Trevor J. Trust

Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori

Helicobacter pylori, one of the most common bacterial pathogens of humans, colonizes the gastric mucosa, where it appears to persist throughout the hosts 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 organisms 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.

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 DH5α 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.

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

ABSTRACT 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 atetO 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 pyloriproteins, 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 ofC. jejuni pathogens.

Comparative Genomics of Helicobacter pylori: Analysis of the Outer Membrane Protein Families

ABSTRACT 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. pylori26695. 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 independentH. pylori isolates. While one pair was present in every strain examined, one allele of the other pair appeared partially deleted in several isolates.

Isolation of motile and non-motile insertional mutants of Campylobacter jejuni: the role of motility in adherence and invasion of eukaryotic cells.

A method of insertional mutagenesis for naturally transformable organisms has been adapted from Haemophilus influenzae and applied to the study of the pathogenesis of Campylobacter jejuni. A series of kanamycin‐resistant Insertional mutants of C. jejuni 81–176 has been generated and screened for loss of ability to invade INT407 cells. Eight noninvasive mutants were identified which showed 18‐200‐fold reductions in the level of invasion compared with the parent. Three of these eight show defects in motility, and five are fully motile. The three mutants with motility defects were further characterized to evaluate the method. One mutant, K2–32, which is non‐adherent and non‐invasive, has an insertion of the kanamycin‐resistance cassette into the flaA flagellin gene and has greatly reduced motility and a truncated flagellar filament typical of flaA mutants. The adherent non‐invasive mutants K2–37 and K2–55 are phenotypically paralysed, i.e. they have a full‐length flagellar filament but are non‐motile. All three mutants show an aberration in flagellar structure at the point at which the filament attaches to the cell. Mutants K2–37 and K2–55 represent overlapping deletions affecting the same gene, termed pflA (paralysed flagella). This gene encodes a predicted protein of 788 amino acid residues and a molecular weight of 90 977 with no significant homology to known proteins. Site‐specific insertional mutants into this open reading frame result in the same paralysed flagellar phenotype and the same invasion defects as the original mutants.

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.

DNA sequence and mutational analyses of the pVir plasmid of Campylobacter jejuni 81-176.

ABSTRACT The circular pVir plasmid of Campylobacter jejuni strain 81-176 was determined to be 37,468 nucleotides in length with a G+C content of 26%. A total of 83% of the plasmid represented coding information, and all but 2 of the 54 predicted open reading frames were encoded on the same DNA strand. There were seven genes on the plasmid in a continguous region of 8.9 kb that encoded orthologs of type IV secretion proteins found in Helicobacter pylori, including four that have been described previously (D. J. Bacon, R. A. Alm, D. H. Burr, L. Hu, D. J. Kopecko, C. P. Ewing, T. J. Trust, and P. Guerry, Infect. Immun. 68:4384-4390, 2000). There were seven other pVir-encoded proteins that showed significant similarities to proteins encoded by the plasticity zones of either H. pylori J99 or 26695. Mutational analyses of 19 plasmid genes identified 5 additional genes that affect in vitro invasion of intestinal epithelial cells. These included one additional gene encoding a component of a type IV secretion system, an ortholog of Cj0041 from the chromosome of C. jejuni NCTC 11168, two Campylobacter plasmid-specific genes, and an ortholog of HP0996 from the plasticity zone of H. pylori 26695.

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 Mr 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 Mr 26 598 with significant homology to CMP‐N‐acetylneuraminic acid synthetase enyzmes 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 Mr 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.

Non‐motile mutants of Helicobacter pylori and Helicobacter mustelae defective in flagellar hook production

Flagellar hooks were purified from Helicobacter pylori and Helicobacter mustelae. The 70 × 16nm H. pylori hook was composed of FIgE subunits of 78kDa, while the 72 × 16nm H. mustelae hook was composed of 87kDa subunits. N‐terminal sequence was obtained for the FIgH proteins of both species, and for an internal H. mustelae FlgE peptide. Degenerate oligonucleotide primers allowed amplification of a 1.2 kb fragment from the H. mustelae chromosome, which carried part of the flgE gene. The corresponding H. pylori gene was cloned by immunoscreening of a genomic library constructed in λZAP Express, The translated H. pylori flgE sequence indicated a protein with limited homology with the hook proteins from Salmonella typhimurium and Treponema phagedenis. Mutants of H. pylori and H. mustelae defective in hook production generated by allele replacement were non‐motile and devoid of flagellar filaments but produced both flagellin subunits, which were localized in the soluble fraction of the cell. The level of flagellin production was unchanged in the mutants, indicating that the regulation of flagellin expression in Helicobacter differs from that in the Enterobacteriaceae.

Cell surface hydrophobicity and macrophage association ofAeromonas salmonicida

Hydrophobic interaction chromatography and salt aggregation were used to compare the call surface hydrophobicity of strains of the fish pathogenAeromonas salmonicida which differed in their ability to produce the surface protein array known as A-layer. Presence of this superficial protein layer is crucial to the virulence of this organism and was found to coincide with a dramatic increase in cell surface hydrophobicity. Assays with in vitro cultured macrophages from either rainbow trout or mice revealed that this hydrophobic A-layer providedA. salmonicida cells with an enhanced ability to associate with phagocytic monocytes. This enhanced association was demonstrated in the absence of opsonizing antibody and may have important implications in the virulence ofA. salmonicida for fish.