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S Suerbaum

Publications and source records attributed to S Suerbaum.

At least 37 records · Page 2Linked to original sources
PubMed1999

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 sigma28 and sigma54 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.

Alleles↗
PubMed1999

In vivo distribution of Helicobacter felis in the gastric mucus of the mouse: experimental method and results.

We describe a method that permits the collection of very small samples (2 nl) from precisely defined positions within the gastric mucus of anesthetized mice. This method was used to study the in vivo local distribution of bacteria within the mucus of Helicobacter felis-infected mice. A total of 200 samples from 40 mice were analyzed. Each sample was microscopically analyzed, within less than 1 min, as a native preparation. To avoid changes in bacterial location within the mucus after collection and to improve the counting accuracy, bacterial motility was blocked by adjusting the pH inside the collecting pipette to 4.5. The mucus in a collected sample was subdivided into three layers, an epithelial layer (the first 25 micron of mucus from the tissue-mucus interface), a luminal layer (the last 25 micron to the mucus-lumen interface), and the remaining central mucus layer. The volume of the analyzed segments in the sample was between 4 and 9 pl. The concentration of bacteria inside the epithelial mucus layer was 3,400 per nl, but it was only 50 per nl inside the central mucus layer. The mean distance of H. felis to the epithelial surface was 16 microm. A total of 75% of all H. felis bacteria resided in the mucus zone between 5 and 20 micron from the tissue surface, with no bacteria closer than 5 micron to the epithelial surface. This method permits the study of factors determining the density of colonization and distribution of bacteria along chemical gradients with a high precision.

Animals↗
PubMed1998

Free recombination within Helicobacter pylori.

Sequences of three gene fragments (flaA, flaB, and vacA) from Helicobacter pylori strains isolated from patients in Germany, Canada, and South Africa were analyzed for diversity and for linkage equilibrium by using the Homoplasy Test and compatibility matrices. Horizontal genetic exchange in H. pylori is so frequent that different loci and polymorphisms within each locus are all at linkage equilibrium. These results indicate that H. pylori is panmictic. Comparisons with sequences from Escherichia coli, Neisseria meningitidis, and Drosophila melanogaster showed that recombination in H. pylori was much more frequent than in other species. In contrast, when multiple family members infected with H. pylori were investigated, some strains were indistinguishable at all three loci. Thus, H. pylori is clonal over short time periods after natural transmission.

Alleles↗
PubMed1998

Green fluorescent protein as a novel marker and reporter system in Helicobacter sp.

In order to be able to study gene regulation in single, live Helicobacter pylori bacteria in vitro or in contact with host cells, we established the green fluorescent protein gene gfp from Aequorea victoria as a reporter gene for use with Helicobacter species. We describe here the construction of genomic transcriptional fusions of the promoterless gfp gene with the flaA and flaB promoters of H. pylori. We have also constructed a Mini-Tn3-km-gfp transposon to be used for shuttle transposon mutagenesis in H. pylori and H. mustelae. A marker strain with wild-type phenotype, carrying multiple plasmid-borne copies of gfp under the control of the H. pylori flaB promoter, was constructed for studies of bacterial distribution and transmission in animal models.

Artificial Gene Fusion↗
PubMed1998

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.

Bacterial Proteins↗
PubMed1998

Primary and acquired Helicobacter pylori resistance to clarithromycin, metronidazole, and amoxicillin--influence on treatment outcome.

