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S Suerbaum

Publications and source records attributed to S Suerbaum.

48 records · Page 3Linked to original sources
PubMed1993

Ultrastructure and biochemical studies of the flagellar sheath of Helicobacter pylori.

Helicobacter pylori flagellar sheaths were isolated by sucrose density-gradient centrifugation and analysed by electronmicroscopy, SDS-PAGE and gas-liquid chromatography. Electronmicroscopy of thin sections of flagella showed an internal electron-dense filament and a surrounding flagellar sheath with the typical bilayer structure of a membrane. The flagellar filaments could be disintegrated by acid treatment and the resulting isolated flagellar sheaths formed vesicles, sometimes with characteristic structures. Centrifugation of flagellar preparations after acid treatment resulted in the enrichment of flagellar sheaths in the pellet. SDS-PAGE analysis of the pellet showed a reduction of the flagellin band and a number of protein bands of 150, 76, 67, 65, 53, 51, 49, 29.5, 18, 17 and 16 kDa. However, there were no major protein bands characteristic for the sheath. Differences between the protein profiles of Sarkosyl-insoluble membranes and flagellar sheaths appeared in the lower M(r) range of 30-14 kDa. Major fatty acids of isolated flagellar sheaths were C 14:0, C 19:0 cyc, C 18:0, and the LPS-specific fatty acids 3-OH C 16:0 and 3-OH C 18:0. The results demonstrate that the flagellar sheaths of H. pylori are membranes and contain LPS and proteins.

Centrifugation, Density Gradient↗
PubMed1993

Cloning and genetic characterization of the Helicobacter pylori and Helicobacter mustelae flaB flagellin genes and construction of H. pylori flaA- and flaB-negative mutants by electroporation-mediated allelic exchange.

Helicobacter pylori is one of the most common human pathogens. It causes chronic gastritis and is involved in the pathogenesis of gastroduodenal ulcer disease and possibly gastric carcinoma. Helicobacter mustelae is a bacterium closely related to H. pylori that causes gastritis and ulcer disease in ferrets and is therefore considered an important animal model of gastric Helicobacter infections. Motility, even in a viscous environment, is conferred to the bacteria by several sheathed flagella and is regarded as one of their principal virulence factors. The flagellar filament of H. pylori consists of two different flagellin species expressed in different amounts. The gene (flaA) encoding the major flagellin has recently been cloned and sequenced. Here we report the cloning and sequencing of two highly homologous new flagellin genes from H. pylori 85P and H. mustelae NCTC 12032. The nucleotide sequence of the H. pylori gene proved that it encoded the second flagellin molecule found in H. pylori flagellar filaments. The genes were named flaB. The H. mustelae and H. pylori flaB genes both coded for proteins with 514 amino acids and molecular masses of 54.0 and 53.9 kDa, respectively. The proteins shared 81.7% identical amino acids. The degree of conservation between H. pylori FlaB and the H. pylori FlaA major flagellin was much lower (58%). Both flaB genes were preceded by sigma 54-like promoter sequences. Mapping of the transcription start site for the H. pylori flaB gene by a primer extension experiment confirmed the functional activity of the sigma 54 promoter. To evaluate the importance of both genes for motility, flaA- and flaB-disrupted mutants of H. pylori N6 were constructed by electroporation-mediated allelic exchange and characterized by Western blot (immunoblot) analysis and motility testing. Both mutations selectively abolished the expression of the targeted gene without affecting the synthesis of the other flagellin molecule. Whereas flaA mutants were completely nonmotile, flaB mutants retained motility.

Amino Acid Sequence↗
PubMed1992

Cloning and genetic characterization of a Helicobacter 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 N-terminal amino acid sequence of purified flagellin resulted in a recombinant plasmid clone carrying the flagellin-encoding gene flaA on a 9.3 kb Bg/II 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-polyacrylamide 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 sigma 28-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.

Amino Acid Sequence↗
PubMed1992

Biochemical studies of Helicobacter mustelae fatty acid composition and flagella.

