Role of Hpn and NixA of Helicobacter pylori in susceptibility and resistance to bismuth and other metal ions.

BACKGROUND: Helicobacter pylori produces Hpn, a 60-amino acid, histidine-rich protein that avidly binds nickel and zinc ions, and NixA, a high-affinity nickel transporter in the cytoplasmic membrane. We tested the hypothesis that Hpn and NixA govern susceptibility to metal ions in H. pylori. MATERIALS AND METHODS: Hpn-negative mutants of four H. pylori strains were constructed by standard allelic exchange techniques to yield isogenic Hpn+/Hpn-deficient pairs. A metal concentration that inhibited growth by 50% (IC50) was calculated for Ni2+, Zn2+, Cu2+, and Co2+ by comparing OD600 of cultures in metal-supplemented and control media. RESULTS: Among all four pairs of isogenic strains, the tolerance for Ni2+ was reduced significantly (p <.001) in the Hpn mutants; the mean IC50 value for wild-type strains was 1.9 mM; for the mutant, it was 0.8 mM. In contrast, growth inhibition by Zn2+ was identical within the fours pairs, as was Cu2+ and Co2+ tolerance in one pair tested. We also found that deletion of the hpn gene increases susceptibility to therapeutic forms of bismuth by testing a mutant and wild-type pair with ranitidine bismuth citrate, bismuth citrate, and four antibiotics. Minimal inhibitory concentrations of ranitidine bismuth citrate dropped from 9.2 to 2.3 microg/ml, and those of bismuth citrate dropped from 7.4 to 3.2 microg/ml (p <.05 for both comparisons), while susceptibility to the antibiotics was unaffected. Disruption of the nixA gene encoding the specific Ni2+ transport protein of H. pylori did not change susceptibility to bismuth. CONCLUSION: We concluded that bacteria lacking Hpn, cultured in vitro, are more susceptible than is the wild type to bismuth and Ni2+.

Unique susceptibility of Helicobacter pylori to simethicone emulsifiers in alimentary therapeutic agents.

Helicobacter pylori is killed in vitro by polyoxyethylene acyl esters and ethers similar to simethicone emulsifiers in therapeutic antifoams. The MBC of these compounds for Helicobacter pylori was less than 20 micrograms/ml, while other gram-negative bacteria were unaffected by much higher concentrations of up to 50 mg/ml.

Studies on the specificity of the IgA-binding lectin, jacalin.

The interactions of IgA with the jackfruit lectin, jacalin, were investigated with regard to the specificity of jacalin for species and subclasses of IgA. It was found that jacalin selectively bound to human IgA1, but not to human IgA2, mouse IgA or rat IgA. Binding studies with human IgA1 fragments produced by different IgA1 proteases revealed that jacalin bound to galactose-terminal oligosaccharides in the hinge region of human IgA1. Affinity chromatography employing jacalin-Sepharose provided a means to separate the subclasses of IgA in human whey.

Inhibition of microbial IgA proteases by human secretory IgA and serum.

Microbial IgA proteases cleave human serum IgA1 immunoglobulin, but human secretory IgA is resistant to hydrolysis. We have found this resistance to be due to an inhibition of protease activity that is mediated by the Fab region of secretory IgA. The IgA proteases of the genus Neisseria are more sensitive to inhibition than is the protease of Streptococcus sanguis. There is also a serum inhibitor of Neisseria proteases that co-chromatographs with IgG. Monoclonal (myeloma) human IgG proteins and plasma protease inhibitors such as alpha-1-antitrypsin and alpha-2-macroglobulin do not inhibit. Human sera do not contain inhibitor to S. sanguis protease activity. We conclude that microbial IgA proteases are subject to inhibition by IgA in secretions and IgG in serum, and this activity is most consistent with being an anti-enzyme antibody. The insensitivity of S. sanguis IgA protease to inhibition is unexplained but provides further evidence that the IgA proteases are structurally diverse.

J chain is covalently bound to both monomer subunits in human secretory IgA.

Previous work has established that the secretory component (SC) in human secretory IgA is covalently linked to only one of the two IgA monomer subunits, but it has not been clear whether the J chain is covalently linked to one or to both of these subunits. In view of the asymmetry in the disulfide bonding between SC and the IgA subunits, an arrangement which follows disulfide interchange, several models for the disulfide linkage of J chain and the bonds between IgA subunits were envisaged and investigated. When sIgA was gel filtered through Sephadex G-200 in acetic acid, a single major symmetrical peak eluted at the front. This material contained SC, alpha and L chains, and all of the J chain. The greater resolution afforded by polyacrylamide gel electrophoresis in detergent confirmed that human sIgA contains no major noncovalently linked components in the 150,000-200,000 molecular weight range. In another series of experiments the Fc monomer, which is not covalently attached to SC, isolated after treatment of sIgA with IgA protease and cyanogen bromide, was investigated to learn whether alpha chain COOH-terminal octapeptides could be released by reduction. The results were negative. The available data thus favor a model in which J chain is disulfide-bonded to both IgA monomer subunits in sIgA.

Evaluation of human oral organisms and pathogenic Streptococcus for production of IgA protease.

IgA protease is a proteolytic enzyme found in whole human saliva and in dental plaque that cleaves both secretory and myeloma IgA of human origin to yield intact Fabalpha and Fcalpha fragments. To determine which bacteria are capable of producing this enzyme, we have examined a variety of strains normally found in the human oral cavity and a number of streptococci of known Lancefield group serotype. Streptococci of groups A, B, C, D, F, G, H, M, and N, Streptococcus mutans, Streptococcus sanguis, Streptococcus mitior, Streptococcus salivarius, Streptococcus faecalis, Veillonella, Lactobacillus, Actinomyces, Propionibacterium, Bacteroides, and Fusobacterium were grown in liquid medium, and fluids were examined for IgA protease activity. Only S. sanguis and clinically isolated group H streptococci elaborated IgA protease under the culture conditions used. Negative strains could not be stimulated to produce the enzyme when cultured in the presence of secretory IgA. Among the natural oral bacteria, capacity to produce IgA protease is restricted to certain species of Streptococcus, notably those of the group H serotype. Since secretory immunity is mediated by the IgA class of antibody, the presence of this enzyme at mucosal surfaces could modify the secretory immune function.