OBJECTIVE: The aim of this study was to evaluate the primary and acquired resistance of H. pylori against clarithromycin, metronidazole, and amoxicillin, and to elucidate the consequential influence on H. pylori eradication. METHODS: A total of 195 patients with positive H. pylori status were consecutively included. In 172 patients, H. pylori could be cultured for evaluation of primary antibiotic resistance. Fifty patients received a 2-wk dual therapy with an acid inhibitor and amoxicillin 2,000 mg daily (A), the other 122 patients a 1-wk modified triple therapy with the acid inhibitor clarithromycin 500-1,000 mg daily, and metronidazole 1,000-1,500 mg daily (B: n = 78), or amoxicillin 2,000 mg daily and metronidazole 1,000 mg daily (C: n = 44), respectively. Acid inhibition was conducted with pantoprazole 40 mg b.i.d. (n = 62), omeprazole 20 mg b.i.d. (n = 50), lansoprazole 30 mg b.i.d. (n = 10), or ranitidine 150 mg t.i.d. (n = 50). After therapy, 36 patients remained H. pylori-positive, 20 after dual therapy (A) and 16 after modified triple therapy (B: n = 7, C: n = 9). In 32 of these patients, H. pylori could be recultured for evaluation of acquired resistance (A: n = 18, B: n = 7, C: n = 7). RESULTS: Primary H. pylori resistance to metronidazole was observed in 36 of 172 patients (21%) and to clarithromycin in three of 172 (2%). Acquired resistance was found in six of 14 (43%) and in two of seven (29%), respectively, whereas neither primary nor acquired H. pylori resistance to amoxicillin was noted. Patients infected with metronidazole resistant H. pylori strains were successfully treated in combination with clarithromycin (eight of nine vs 63 of 67 with sensitive strains, NS), but not with amoxicillin (one of eight vs 32 of 34 with sensitive strains, p < 0.0001). In two patients with acquired combined clarithromycin and metronidazole resistance, modified triple therapy failed. CONCLUSION: The value of modified triple therapy with amoxicillin and metronidazole is significantly limited by metronidazole resistance. However, metronidazole resistance does not negatively influence treatment outcome in modified triple therapy including clarithromycin. H. pylori resistance to amoxicillin still is not present.

Amoxicillin↗
PubMed1998

Helicobacter pylori induces apoptosis in mucosal lymphocytes in patients with gastritis.

BACKGROUND: Previous studies suggest that Helicobacter pylori (H. pylori) induces apoptosis and compensatory hyperproliferation in gastric epithelial cells possibly explaining the carcinogenic capacity of the bacteria. The aim of this study was to measure the effect of H. pylori on apoptosis of gastric lymphoid cells in view of the development of gastric lymphoma. METHODS: 16 H. pylori-positive and 19 H. pylori-negative individuals were enrolled. Single cell suspensions were prepared from antral biopsies and apoptosis was measured by staining with the TUNEL-assay and the fluorochrome Hoechst 33342. Lymphocyte subsets were simultaneously identified by immunocytochemistry. RESULTS: The apoptotic index of all gastric mucosal cells was significantly higher in H. pylori-positive mucosa compared to negative controls. Additionally, H. pylori-infected patients showed a significant increase in apoptosis of mucosal B-lymphocytes. Apoptosis of T cells and plasma cells was unaffected by H. pylori. CONCLUSION: H. pylori induces apoptosis in mucosal B cells which might be important in the development of gastritis and possibly B-cell lymphoma of the mucosa-associated lymphoid tissue (MALT).

Adult↗
PubMed1997

Infection of the ferret stomach by isogenic flagellar mutant strains of Helicobacter mustelae.

Helicobacter mustelae, like Helicobacter pylori, possesses two flagellin proteins, FlaA and FlaB. Isogenic mutant strains of H. mustelae have been constructed by disruption of the flaA or flaB gene with a kanamycin resistance cassette or by introduction of both a kanamycin and a chloramphenicol resistance gene to produce a double mutant. To determine whether one or both flagellin proteins are necessary for colonization and persistence of infection with H. mustelae, 19 ferrets, specific pathogen free for H. mustelae, were given either the HMF1 flaA::km (weakly motile), ATCC 43772 flaB::km (moderately motile), or HMF1 flaA::cat flaB::km (non-motile) mutant strain, the wild-type parent strains, or sterile broth. Gastric tissue samples were obtained during sequential gastric biopsies beginning at 3 weeks postinoculation and ending at necropsy at 3 months postinoculation. H. mustelae infection status was determined by culture, histology, and serology. The wild-type parent strains of H. mustelae infected all ferrets at all time points. The double-mutant strain was unable to colonize; the flaA and flaB single-mutant strains were able to initially colonize at a low level and establish persistent infection with increasing numbers of organisms over time. The severity of gastritis produced by infection with these strains of H. mustelae correlated with the number of organisms present in the gastric mucosa. Flagellar motility is an important virulence factor for colonization and pathogenesis in the H. mustelae ferret model.