The fatty acid compositions of Helicobacter mustelae whole cells, isolated phospholipids, and isolated lipopolysaccharides were analyzed by gas-liquid chromatography. Major phospholipid fatty acids were C16:0, C18:0, C18:1, and C19:0 cyc. In isolated lipopolysaccharides, 3-OH-C16:0, 3-OH-C14:0, C14:0, C16:0, and C18:0 were found. The lipid composition of H. mustelae thus showed pronounced differences from that of H. pylori. Flagella were purified by mechanical shearing and centrifugation steps. In all H. mustelae strains, the flagellin had an apparent molecular mass of 53 kDa and was thus the same size as H. pylori flagellin. The flagellin of strain NCTC 12032 was further purified and subjected to N-terminal amino acid sequence analysis. The first 10 amino acids were identical to those of H. pylori flagellin, but the next 5 were different. Significant homology was also found with flagellins of other bacteria.

Amino Acid Sequence↗
PubMed1990

Influence of beta-lactam antibiotics on serum resistance of K1-positive blood culture isolates of Escherichia coli.

The K1-positive strains of Escherichia coli are a group with considerable clinical importance, serum resistance being a common virulence factor of these strains. In the present paper, the influences of cephaloridine, imipenem, and ceftazidime on the serum resistance of eight serum-resistant K1-positive E. coli blood culture isolates with smooth-type lipopolysaccharide were studied. All strains were rendered more serum sensitive by treatment with subinhibitory concentrations of antibiotics. The amount of the reduction of serum resistance was dependent on the concentration of the antibiotic. Amounts of K1 produced under the influence of the antibiotics were measured and were found to be reduced for almost all strains tested. To further test the hypothesis that antibiotic-induced reduction of serum resistance is mediated by inhibition of K1 expression, isogenic mutants of one strain were produced by selection for resistance against infection with K1-specific bacteriophages. These mutants were found to be highly serum sensitive. We conclude from this study that beta-lactam antibiotics can render K1-positive serum-resistant strains of E. coli highly serum sensitive and that this effect is mediated by inhibition of K1 expression.

Ceftazidime↗
PubMed1990

The capsular polysaccharide is a major determinant of serum resistance in K-1-positive blood culture isolates of Escherichia coli.

Serum resistance is a major virulence factor of gram-negative bacteria, and K-1 polysaccharide has been shown to contribute to serum resistance in selected strains. To obtain further information about the role of K-1 in serum resistance and to find out whether loss of the ability to produce K-1 can induce loss of serum resistance, we studied the serum resistance of mutants derived from completely serum-resistant, K-1-positive blood culture isolates of Escherichia coli by selection for resistance to infection with K-1 specific bacteriophages. The amounts of K-1 polysaccharide produced by wild-type strains and mutants were measured, and outer membrane protein and lipopolysaccharide (LPS) patterns were analyzed. In each group of mutants, several highly serum-sensitive strains were found. All mutant strains expressed less K-1 than did the corresponding wild-type strains. Mutants that became highly serum sensitive always had less K-1 than did mutants with less-pronounced changes of serum resistance. A few mutants derived from different wild-type strains showed increased expression of outer membrane proteins with molecular weights of about 46,000 and 67,000. All of the wild-type strains examined had smooth-type LPS, and only two mutants had altered LPS structures; alterations of mutants in outer membrane proteins and LPS could not be correlated with alterations of serum resistance. The results indicate that for K-1-positive blood culture strains of E. coli, K-1 expression is a prerequisite for serum resistance, and loss of ability to synthesize K-1 leads to loss of serum resistance.

Antigens, Bacterial↗
PubMed1990

Unusual fatty acid substitution in lipids and lipopolysaccharides of Helicobacter pylori.

Cellular fatty acids, phospholipid fatty acids, and lipopolysaccharide fatty acids of four strains of Helicobacter pylori were analyzed by gas-liquid chromatography. The presence of myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, 19-carbon cyclopropane fatty acid, beta-hydroxypalmitic acid, and beta-hydroxystearic acid was confirmed. In phospholipids, myristic acid and 19-carbon cyclopropane fatty acid were the major fatty acids. Hydroxy fatty acids and unsaturated fatty acids were not detected or occurred only in small amounts. The major fatty acids of lipopolysaccharides were stearic acid, beta-hydroxypalmitic acid, and beta-hydroxystearic acid. Unsaturated fatty acids and 19-carbon cyclopropane fatty acid were not found. The unusual compositions of H. pylori phospholipid and lipopolysaccharide fatty acids may have important implications for the taxonomy, physicochemical membrane properties, and biological activity of lipopolysaccharides.

Campylobacter↗
PubMed1989

Ultrastructure and chemical analysis of Campylobacter pylori flagella.

