Sebastian Suerbaum

Recombination and clonal groupings within Helicobacter pylori from different geographical regions

A collection of 20 strains of Helicobacter pylori from several regions of the world was studied to better understand the population genetic structure and diversity of this species. Sequences of fragments from seven housekeeping genes (atpA, efp, mutY, ppa, trpC, ureI, yphC ) and two virulence‐associated genes (cagA, vacA) showed high levels of synonymous sequence variation (mean percentage Ks of 10–27%) and lower levels of non‐synonymous variation (mean percentage Ka of 0.2–5.6%). Cluster analysis of pairwise differences between alleles revealed the existence of two weakly clonal groupings, which included half of the strains investigated. All six strains isolated from Japanese and coastal Chinese were assigned to the ‘Asian’ clonal grouping, probably reflecting descent from a distinct common ancestor. The clonal groupings were not totally uniform; recombination, as measured by the homoplasy test and compatibility matrices, was extremely common within all genes tested, except cagA. The fact that clonal descent could still be discerned despite such frequent recombination possibly reflects founder effects and geographical separation and/or selection for particular alleles of these genes.

Cloning and genetic characterization of a Hellcobacter pylori flagellin gene

Helicobacter pylori produces polar sheathed flagella, which are believed to be essential for the bacterial colonization of the human gastric mucosa. Here we report on the cloning and genetic characterization of a H. pylori gene encoding the subunit of the flagellar filament, the flagellin. Screening of a genomic library of H. pylori with an oligonucleotide probe derived from the W‐terminal amino acid sequence of purified flagellin resulted in a recombinant plasmid clone carrying the flagellin‐encoding gene flaA on a 9.3 kb Bglll fragment. The nucleotide sequence of flaA revealed an open reading frame of 1530 nucleotides, encoding a protein with a predicted molecular mass of 53.2 kDa, which is similar in size with the purified flagellin protein in SDS‐potyacrylamide gel electrophoresis. Sequence alignment of H. pylori flagellin (FlaA) with other bacterial flagellins demonstrates a high degree of similarity in the amino‐terminal and carboxy‐terminal regions, including those of the closely related genus Campylobacter (56% overall identity with Campylobacter coli flaA), but little homology in the central domain. Southern hybridizations of chromosomal DNA with flaA‐specific probes did not reveal the presence of additional homologous flagellin genes in H. pylori. Sequence analysis of the flaA flanking regions and mapping of the flaA mRNA start site by a primer extension experiment indicated that transcription of the gene is under the control of a σ28‐specific promoter sequence in H. pylori. The region upstream of the flaA promoter is subject to local DNA modification, resulting in the masking of two out of three closely linked HindIII restriction sites in the chromosome of strain 898–1. Escherichia coli strains harbouring the recombinant plasmid did not produce full‐length flagellin and data obtained with FlaA fusion proteins using an E. coli plasmid expression system suggest that a distinct nucleotide sequence in the gene interferes with productive translation of this protein in E. coli.

Helicobacter pylori infection and polymorphisms in the tumor necrosis factor region.

Twin studies evidence that genetic factors of the host influence the acquisition and the clinical outcome of Helicobacter pylori infections in addition to bacterial and environmental factors. In the tumor necrosis factor (TNF) α‐gene, allelic frequencies of the polymorphic microsatellite TNFa and the promoter polymorphism TNF‐308 were studied for 209 H. pylori+ patients and compared to 184 H. pylori— controls. In the H. pylori+ group 34 individuals suffered from duodenal ulcer and 45 from gastric ulcer. Genotyping of the TNFa microsatellite and TNF‐308 polymorphisms was performed after polymerase chain reaction by polyacrylamide gel electrophoresis (PAGE) and allele‐specific oligonucleotide hybridizations, respectively. The phenotype frequency of microsatellite allele TNFa6 was lower in the H. pylori+ females as well as infected females with gastric ulcer compared to uninfected controls. Infected men with duodenal ulcer had a decreased frequency of allele TNFa10. The genotype TNF1/TNF1 of the polymorphism TNF‐308 is a risk factor for duodenal ulcer in H. pylori+ females; p = 0.01; relative risk (RR) = 10.7; corrected p‐value (pc) = 0.05. Thus, the TNF region is crucial in the complex genetic predisposition for H. pylori infection for certain patient subgroups.

