1994

Identification and molecular characterization of a major ring-forming surface protein from the gastric pathogen Helicobacter mustelae.

The spiral microaerophilic bacterium Helicobacter mustelae is linked to gastritis and gastric ulcers in ferrets. Electron microscopy of H. mustelae showed the presence of a laterally extensive array of 8.5-nm-diameter rings on the cell surface, which was shown to be composed of a 150kDa protein. This protein was purified, and the sequence of 10 amino-terminal residues was determined. Polyclonal antibody against the purified 150 kDa protein labelled the ring structures on the homologous strain by means of immunogold. Cross-reactive proteins were identified in three H. mustelae strains, but not in Helicobacter pylori or Helicobacter felis. The hsr gene encoding this protein was cloned, and the protein expressed in Escherichia coli independently of vector promoters. The 1519-codon nucleotide sequence of the gene was determined, and comparison with the chemically derived protein sequence indicated a 47-residue leader peptide, and a mature protein with a molecular weight of 152,300. Thus the cell surface of H. mustelae differs markedly from other members of the genus Helicobacter in being covered by an array of 8.5 nm rings composed of a 150kDa protein.

Amino Acid Sequence↗