ABSTRACT
Helicobacter mustelae, the gastric pathogen of ferrets, produces an array of surface ring structures which have not been described for any other member of the genus Helicobacter, including H. pylori. The unique ring structures are composed of a protein named Hsr. To investigate whether the Hsr rings are important for colonization of the ferret stomach, ferrets specific pathogen free for H. mustelae were inoculated with an Hsr-deficient mutant strain or the wild-type H. mustelae strain. Quantitative cultures from antral biopsy specimens obtained at 3, 6, and 9 weeks postinoculation demonstrated no significant difference in the levels of bacteria in the ferrets that received the Hsr-negative strain and the ferrets infected with the parent strain. However, when the ferrets were biopsied at 12 and 15 weeks and necropsied at 18 weeks after infection, the levels of bacteria of the Hsr-negative strain in the stomach antrum were significantly reduced. This decline contrasted the robust antral colonization by the wild-type strain. The Hsr-negative strain did not efficiently colonize the gastric body of the study ferrets. Histological examination at 18 weeks postinoculation revealed minimal gastric inflammation in the animals that received the mutant H. mustelae strain, a finding consistent with its waning infection status, whereas lesions characteristic of helicobacter infection were present in ferrets infected with the wild-type strain. Scant colonization by the Hsr-negative H. mustelae strain at the end of the 18-week study, despite initial successful colonization, indicates an inability of the mutant to persist, perhaps due to a specific host response.
Subject(s)
Bacterial Proteins/physiology , Ferrets/microbiology , Helicobacter/physiology , Stomach/microbiology , Animals , Bacterial Proteins/chemistry , Helicobacter/chemistry , Helicobacter Infections/pathology , Mutation , Stomach/pathologyABSTRACT
Helicobacter pylori induces chronic superficial gastritis which in some patients may lead to peptic ulcer disease, while a subset of infected individuals develop gastric cancer or gastric lymphoma. Consensus guidelines recommend that patients with a known H. pylori infection receive eradication treatment. Successful treatment requires that antibiotics be used in combination with acid suppressants or bismuth, and although the list of effective antibacterials is short, regimens such as amoxicillin and clarithromycin or metronidazole and clarithromycin with the proton pump inhibitor omeprazole have achieved eradication rates of approximately 90% in trials. However lower eradication rates are probably more common, and strains resistant to clarithromycin or metronidazole, or both, are of concern. Stable amoxycillin resistance has also been reported. Efforts are underway to discover and develop novel therapeutics, both H. pylori specific antibacterial drugs and a therapeutic vaccine. Impetus to these efforts has been provided by the availability of the genome sequences of two different H. pylori isolates. In the case of drug discovery, a genome-based strategy facilitates the expeditious selection of novel lethal targets not used by today's antibiotics, providing the opportunity to identify novel classes of antibacterials. Vaccine discovery and development has largely focused on a small number of antigens selected by conventional means. Recent reports that mucosal and serum antibody titers do not appear to be essential for protection against H. pylori in murine models suggest that that a wider range of H. pylori proteins than those previously considered may be able to induce protective immunity. Progress towards development of new H. pylori therapeutics is discussed.
Subject(s)
Helicobacter Infections/therapy , Helicobacter pylori , Antigens, Bacterial , Bacterial Vaccines/therapeutic use , Forecasting , HumansABSTRACT
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.
Subject(s)
Campylobacter jejuni/chemistry , Flagellin/chemistry , Glycopeptides/analysis , Amino Acid Sequence , Glycosylation , Mass Spectrometry , Molecular Sequence DataABSTRACT
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.
Subject(s)
Campylobacter jejuni/pathogenicity , Plasmids/genetics , Virulence Factors , Animals , Bacterial Adhesion , Bacterial Proteins/genetics , Conjugation, Genetic , Diarrhea , Female , Ferrets , Molecular Sequence Data , Mutagenesis, Insertional , Mutagenesis, Site-Directed , Open Reading Frames , R Factors/genetics , Sequence Analysis, DNA , Species Specificity , Tetracycline Resistance/genetics , Transformation, BacterialABSTRACT
The two complete genomic sequences of Helicobacter pylori J99 and 26695 were used to compare the paralogous families (related genes within one genome, likely to have related function) of genes predicted to encode outer membrane proteins which were present in each strain. We identified five paralogous gene families ranging in size from 3 to 33 members; two of these families contained members specific for either H. pylori J99 or H. pylori 26695. Most orthologous protein pairs (equivalent genes between two genomes, same function) shared considerable identity between the two strains. The unusual set of outer membrane proteins and the specialized outer membrane may be a reflection of the adaptation of H. pylori to the unique gastric environment where it is found. One subfamily of proteins, which contains both channel-forming and adhesin molecules, is extremely highly related at the sequence level and has likely arisen due to ancestral gene duplication. In addition, the largest paralogous family contained two essentially identical pairs of genes in both strains. The presence and genomic organization of these two pairs of duplicated genes were analyzed in a panel of independent H. pylori isolates. While one pair was present in every strain examined, one allele of the other pair appeared partially deleted in several isolates.