Evolution of Helicobacter pylori: the role of recombination

New insight into the diversity of H. pylori has also come from recent studies using genomic approaches. In January 1999, H. pylori became the first bacterial species for which two complete genome sequences are publicly available18xAlm, R.A. et al. Nature. 1999; 397: 176–180Crossref | PubMed | Scopus (1403)See all References18. The genome of H. pylori strain J99 was sequenced by Genome Therapeutics, Inc., and the sequence was then acquired by the pharmaceutical company Astra. A research group at Astra Research Center, Boston, MA, USA, led by Richard Alm and Trevor Trust, completed the annotation and compared the genome of strain J99 with that of H. pylori 26695, the strain whose sequence was published by The Institute of Genomic Research (TIGR) in 1997 (Ref. 17xTomb, J-F. et al. Nature. 1997; 388: 539–547Crossref | PubMed | Scopus (2557)See all ReferencesRef. 17). Alm and coworkers18xAlm, R.A. et al. Nature. 1999; 397: 176–180Crossref | PubMed | Scopus (1403)See all References18 have now published the comparative analysis of these two genomes, and a comparative interactive database for both genomes is publicly available (http://www.astra-boston.com/hpylori/). This work reveals further details of the amazing diversity within H. pylori. A large part of the genome and proteome is highly conserved. However, ∼7% of the genes of J99 and 26695 are unique to each of these strains (89 and 117 genes, respectively). Almost half of these strain-specific genes are located in a single region of the chromosome (termed the ‘plasticity zone’). The authors note that in strain 26695, the plasticity zone contains sequences that have also been reported to be part of an H. pylori plasmid (pHPM186)19xSee all References19, suggesting that plasmids might play a role in the transfer of novel DNA into the H. pylori chromosome.Alm et al.18xAlm, R.A. et al. Nature. 1999; 397: 176–180Crossref | PubMed | Scopus (1403)See all References18 stress the degree of conservation of genome organization and protein sequences and conclude that the degree of diversity in H. pylori might have been overestimated by typing studies such as those cited above1xMajewski, S.I. and Goodwin, C.S. J. Infect. Dis. 1988; 157: 465–471Crossref | PubMed | Scopus (88)See all References, 2xAkopyanz, N. et al. Nucleic Acids Res. 1992; 20: 5137–5142Crossref | PubMed | Scopus (585)See all References, 3xKansau, I. et al. Res. Microbiol. 1996; 147: 661–669Crossref | PubMed | Scopus (98)See all References, 4xTaylor, D.E. et al. J. Bacteriol. 1992; 174: 6800–6806PubMedSee all References. However, while the fact that the majority of the proteins are conserved might be reassuring for vaccine development and the identification of novel targets for antibiotic therapy, the amount of DNA that is unique to each of the two strains is amazing. Currently, nothing is known about the distribution or functions of the strain-specific genes in different populations of H. pylori. An obvious question raised by these data is how many additional genes will be found in other strains. It seems unlikely that additional H. pylori genome sequences will become available in the immediate future. Other approaches, such as PCR-based subtractive hybridization methods (the application of one such method to H. pylori has recently been reported20xAkopyants, N.S. et al. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13108–13113Crossref | PubMed | Scopus (184)See all References20) or the use of high-throughput robotics, will facilitate addressing such genomic issues without the need to completely sequence additional genomes.

Cloning and allelic exchange mutagenesis of two flagellin genes of Helicobacter felis

Helicobacter felis has been used extensively in animal model studies of gastric Helicobacter infections. Attempts to manipulate H. felis genetically have, however, been unsuccessful and, consequently, little is known about the pathogenic mechanisms of this bacterium. In common with other Helicobacter spp., H. felis is a highly motile organism. To characterize the flagellar structures responsible for this motility, we cloned and sequenced the two flagellin‐encoding genes, flaA and flaB, from H. felis. These genes encode two flagellin proteins that are expressed simultaneously under the control of putative σ28 and σ54 promoters respectively. Isogenic mutants of H. felis in flaA and flaB were generated by electroporation‐mediated allelic disruption and replacement, showing for the first time that H. felis could be manipulated genetically. Both types of H. felis flagellin mutants exhibited truncated flagella and were poorly motile. H. felis flaA mutants were unable to colonize the gastric mucosa in a mouse infection model.

