Showing papers by "Martin J. Blaser published in 1992"

Campylobacter Jejuni: Current Status and Future Trends

Abstract: Clinical and Epidemiologic Aspects Reservoirs and Antimicrobial Resistance Clinical Microbiology Pathogenesis of Campylobacter Infections Immune Responses and Antigenic Analysis Molecular Pathogenesis

Helicobacter pylori and gastroduodenal disease.

Abstract: Helicobacter pylori infection is now recognized as the primary cause of active chronic gastritis in humans. Most infected persons remain asymptomatic, but are at increased risk for the development of peptic ulcer disease and possibly gastric cancer. The pathogenesis of this infection is not well understood, but motility and urease activity are virulence factors in an animal model. The eradication of H. pylori infection is associated with resolution of gastritis and a decreased rate of duodenal ulcer recurrence.

Helicobacter pylori: its role in disease.

Abstract: Since its discovery in 1982, Helicobacter (formerly Campylobacter) pylori has been shown to be an important pathogen of humans. This bacterium colonizes the stomach for years or decades, not days or weeks, as we usually expect for bacterial pathogens. Unlike Mycobacteriuini tuberculosis, which persists in infected hosts for extended periods but is mostly in a dormant or latent state, H. pylori causes continuous inflammation. It is this longevity and persistent lowgrade gastric inflammation that suggest that H. pylori should be considered the prototypic "slow" bacterium. In this review, I will provide a brief overview of the microbiology, epidemiology, and pathogenesis of H. pylori infection, topics that have been recently reviewed elsewhere [1, 2]. Subsequently, I will outline the relationship of this organism to peptic ulcer disease and gastric cancer, two important clinical consequences of infection, and then concentrate on a clinical approach to H. pylori infection.

Potentiation of Helicobacter pylori vacuolating toxin activity by nicotine and other weak bases.

Abstract: About 50% of Helicobacter pylori isolates produce a vacuolating toxin in vitro, which may be an important determinant of virulence. Because ammonium salts potentiate H. pylori toxin activity, the effect of other weak bases upon toxin activity was determined. Vacuolation of HeLa cells was quantitated using a neutral red uptake assay. As expected, ammonium chloride, trimethylamine, triethanolamine, and nicotine each induced vacuolation of HeLa cells when tested independently. In addition, each of these weak bases potentiated H. pylori vacuolating toxin activity, whereas sodium chloride or sodium hydroxide did not. Sequential incubation of cells with toxin followed by nicotine resulted in potentiation of vacuolation, whereas sequential incubation in the reverse order did not lead to potentiation. Monensin inhibited the formation of vacuoles by either H. pylori vacuolating toxin or nicotine. The potentiation of H. pylori toxin activity by ammonia and nicotine may contribute to gastroduodenal mucosal injury associated with this infection.

Characteristics of Helicobacter pylori variants selected for urease deficiency.

Abstract: The urease of Helicobacter pylori is suspected to play a role in the pathogenesis of gastritis. Although all clinical isolates of H. pylori are urease positive (U+), we have selected and characterized several spontaneously arising urease-negative (U-) variants from wild-type strain 60190. Urease-negative variants were identified by growth in medium containing 60 mM urea and arose at a frequency of 10(-5) to 10(-6). The urease activity of the wild-type strain inhibited growth of this strain in the presence of 60 mM urea. U- variants retained the U- phenotype for more than 100 passages on medium with or without urea. The urease activities of the original U+ and derived U- cells were 9.55 to 16.7 and 0.01 to 0.17 U/mg of protein, respectively. Colonial growth and other biochemical characteristics were identical for the strains. U- variants showed three classes of whole-cell sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles: (i) identical to U+; (ii) change in the migration of the 61-kDa urease subunit; and (iii) lack of 61- and 30-kDa subunits. These differences were confirmed by immunoblotting and by protein separation using fast protein liquid chromatography. The U+ strain but not U- variants tolerated exposure to pH 4.0 for 60 min in the presence of urea. Supernatants of the U+ strain and U- variants contained vacuolating cytotoxin activity for HeLa cells in similar titers. By enzyme-linked immunosorbent assay, human serum samples recognized water extract from the U+ strain significantly better than extract from a U- variant lacking urease subunits. In conclusion, this study demonstrates that U- H. pylori variants may arise spontaneously, that urease activity enhances survival at acid pH, and that urease and cytotoxin activities are disparate phenotypes. Images

Serum neutralizing antibody response to the vacuolating cytotoxin of Helicobacter pylori

Abstract: Approximately 50% of Helicobacter pylori isolates produce a cytotoxin in vitro that induces vacuolation of eukaryotic cells. To determine the in vivo relevance of this phenomenon, we sought to detect cytotoxin-neutralizing antibodies in sera from H. pylori-infected persons. As a group, sera from 29 H. pylori-infected patients neutralized the activity of the purified cytotoxin to a significantly greater extent than sera from 24 uninfected persons (P = 0.007). The cytotoxin neutralizing activity in sera from H. pylori-infected persons was mediated predominantly by the purified IgG fraction. Sera from H. pylori-infected persons neutralized the cytotoxins produced by multiple H. pylori strains, but failed to neutralize trimethylamine-induced cell vacuolation. Neutralization of cytotoxin activity by human or immune rabbit sera was associated with immunoblot IgG recognition of an 87-kD H. pylori protein. Similarly, neutralization of the toxin by sera was associated with IgG recognition of the purified cytotoxin in an enzyme-linked immunosorbent assay (P less than 0.0001). The presence of cytotoxin-neutralizing antibodies in sera from H. pylori-infected persons indicates that the cytotoxin is synthesized in vivo.