Subject(s)
Bacterial Outer Membrane Proteins/genetics , Genome, Bacterial , Helicobacter pylori/genetics , Alleles , Amino Acid Sequence , Base Sequence , Binding Sites/genetics , Conserved Sequence , DNA Primers/genetics , Evolution, Molecular , Gene Duplication , Helicobacter pylori/classification , Helicobacter pylori/metabolism , Humans , Molecular Sequence Data , Multigene Family , Phylogeny , Protein Sorting Signals/genetics , Ribosomes/metabolism , Sequence Homology, Amino Acid , Species SpecificityABSTRACT
We previously demonstrated (M. M. Exner, P. Doig, T. J. Trust, and R. E. W. Hancock, Infect. Immun. 63:1567-1572, 1995) that Helicobacter pylori has at least one nonspecific porin, HopE, which has a low abundance in the outer membrane but forms large channels. H. pylori is relatively susceptible to most antimicrobial agents but less susceptible to the polycationic antibiotic polymyxin B. We demonstrate here that H. pylori is able to take up higher basal levels of the hydrophobic fluorescent probe 1-N-phenylnaphthylamine (NPN) than Pseudomonas aeruginosa or Escherichia coli, consistent with its enhanced susceptibility to hydrophobic agents. Addition of polymyxin B led to a further increase in NPN uptake, indicative of a self-promoted uptake pathway, but it required a much higher amount of polymyxin B to yield a 50% increase in NPN uptake in H. pylori (6 to 8 microg/ml) than in P. aeruginosa or E. coli (0.3 to 0.5 microg/ml), suggesting that H. pylori has a less efficient self-promoted uptake pathway. Since intrinsic resistance involves the collaboration of restricted outer membrane permeability and secondary defense mechanisms, such as periplasmic beta-lactamase (which H. pylori lacks) or efflux, we examined the possible role of efflux in antibiotic susceptibility. We had previously identified in H. pylori 11637 the presence of portions of three genes with homology to potential restriction-nodulation-division (RND) efflux systems. It was confirmed that H. pylori contained only these three putative RND efflux systems, named here hefABC, hefDEF, and hefGHI, and that the hefGHI system was expressed only in vivo while the two other RND systems were expressed both in vivo and in vitro. In uptake studies, there was no observable energy-dependent tetracycline, chloramphenicol, or NPN efflux activity in H. pylori. Independent mutagenesis of the three putative RND efflux operons in the chromosome of H. pylori had no effect on the in vitro susceptibility of H. pylori to 19 antibiotics. These results, in contrast to what is observed in E. coli, P. aeruginosa, and other clinically important gram-negative bacteria, suggest that active efflux does not play a role in the intrinsic resistance of H. pylori to antibiotics.
Subject(s)
Anti-Bacterial Agents/pharmacology , Genes, Bacterial , Helicobacter pylori/drug effects , 1-Naphthylamine/analogs & derivatives , 1-Naphthylamine/metabolism , Anti-Bacterial Agents/metabolism , Biological Transport , Chloramphenicol/metabolism , Chloramphenicol/pharmacology , Fluorescent Dyes/metabolism , Helicobacter pylori/genetics , Helicobacter pylori/metabolism , Humans , Microbial Sensitivity Tests , Mutagenesis , Operon , Tetracycline/metabolism , Tetracycline/pharmacology , TritiumABSTRACT
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.