Virulence factors of Helicobacter pylori: implications for vaccine development.

Helicobacter pylori is one of the most common infectious diseases in humans and causes gastritis, peptic ulcer disease and malignant tumours of the stomach. This review discusses how H. pylori can colonize the human stomach, an ecological niche that is protected against all other bacteria. Knowledge about the virulence factors of H. pylori has accumulated rapidly over the last decade. Together with the information contained in the complete H. pylori genome sequence, this knowledge is now being applied in the search for a vaccine against this global pathogen.

Topoisomerase I of Helicobacter pylori: juxtaposition with a flagellin gene (flaB) and functional requirement of a fourth zinc finger motif

Cloning and nucleotide sequence analysis showed that in Helicobacter pylori the gene encoding topoisomerase I (topA) lies about 170 nucleotides upstream from flaB, a gene encoding one of the two flagellin proteins that is required for virulence. The topA and flaB genes are divergently transcribed. The orientation and spatial relationship between flaB and topA are remarkably conserved among strains of a bacterium in which genomic rearrangements are common. The deduced amino acid sequence of topoisomerase I revealed four zinc finger motifs, one more than has been reported previously for the Escherichia coli homologue. The additional motif, which is near the C-terminus of the protein, appears to be essential for function since mutations in that region are lethal. These data show that TopA proteins can be divided into several classes on the basis of zinc finger motifs and raise the interesting possibility that the H. pylori enzyme has local topological effects focussed on a flagellin gene.

Expression of capsular polysaccacharide determines serum resistance in Escherichia coli k92

The amount of capsular polysaccharide expression has been shown to be the major determinant of serum resistance in Escherichia coli K1. E. coli K92, like K1, is a polymer of sialic acid molecules. It differs from K1 by containing both alpha (2.8) and alpha (2.9) linkages. Four strains of E. coli K92 were tested for serum resistance. Three strains were serum-resistant (50% normal human serum), one strain was moderately serum-sensitive. The serum-resistant strains expressed significantly more capsular polysaccharide than did the serum-sensitive strain. For each of the serum-resistant strains, six mutants were isolated by selection for resistance against infection with a K92-specific bacteriophage. All of the mutants expressed less capsular polysaccharide than the respective wild-type strains. All mutants were more sensitive to serum killing than the wild-type strains. In all groups, the mutants with lowest expression of capsular polysaccharide were highly serum-sensitive. Changes of outer membrane proteins or lipopolysaccharide patterns that were present in some mutants did not correlate with serum resistance properties of the mutants. Furthermore, it was investigated whether the presence of active serum had an influence on capsule expression. In the serum-sensitive strain, the presence of serum induced a significant and concentration-dependent increase of capsule expression. Serum had no effect on capsule expression by the serum-resistant strains. We conclude from the data that the expression of K92 capsular polysaccharide determines serum resistance in the strains examined.

Influence of Antibiotics on the Cell Surface of Escherichia coli

Many studies have shown that various host-parasite interaction processes like adherence, phagocytosis, serum resistance, or immune response to outer membrane (OM) components can be altered by preincubation of bacteria in sub-minimal inhibitory concentrations (sub-MICs) of antibiotics [6, 18, 24, 26, 27, 29, 30]. Though all these effects appear to be cell surface mediated, only few authors have investigated the influence of antibiotics on cell surface properties. James [12] reported that mecillinam selectively increased the formation of certain OM proteins. Kadurugamuwa et al. [13, 14] and Taylor et al. [30] observed an influence of antibiotics on the production of capsular polysaccharides in Escherichia coli and Klebsiella pneumoniae.

The Influence of Subinhibitory Concentrations of Antibiotics on the Bacterial Surface with Respect to Host Defense Mechanisms

It is well known that a successful treatment of bacterial infections depends on an effective host defense system. This observation presupposes either that the antibiotic treatment reduces the number of bacteria in a given infection and that the remaining bacteria are subsequently eliminated by the host-defense system, or that antibiotics render bacteria more susceptible to host defense mechanisms. In addition, it is well established that β-lactam antibiotics interfere with peptidoglycan synthesis and that this is combined with changes in the shape of bacteria. Several authors (2,3,12) have pointed out that there is a mutual influence between peptidoglycan and membrane synthesis, but the arrangement and composition of the outer membrane under the influence of (β-lactam antibiotics have so far not been investigated adequately.