Animals↗
PubMed1997

Cloning and characterization of the Helicobacter pylori flbA gene, which codes for a membrane protein involved in coordinated expression of flagellar genes.

Flagellar motility has been shown to be an essential requirement for the ability of Helicobacter pylori to colonize the gastric mucosa. While some flagellar structural components have been studied in molecular detail, nothing was known about factors that play a role in the regulation of flagellar biogenesis. We have cloned and characterized an H. pylori homolog (named flbA) of the lcrD/flbF family of genes. Many proteins encoded by these genes are known to be involved in flagellar biogenesis or secretion of virulence-associated proteins via type III secretion systems. The H. pylori flbA gene (2,196 bp) is capable of coding for a predicted 732-amino-acid, 80.9-kDa protein that has marked sequence similarity with other known members of the LcrD/FlbF protein family. An isogenic strain with a mutation in the flbA gene was constructed by disruption of the gene with a kanamycin resistance cassette and electroporation-mediated allelic exchange mutagenesis. The mutant strain expressed neither the FlaA nor the FlaB flagellin protein. The expression of the FlgE hook protein was reduced in comparison with the wild-type strain, and the extent of this reduction was growth phase dependent. The flbA gene disruption was shown to downregulate the expression of these flagellar genes on the transcriptional level. The flbA mutants were aflagellate and completely nonmotile. Occasionally, assembled hook structures could be observed, indicating that export of axial flagellar filament components was still possible in the absence of the flbA gene product. The hydrophilic part of the FlbA protein was expressed in Escherichia coli, purified, and used to raise a polyclonal rabbit antiserum against the FlbA protein. Western blot experiments with this antiserum indicated that the FlbA protein is predominantly associated with the cytoplasmic membrane in H. pylori. The antiserum cross-reacted with two other proteins (97 and 43 kDa) whose expression was not affected by the flbA gene disruption and which might represent further H. pylori homologs of the LcrD/FlbF protein family.

Amino Acid Sequence↗
PubMed1996

Colonization of gnotobiotic piglets by Helicobacter pylori deficient in two flagellin genes.

Helicobacterpylori possesses two flagellin molecules, MA, the major species, and FlaB, which is expressed in minor amounts. This study sought to determine if one or both flagellin species are necessary for colonization or persistence by H. pylon. Thirty-six gnotobiotic piglets from six litters were given one of four isogenic strains of H. pylon orally. The bacterial strains used were strain N6, the wild type, which produced both FlaA and FlaB and was fully motile; N6flaB::km, which produced FlaA but not FlaB and was weakly motile; N6flaA::km, which expressed FlaB but not FlaA and was nonmotile; and N6flaA::cat/flaB::km, which produced neither flagellin and was nonmotile. Strain N6 colonized all piglets and persisted for 2, 4, and 10 days after inoculation. Both N6flaA::km and N6flaB::km colonized for 2 and 4 but not 10 days, and colonization was weak. N6flaA::cat/flaB:: km colonized for 2 days but did not persist for 4 or 10 days after inoculation. These findings demonstrate that both flagellin species are necessary for full colonization by H. pylon. Colonization for up to 4 days is possible in the absence of either flagellin species but not both.

Animals↗
PubMed1995

Construction and characterization of an isogenic urease-negative mutant of Helicobacter mustelae.

Helicobacter mustelae infects the ferret stomach and provides an opportunity to study pathogenic determinants of a Helicobacter species in its natural host. We constructed an isogenic urease-negative mutant of H. mustelae which produced no detectable urease and showed a reduced acid tolerance. This mutant provides an opportunity to further evaluate the role of urease in the pathogenesis of Helicobacter infection.

Amino Acid Sequence↗
PubMed1995

Comparative ultrastructural and functional studies of Helicobacter pylori and Helicobacter mustelae flagellin mutants: both flagellin subunits, FlaA and FlaB, are necessary for full motility in Helicobacter species.

