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.

For 70 years antibiotics have saved countless lives and enabled the development of modern medicine, but it is becoming clear that the success of antibiotics may have only been temporary and we now anticipate a long-term, generational and perhaps never-ending challenge to find new therapies to combat antibiotic-resistant bacteria. As the search for new conventional antibiotics has become less productive and there are no clear strategies to improve success, a broader approach to address bacterial infection is needed. This review of potential alternatives to antibiotics (A2As) was commissioned by the Wellcome Trust, jointly funded by the Department of Health, and involved scientists and physicians from academia and industry. For the purpose of this review, A2As were defined as non-compound approaches (that is, products other than classical antibacterial agents) that target bacteria or approaches that target the host. In addition, the review was limited to agents that had potential to be administered orally, by inhalation or by injection for treatment of systemic/invasive infection. Within these criteria, the review has identified 19 A2A approaches now being actively progressed. The feasibility and potential clinical impact of each approach was considered. The most advanced approaches (and the only ones likely to deliver new treatments by 2025) are antibodies, probiotics, and vaccines now in Phase II and Phase III trials. These new agents will target infections caused by P. aeruginosa, C. difficile and S. aureus. However, other than probiotics for C. difficile, this first wave will likely best serve as adjunctive or preventive therapies. This suggests that conventional antibiotics will still be needed. The economics of pathogen-specific therapies must improve to encourage innovation, and greater investment into A2As with broad-spectrum activity (e.g. antimicrobial-, host defense-and, anti-biofilm peptides) is needed. Increased funding, estimated at >£1.5 bn over 10 years is required to validate and then develop these A2As. Investment needs to be partnered with translational expertise and targeted to support the validation of these approaches at Clinical Phase II proof of concept. Such an approach could transform our understanding of A2As as effective new therapies and should provide the catalyst required for both active engagement and investment by the pharma/biotech industry. Only a sustained, concerted and coordinated international effort will provide the solutions needed for the next decade.

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-Lglycero-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.

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.

Novel fimbriae were isolated and purified from the human enteropathogen SalmoneUa enteritidis 27655. These fimbriae were thin (measuring 3 to 4 nm in diameter), were extremely aggregative, and remained cell associated despite attempts to separate them from blended cells by centrifugation. The thin fimbriae were not solubilized in 5 M NaOH or in boiling 0.5% deoxycholate, 8 M urea, or 1 to 2% sodium dodecyl sulfate (SDS) with or without 5% 13-mercaptoethanol. Therefore, an unconventional purification procedure based on the removal of contaminating cell macromolecules in sonicated cell extracts by enzymatic digestion and preparative SDS-polyacrylamide gel electrophoresis (PAGE) was used. The insoluble fimbriae recovered from the well of the gel required depolymerization in formic acid prior to analysis by SDS-PAGE. Acid depolymerization revealed that the fimbriae were composed of fimbrin subunits, each with an apparent molecular mass of 17 kDa. Although their biochemical characteristics and amino acid composition were typical of fimbriae in general, these thin fimbriae were clearly distinct from other previously characterized fimbriae. Moreover, their fimbrin subunits had a unique N-terminal amino acid sequence. Native fimbriae on whole cells were specifically labeled with immune serum raised to the purified fimbriae. This immune serum also reacted with the denatured 17-kDa fimbrin protein in Western blots. The polyclonal immune serum did not cross-react with the other two native fimbrial types produced by this strain or with their respective fimbrins on Western blots (immunoblots). Therefore, these fimbriae represent the third fimbrial type produced by the enteropathogen S. enteritidis.

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.Although Campylobacter jejuni is one of the major causes of bacterial diarrhea worldwide (51, 59), the details of its molecular pathogenesis are not well understood. The clinical symptoms of campylobacter infection can range from a mild, watery diarrhea to a dysentery-like illness with fecal blood and leukocytes (2). Although there are reports of numerous cytotoxins, only the cytolethal distending toxin, which arrests eukaryotic cells at the G 2 phase of the cell cycle (64), has been characterized in detail. There are numerous reports that C. jejuni strains can invade intestinal epithelial cells in vitro (20,21,24,29,30,38), although levels of invasion by different strains vary considerably (20,28,38,52). Strain 81-176, originally isolated from a diarrheal outbreak associated with raw-milk consumption (31), is one of the best-characterized strains of C. jejuni. This strain has been shown to cause an inflammatory diarrhea in two human feeding studies (8; D. Tribble, unpublished data) and to cause disease in experimental models using primates (40) and ferrets (19,67). Further, C. jejuni strain 81-176 invades INT407 cells at levels higher than those of most other C. jejuni strains (28, 38).Plasmids have been found in between 19 and 53% of C. jejuni strains (5, 9-11, 41, 53-58), and many of these have been reported to be R plasmids that are transmissible among Campylobacter spp. but not to Escherichia coli (53)(54)(55)(56)(57)(58)60). Despite the importance of plasmids to virulence in numerous other pathogens, it is generally believed that plasmids play no role in Campylobacter pathogenicity. This paradigm is based on the rather low level at which plasmids ...

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.

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. The differences in adherence between the two classes of flagellar mutant suggest that flagellin can serve as a secondary adhesion, although other adhesins mediate a motility-dependent internalization process. Characterization of the mutants at the molecular level and in animal models should further contribute to our understanding of the pathogenicity of these organisms.