Subject(s)
Bacterial Vaccines/immunology , Campylobacter jejuni/immunology , Flagellin/immunology , Peptide Fragments/immunology , Recombinant Fusion Proteins/immunology , Vaccines, Synthetic/immunology , Amino Acid Sequence , Animals , Flagellin/chemistry , Humans , Immunization , Mice , Mice, Inbred BALB C , Molecular Sequence DataABSTRACT
Helicobacter pylori is a gram-negative bacteria which colonizes the gastric mucosa of humans and is implicated in a wide range of gastroduodenal diseases. This paper reviews the physiology of this bacterium as predicted from the sequenced genomes of two unrelated strains and reconciles these predictions with the literature. In general, the predicted capabilities are in good agreement with reported experimental observations. H. pylori is limited in carbohydrate utilization and will use amino acids, for which it has transporter systems, as sources of carbon. Energy can be generated by fermentation, and the bacterium possesses components necessary for both aerobic and anaerobic respiration. Sulfur metabolism is limited, whereas nitrogen metabolism is extensive. There is active uptake of DNA via transformation and ample restriction-modification activities. The cell contains numerous outer membrane proteins, some of which are porins or involved in iron uptake. Some of these outer membrane proteins and the lipopolysaccharide may be regulated by a slipped-strand repair mechanism which probably results in phase variation and plays a role in colonization. In contrast to a commonly held belief that H. pylori is a very diverse species, few differences were predicted in the physiology of these two unrelated strains, indicating that host and environmental factors probably play a significant role in the outcome of H. pylori-related disease.
Subject(s)
Bacterial Proteins/metabolism , Genome, Bacterial , Helicobacter Infections/microbiology , Helicobacter pylori/physiology , Bacterial Proteins/genetics , Energy Metabolism , Genes, Bacterial , Helicobacter pylori/genetics , HumansABSTRACT
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.
Subject(s)
Bacterial Proteins/metabolism , Campylobacter jejuni/genetics , Campylobacter jejuni/metabolism , Membrane Proteins/metabolism , Antigens, Bacterial/immunology , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Campylobacter jejuni/immunology , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Enzyme-Linked Immunosorbent Assay , Flagellin/genetics , Flagellin/metabolism , Genes, Bacterial , Glycosylation , Immunoblotting , Lipopolysaccharides/immunology , Lipopolysaccharides/isolation & purification , Lipopolysaccharides/metabolism , Membrane Proteins/genetics , Membrane Proteins/immunology , Molecular Sequence Data , Mutation , Plasmids/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Sequence Analysis, DNA , Wheat Germ Agglutinins/metabolismABSTRACT
Helicobacter pylori, one of the most common bacterial pathogens of humans, colonizes the gastric mucosa, where it appears to persist throughout the host's life unless the patient is treated. Colonization induces chronic gastric inflammation which can progress to a variety of diseases, ranging in severity from superficial gastritis and peptic ulcer to gastric cancer and mucosal-associated lymphoma. Strain-specific genetic diversity has been proposed to be involved in the organism's ability to cause different diseases or even be beneficial to the infected host and to participate in the lifelong chronicity of infection. Here we compare the complete genomic sequences of two unrelated H. pylori isolates. This is, to our knowledge, the first such genomic comparison. H. pylori was believed to exhibit a large degree of genomic and allelic diversity, but we find that the overall genomic organization, gene order and predicted proteomes (sets of proteins encoded by the genomes) of the two strains are quite similar. Between 6 to 7% of the genes are specific to each strain, with almost half of these genes being clustered in a single hypervariable region.
Subject(s)
Genome, Bacterial , Helicobacter pylori/genetics , Duodenal Ulcer/microbiology , Gene Expression Regulation, Bacterial , Helicobacter Infections/microbiology , Helicobacter pylori/classification , Humans , Molecular Sequence Data , Sequence Alignment , Sequence Analysis, DNA , Species SpecificityABSTRACT
Infection with Helicobacter pylori has been linked to numerous severe gastroduodenal diseases including peptic ulcer and gastric cancer. Several techniques have been used to measure the genetic heterogeneity of H. pylori at several different levels and to determine whether there is any correlation with severity of disease. The availability of two completed genome sequences from unrelated strains (J99 and 26,695) has allowed an analysis of the level of diversity from a large-scale yet detailed perspective. Although the two chromosomes are organized differently in a limited number of discrete regions, the genome size and gene order of these two "high-virulence" (cagA+ and vacA+) H. pylori isolates was found to be highly similar. The regions of organizational difference are associated with insertion sequences, DNA restriction/modification genes, repeat sequences, or a combination of the above. A significant level of variation at the nucleotide level is seen across the genome, providing an explanation for why the nucleotide-based typing techniques have such high discriminatory power among independent H. pylori isolates. This nucleotide variation together with the organizational rearrangements appears to have provided an over-estimation of the gene order diversity of H. pylori as assessed by pulse-field gel electrophoresis. Functional assignments are assigned to approximately only 60% of the gene products in each strain, with one-half of the remaining gene products of unknown function having homologues in other bacteria, while the remainder appear to be H. pylori-specific. Between 6% and 7% of the coding capacity of each strain are genes that are absent from the other strain, with almost one-half of these strain-specific genes located in a single hypervariable region called the plasticity zone. The majority of the strain-specific genes in each strain are also H. pylori-specific, with no homologues being identified in the public databases. Significantly, over one-half of the functionally assigned strain-specific genes in both H. pylori J99 and 26695 encode DNA restriction/modification enzymes. Analysis of the level of conservation between orthologues from the two strains indicates that the H. pylori specific genes have a lower level of conservation than those orthologues to which a putative function can be assigned. The plasticity zone represents one of several regions across each genome that is comprised of lower (G+C)% content DNA, some of which has been detected in self-replicating plasmids, suggesting that both horizontal transfer from other species and plasmid integration are responsible for the strain-specific diversity at this locus. These analyses have yielded results with important implications for understanding the genetic diversity of H. pylori and its associated diseases, and imply a need to reassess the respective roles of bacterial and host factors in H. pylori associated diseases.