Helicobacter mustelae causes chronic gastritis and ulcer disease in ferrets. It is therefore considered an important animal model of human Helicobacter pylori infection. High motility even in a viscous environment is one of the common virulence determinants of Helicobacter species. Their sheathed flagella contain a complex filament that is composed of two distinctly different flagellin subunits, FlaA and FlaB, that are coexpressed in different amounts. Here, we report the cloning and sequence determination of the flaA gene of H. mustelae NCTC12032 from a PCR amplification product. The FlaA protein has a calculated molecular mass of 53 kDa and is 73% homologous to the H. pylori FlaA subunit. Isogenic flaA and flaB mutants of H. mustelae F1 were constructed by means of reverse genetics. A method was established to generate double mutants (flaA flaB) of H. mustelae F1 as well as H. pylori N6. Genotypes, motility properties, and morphologies of the H. mustelae flagellin mutants were determined and compared with those of the H. pylori flaA and flaB mutants described previously. The flagellar organizations of the two Helicobacter species proved to be highly similar. When the flaB genes were disrupted, motility decreased by 30 to 40%. flaA mutants retained weak motility by comparison with strains that were devoid of both flagellin subunits. Weakly positive motility tests of the flaA mutants correlated with the existence of short truncated flagella. In H. mustelae, lateral as well as polar flagella were present in the truncated form. flaA flaB double mutants were completely nonmotile and lacked any form of flagella. These results show that the presence of both flagellin subunits is necessary for complete motility of Helicobacter species. The importance of this flagellar organization for the ability of the bacteria to colonize the gastric mucosa and to persist in the gastric mucus remains to be proven.

Amino Acid Sequence↗
PubMed1994

Expression of capsular polysaccharide 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.

Bacterial Capsules↗
PubMed1994

Helicobacter pylori hspA-hspB heat-shock gene cluster: nucleotide sequence, expression, putative function and immunogenicity.

All Helicobacter pylori isolates synthesize a 54 kDa immunodominant protein that was reported to be associated with the nickel-dependent urease of H. pylori. This protein was recently recognized as a homologue of the heat-shock protein of the GroEL class. The gene encoding the GroEL-like protein of H. pylori (HspB) was cloned (pILL689) and was shown to belong to a bicistronic operon including the hspA and hspB genes. In Escherichia coli, the constitutive expression of the hspA and hspB genes was initiated from a promoter located within an IS5 insertion element that mapped upstream to the two open reading frames (ORFs). IS5 was absent from the H. pylori genome, and was thus acquired during the cosmid cloning process. hspA and hspB encoded polypeptides of 118 and 545 amino acid residues, corresponding to calculated molecular masses of 13.0 and 58.2 kDa, respectively. Amino acid sequence comparison studies revealed that, although H. pylori HspA and HspB proteins were highly similar to their bacterial homologues, the H. pylori HspA featured a striking motif at the C-terminus. This unique motif consists of a series of cysteine and histidine residues resembling a nickel-binding domain, which is not present in any of the other bacterial GroES homologues so far characterized. When the pILL689 recombinant plasmid was introduced together with the H. pylori urease gene cluster (pILL763) into an E. coli host strain, an increase of urease activity was observed. This suggested a close interaction between the HspA and HspB proteins and the urease enzyme, and a possible role for HspA in the chelation of nickel ions. The genes encoding each of the HspA and HspB polypeptides were cloned, expressed independently as proteins fused to the maltose-binding protein (MBP) and purified in large scale. The MBP-HspA and MBP-HspB fusion proteins were shown to retain their antigenic properties. Both HspA and HspB represent antigens that are specifically recognized by the sera from H. pylori-infected patients. Whereas HspB was known to be immunogenic in humans, this is the first demonstration that HspA per se is also immunogenic.

Amino Acid Sequence↗
PubMed1994

[Helicobacter pylori and molecular biology--virulence, diagnosis and vaccine development].

The discovery of H. pylori as the etiologic agent of chronic antral type B gastritis and the finding that H.pylori is involved in the pathogenesis of gastroduodenal ulcer disease and gastric carcinoma have triggered intensive research about this organism. After overcoming considerable initial difficulties, researchers succeeded in adapting the instruments of molecular biology to the study of this highly fastidious organism. This has led to a rapid increase of knowledge concerning the basis of H.pylori virulence (as well as that of the related animal pathogens H. mustelae and H. felis) and to the development of molecular methods for the purposes of diagnosis and epidemiological research. The latest application of molecular biology in this area is the use of recombinant proteins for the development of an H.pylori vaccine. This review gives an overview of this rapidly developing field.

Bacterial Vaccines↗