Flagella of Campylobacter pylori were analyzed by electron microscopy and purified, and the molecular weight of the flagellin was determined. Isolation of flagella was performed by mechanical shearing from the cell surface, sucrose density gradient centrifugation, and Sepharose CL-4B gel chromatography. The flagella of C. pylori differ from those of other Campylobacter species and of most other bacteria by the presence of a flagellar sheath. The sheath narrows at the end and is linked to a club-shaped terminal structure. The molecular weight of C. pylori flagellin was 51,000.

Campylobacter↗
PubMed1987

Influence of beta-lactam antibiotics and ciprofloxacin on cell envelope of Escherichia coli.

The effects of subinhibitory concentrations of different beta-lactam antibiotics and one quinolone on the quantitative composition of the outer membrane (OM) of two strains of Escherichia coli, on lipid translocation into the OM, and on the production of capsular K1 polysaccharide were studied. The phospholipid/amino acid ratio was reduced in almost all OM preparations from antibiotic-treated bacteria. In one strain, antibiotic treatment increased the lipopolysaccharide/amino acid ratio. The amount of peptidoglycan fragments bound to the OM was increased by all the antibiotics. In pulse-chase experiments with a radioactive lipid precursor, ciprofloxacin, imipenem, and aztreonam inhibited phospholipid translocation into the OM. Furthermore, imipenem, cephaloridine, and ciprofloxacin induced a pronounced reduction of the production of capsular K1 polysaccharide. Thus, antibiotics seem to induce marked changes of the quantitative composition of the cell envelope of E. coli. Possible connections of these data with findings on the influence of antibiotics on functional parameters of the host-parasite relationship such as OM immunogenicity and serum resistance are discussed.

Amino Acids↗
PubMed1986

Influence of beta-lactam antibiotics and ciprofloxacin on composition and immunogenicity of Escherichia coli outer membrane.

The effects of subinhibitory concentrations of different beta-lactam antibiotics and one quinolone on the sedimentation of outer membranes (OMs) of Escherichia coli and on the qualitative properties and immunogenicity of OM components were studied. Membranes were prepared by osmotic lysis of plasmolyzed bacteria. OM and cytoplasmic membrane vesicles were separated by sucrose density ultracentrifugation. Two peaks of OM vesicles with different buoyant densities could be isolated; the quantitative contribution of these to the total OM varied, depending upon the growth phase. In early log phase, the OM consisted mainly of lighter material; in late log and stationary phases, the OM consisted mainly of heavier material. Moxalactam, imipenem, and ciprofloxacin inhibited the formation of heavier material in all growth phases. The immunogenicity of OM vesicles was tested in mice by the hemolytic plaque test. The lighter OM material was markedly less immunogenic than the heavier OM material. The vesicles from antibiotic-treated bacteria and those from early-log-phase cells were less immunogenic than vesicles from untreated late-log-phase and stationary-phase bacteria. These changes were found for the immune response against lipopolysaccharides, as well as against OM proteins. Thus, the immunogenicity of OM components seems to be dependent upon the quantitative composition of lighter and heavier compounds, which is strongly influenced by growth phase and treatment with certain antibiotics.

Animals↗
PubMedDate not supplied

Exacerbation of Whipple's disease associated with infliximab treatment.

A 34-year-old man with chronic inflammatory joint disease and recurrent fever over 6 years was diagnosed as having Still's disease. Treatment with corticosteroids and azathioprine was ineffective. Therefore, infliximab/ methotrexate was started. The patient subsequently developed a wasting disease with rapid weight loss, erythema nodosum, diarrhoea, progressive lymph node enlargement, and a sigmoido-vesical fistula. Histological analysis of several enlarged lymph nodes, the margins of the fistula, and the small bowel established the diagnosis of Whipple's disease (WD). The presence of Tropheryma whipplei (Tw) DNA in the tissues was confirmed by polymerase chain reaction (PCR). Careful re-evaluation of biopsies taken from the ileum and the liver 2 years earlier, which at that time was not judged to be diagnostic for WD, retrospectively showed subtle histological signs of WD and were positive for Tw DNA. In summary, infliximab treatment seems to increase the risk of exacerbation of WD. WD should be carefully ruled out prior to application of tumour necrosis factor-alpha (TNF-alpha) blockade.

Adult↗