Subject(s)
Genetic Variation , Genome, Bacterial , Helicobacter pylori/genetics , Genes, Bacterial , Helicobacter Infections/genetics , Helicobacter Infections/microbiology , Humans , PhenotypeABSTRACT
Two tandemly located flagellin genes, flaA and flaB, with 79% nucleotide sequence identity were identified in Aeromonas salmonicida A449. The fla genes are conserved in typical and atypical strains of A. salmonicida, and they display significant divergence at the nucleotide level from the fla genes of the motile species Aeromonas hydrophila and Aeromonas veronii biotype sobria. flaA and flaB encode unprocessed flagellins with predicted Mrs of 32,351 and 32,056, respectively. When cloned under the control of the Ptac promoter, flaB was highly expressed when induced in Escherichia coli DH5alpha, and the FlaB protein was detectable even in the uninduced state. In flaA clones containing intact upstream sequence, FlaA was barely detectable when uninduced and poorly expressed on induction. The A. salmonicida flagellins are antigenically cross-reactive with the A. hydrophila TF7 flagellin(s) and evolutionarily closely related to the flagellins of Pseudomonas aeruginosa and Vibrio anguillarum. Electron microscopy showed that A. salmonicida A449 expresses unsheathed polar flagella at an extremely low frequency under normal laboratory growth conditions, suggesting the presence of a full complement of genes whose products are required to make flagella; e.g., immediately downstream of flaA and flaB are open reading frames encoding FlaG and FlaH homologs.
Subject(s)
Aeromonas/genetics , Flagellin/genetics , Genes, Bacterial/genetics , Aeromonas/immunology , Amino Acid Sequence , Antibodies, Bacterial , Cloning, Molecular , Cross Reactions , DNA, Bacterial/genetics , Flagella/genetics , Flagella/ultrastructure , Gene Expression Regulation, Bacterial/genetics , Molecular Sequence Data , Open Reading Frames/genetics , Phylogeny , Restriction Mapping , Sequence Analysis , Sequence Analysis, DNA , Sequence Homology, Amino AcidABSTRACT
The S-layers of the Aeromonas spp. studied to date are composed of identical protein subunits which are translocated across the cytoplasmic membrane, periplasm and outer membrane to the cell surface, where they are assembled and tethered to the cell via an interaction with the O-polysaccharide side chains of the lipopolysaccharide. Aeromonas S-layers have the ability to bind a number of host factors such as fibronectin, laminin and vitronectin as well as providing resistance to serum killing and protease digestion. Aeromonas mutants unable to produce an S-layer are altered in their ability to cause disease. In the case of Aeromonas salmonicida, the loss of ability to produce an S-layer effectively abolishes virulence. However, in the case of A. hydrophila, the reduction in virulence caused by the loss of the S-layer is less significant.
Subject(s)
Aeromonas hydrophila/metabolism , Aeromonas hydrophila/pathogenicity , Bacterial Outer Membrane Proteins/biosynthesis , Bacterial Outer Membrane Proteins/metabolism , Aeromonas hydrophila/chemistry , Crystallization , VirulenceABSTRACT
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.
Subject(s)
Bacterial Proteins/genetics , Campylobacter coli/genetics , DNA-Binding Proteins , DNA-Directed RNA Polymerases/physiology , Flagella , Gene Expression Regulation, Bacterial , Sigma Factor/physiology , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA, Bacterial , Flagellin , Genes, Bacterial , Molecular Sequence Data , Mutagenesis, Site-Directed , Promoter Regions, Genetic , RNA Polymerase Sigma 54 , Rabbits , Sequence Homology, Amino AcidABSTRACT
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.