Reattachment of surface array proteins to Campylobacter fetus cells

Abstract: Campylobacter fetus strains may be of serotype A or B, a property associated with lipopolysaccharide (LPS) structure. Wild-type C. fetus strains contain surface array proteins (S-layer proteins) that may be extracted in water and that are critical for virulence. To explore the relationship of S-layer proteins to other surface components, we reattached S-layer proteins onto S- template cells generated by spontaneous mutation or by serial extractions of S+ cells with water. Reattachment occurred in the presence of divalent (Ba2+, Ca2+, Co2+, and Mg2+) but not monovalent (H+, NH4+, Na+, K+) or trivalent (Fe3+) cations. The 98-, 125-, 127-, and 149-kDa S-layer proteins isolated from strains containing type A LPS (type A S-layer protein) all reattached to S- template cells containing type A LPS (type A cells) but not to type B cells. The 98-kDa type B S-layer protein reattached to SAP- type B cells but not to type A cells. Recombinant 98-kDa type A S-layer protein and its truncated amino-terminal 65- and 50-kDa segments expressed in Escherichia coli retained the full and specific determinants for attachment. S-layer protein and purified homologous but not heterologous LPS in the presence of calcium produced insoluble complexes. By quantitative enzyme-linked immunosorbent assay, the S-layer protein copy number per C. fetus cell was determined to be approximately 10(5). In conclusion, C. fetus cells are encapsulated by a large number of S-layer protein molecules which may be specifically attached through the N-terminal half of the molecule to LPS in the presence of divalent cations. Images

Characterization of the Campylobacter fetus sapA promoter: evidence that the sapA promoter is deleted in spontaneous mutant strains.

Abstract: Wild-type Campylobacter fetus cells possess S-layer proteins (S+ phenotype), whereas after laboratory passage, spontaneous stable mutants that do not express these proteins (S- phenotype) arise. To determine the molecular mechanisms by which C. fetus changes to the S- phenotype, we studied wild-type strain 23D, from which the sapA gene encoding the 97-kDa S-layer protein has been cloned, and strain 23B, a spontaneous S- mutant. We compared these strains with another pair of strains, LP (S+) and HP (S-). Southern analysis with the cloned sapA gene as a probe indicated that both pairs of strains have multiple sapA homologs. Using gene disruption and replacement techniques, we constructed an isogenic strain of 23D that differed only in sapA expression (strain 23D:401:1). A 6.0-kb HindIII fragment from 23D:401:1 containing 3.4 kb of sapA upstream region then was cloned into pBluescript to produce pBG101. Nucleotide sequence analysis of sapA upstream region revealed a consensus promoter at -121 bp from the translational start site. Primer extension analysis placed a single in vivo transcription initiation site at the -114-bp position of sapA. A DNA probe derived from the sapA promoter region hybridized to a 5.5-kb HindIII fragment of chromosomal DNA from strain 23D but not to DNA from strain 23B. Northern RNA blot analysis showed no sapA mRNA in strain 23B. These data indicate that the lack of S-layer protein expression in spontaneous mutant strains is caused by the deletion of promoter sequences.

An outbreak of bacteremic Campylobacter jejuni infection.

Abstract: : During September 1980, an outbreak of bacteremic Campylobacter jejuni infection occurred in metropolitan Los Angeles. The outbreak was recognized when blood cultures obtained from 11 previously healthy persons with acute febrile illnesses (characterized in over 80% by fever, diarrhea, and headaches) were positive for C . jejuni. All recovered after an illness that lasted a mean of 8 days. A surveillance system failed to reveal a concomitant outbreak of gastroenteritis. Isolates had identical biochemical characteristics, susceptibility patterns to antimicrobial agents, and serotypes. Isolates from 2 patients were found to be susceptible to bactericidal activity of normal human serum. When bacteremic case-patients were matched with healthy controls, a significant association (p < 0.05, odds ratio 10) between illness and consumption of processed turkey was established. Although turkey was not available for culture, and processing of turkey theoretically destroys Campylobacter, turkey carcasses are known to be heavily contaminated with the pathogen.

Molecular probe analysis of Shigella dysenteriae type 1 isolates from 1940 to 1987.

Abstract: Fourteen strains of Shigella dysenteriae type 1 (Shiga bacillus) isolated from people in diverse locations from 1940 to 1987 were studied. Southern hybridization with three cloned Escherichia coli genes, Shiga-like toxin I (SLTI), frd, and ompF, was used to determine restriction fragment length polymorphism (RFLP) of the genomic DNA of these strains. Digestion with each of four restriction endonucleases generated fragments of identical size to which the frd and ompF hybridized for each of the 14 strains, indicating the conservation of these genes and their flanking sequences. In contrast, after digestion with HindIII, EcoRV, and ClaI and probing with SLTI, there were RFLP among the strains. The results showed three clones of the Shiga bacillus, and suggested that dissemination of a single clone may continue for decades within a wide geographical area.