Subject(s)
Campylobacter/genetics , Genes, Bacterial , Iron/metabolism , Amino Acid Sequence , Campylobacter/metabolism , Chromosome Mapping , DNA, Bacterial/analysis , Molecular Sequence Data , Mutagenesis, Site-Directed , Nucleic Acid Hybridization , Open Reading FramesABSTRACT
Despite its clinical significance, relatively little is known about the components of Helicobacter pylori that allow it to colonize, persist, and elicit an inflammatory response within the host. Bacterial surface components frequently influence colonization and persistence of a pathogen, as well as the disease process (1). In the case of H. pylori, one macromolecular assembled protein component that is unequivocally located on the surface of the bacterium is the sheathed flagellum. Two other proteins that can be isolated in abundance from suspensions of H. pylori cells, but that are normally regarded as intracellular in other bacterial species, are urease and a GroEL analog, Hp60K (2-6). Whether these large macromolecular assemblies are surface proteins on H. pylori or are released as a result of cellular lysis is uncertain. However, numerous other intracellular proteins can be readily isolated from culture supernatants after mild shearing and extraction procedures, and these released proteins have initially been incorrectly interpreted as representing surface proteins (7-9).
ABSTRACT
The Aeromonas salmonicida A449 recA gene has been cloned, sequenced and expressed in vitro. The predicted amino acid sequence of A. salmonicida RecA was determined and, when compared to other RecA, was found to possess a number of domains identical to those characterized in Escherichia coli RecA. The A. salmonicida recA was mobilized into an E. coli recA mutant strain and was shown to allow increased survival in the presence of the chemical mutagen MMS and after ultraviolet (UV) irradiation. The A. salmonicida recA also possesses a potential regulatory SOS box in the DNA 5' of the gene. The rate of A. salmonicida-mediated recombination in E. coli was increased by exposure to UV light, which suggests that SOS induction in A. salmonicida parallels that of E. coli.
Subject(s)
Aeromonas/genetics , Escherichia coli/genetics , Rec A Recombinases/genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Escherichia coli/radiation effects , Gene Expression Regulation, Bacterial/genetics , Genetic Complementation Test , Molecular Sequence Data , Rec A Recombinases/isolation & purification , Rec A Recombinases/metabolismABSTRACT
The asoB gene of Aeromonas salmonicida is located approximately 9 kb downstream of the structural gene (vapA) for the surface layer (A-layer). The nucleotide sequence of asoB was determined and found to encode a putative polytopic cytoplasmic membrane protein which exhibited homology to a number of bacterial transport proteins. Allele exchange mutagenesis of asoB resulted in a mutant (A449-D) which was avirulent when administered by bath immersion. However, when administered by intraperitoneal injection, A449-D is as lethal as wild type. Characterization of the phenotype of A449-D showed that there were pleiotropic effects on VapA secretion, haemolysis and outer membrane protein composition. Mobilization of cloned asoB on a broad-host-range plasmid into A449-D resulted in the complementation of VapA translocation, haemolytic activity and virulence.
Subject(s)
Aeromonas/genetics , Antigens, Bacterial , Bacterial Outer Membrane Proteins/genetics , Genes, Bacterial/genetics , Gram-Negative Bacterial Infections/veterinary , Oncorhynchus mykiss/microbiology , Open Reading Frames/genetics , Aeromonas/pathogenicity , Amino Acid Sequence , Animals , Bacterial Outer Membrane Proteins/chemistry , Bacterial Proteins/genetics , Base Sequence , Gram-Negative Bacterial Infections/genetics , Gram-Negative Bacterial Infections/microbiology , Membrane Proteins/genetics , Molecular Sequence Data , Mutagenesis, Insertional , Open Reading Frames/physiology , Virulence/geneticsABSTRACT
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.
Subject(s)
Campylobacter/pathogenicity , Escherichia coli Proteins , Fimbriae, Bacterial/genetics , Serine Endopeptidases , Amino Acid Sequence , Animals , Bacterial Outer Membrane Proteins/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bile Acids and Salts/pharmacology , Campylobacter/ultrastructure , Chromosomes, Bacterial/chemistry , Cloning, Molecular , DNA Probes/metabolism , Disease Models, Animal , Female , Ferrets , Fimbriae Proteins , Fimbriae, Bacterial/ultrastructure , Heat-Shock Proteins/chemistry , Microscopy, Electron , Molecular Sequence Data , Molecular Weight , Open Reading Frames , Peptide Hydrolases/chemistry , Sequence AlignmentABSTRACT
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.