Multiplex PCR assay for ureC and 16S rRNA genes clearly discriminates between both subspecies of Photobacterium damselae.
(57/970)
A multiplex-PCR approach, employing 2 primer pairs directed to internal regions of the 16S rRNA and ureC genes, was utilized to analyze a collection of Photobacterium damselae strains, including 25 isolates of subspecies piscicida and 15 isolates of subspecies damselae. With this procedure, all the P. damselae subsp. damselae strains yielded 2 amplification products, one of 267 bp and the other of 448 bp, corresponding to internal fragments of the 16S rRNA and ureC genes, respectively. However, P. damselae subsp. piscicida isolates only showed the PCR product of 267 bp (16S rRNA fragment), indicating the absence of the urease gene in its genome. We have constructed a DNA probe directed to an internal region of the ureC gene, and corroborated by dot blot hybridization that the P. damselae subsp. piscicida lacks this gene, whereas it is present in the subspecies damselae. This constitutes the first successful discrimination between both subspecies using a PCR procedure, which could become a useful tool for diagnosis of pasteurellosis in the field. In addition, since these 2 subspecies have been shown to share nearly the same rrn operon sequence, our results provided evidence that one of the steps in the P. damselae speciation proccess included gain/loss events associated with the ure operon. (+info)
Helicobacter pylori urease suppresses bactericidal activity of peroxynitrite via carbon dioxide production.
(58/970)
Helicobacter pylori can produce a persistent infection in the human stomach, where chronic and active inflammation, including the infiltration of phagocytes such as neutrophils and monocytes, is induced. H. pylori may have a defense system against the antimicrobial actions of phagocytes. We studied the defense mechanism of H. pylori against host-derived peroxynitrite (ONOO(-)), a bactericidal metabolite of nitric oxide, focusing on the role of H. pylori urease, which produces CO(2) and NH(3) from urea and is known to be an essential factor for colonization. The viability of H. pylori decreased in a time-dependent manner with continuous exposure to 1 microM ONOO(-), i.e., 0.2% of the initial bacteria remained after a 5-min treatment without urea. The bactericidal action of ONOO(-) against H. pylori was significantly attenuated by the addition of 10 mM urea, the substrate for urease, whereas ONOO(-)-induced killing of a urease-deficient mutant of H. pylori or Campylobacter jejuni, another microaerophilic bacterium lacking urease, was not affected by the addition of urea. Such a protective effect of urea was potentiated by supplementation with exogenous urease, and it was almost completely nullified by 10 microM flurofamide, a specific inhibitor of urease. The bactericidal action of ONOO(-) was also suppressed by the addition of 20 mM NaHCO(3) but not by the addition of 20 mM NH(3). In addition, the nitration of L-tyrosine of H. pylori after treatment with ONOO(-) was significantly reduced by the addition of urea or NaHCO(3), as assessed by high-performance liquid chromatography with electrochemical detection. These results suggest that H. pylori-associated urease functions to produce a potent ONOO(-) scavenger, CO(2)/HCO(3)(-), that defends the bacteria from ONOO(-) cytotoxicity. The protective effect of urease may thus facilitate sustained bacterial colonization in the infected gastric mucosa. (+info)
Dual functions of Streptococcus salivarius urease.
(59/970)
A urease-deficient derivative of Streptococcus salivarius 57.I was constructed by allelic exchange at the ureC locus. The wild-type strain was protected against acid killing through hydrolysis of physiologically relevant concentrations of urea, whereas the mutant was not. Also, S. salivarius could use urea as a source of nitrogen for growth exclusively through a urease-dependent pathway. (+info)
Helicobacter pylori urease binds to class II MHC on gastric epithelial cells and induces their apoptosis.
(60/970)
Infection by Helicobacter pylori leads to injury of the gastric epithelium and a cellular infiltrate that includes CD4+ T cells. H. pylori binds to class II MHC molecules on gastric epithelial cells and induces their apoptosis. Because urease is an abundant protein expressed by H. pylori, we examined whether it had the ability to bind class II MHC and induce apoptosis in class II MHC-bearing cells. Flow cytometry revealed the binding of PE-conjugated urease to class II MHC+ gastric epithelial cell lines. The binding of urease to human gastric epithelial cells was reduced by anti-class II MHC Abs and by staphylococcal enterotoxin B. The binding of urease to class II MHC was confirmed when urease bound to HLA-DR1-transfected COS-1 (1D12) cells but not to untransfected COS-1 cells. Urease also bound to a panel of B cell lines expressing various class II MHC alleles. Recombinant urease induced apoptosis in gastric epithelial cells that express class II MHC molecules, but not in class II MHC- cells. Also, Fab from anti-class II MHC and not from isotype control Abs blocked the induction of apoptosis by urease in a concentration-dependent manner. The adhesin properties of urease might point to a novel and important role of H. pylori urease in the pathogenesis of H. pylori infection. (+info)
The mucosal adjuvanticity of two nontoxic mutants of Escherichia coli heat-labile enterotoxin varies with immunization routes.
(61/970)
Escherichia coli heat-labile enterotoxin (LT), which causes a characteristic diarrhea in humans and animals, is a strong mucosal immunogen and has powerful mucosal adjuvant activity towards coadministered unrelated antigens. Here we report the different mucosal adjuvanticity of nontoxic LT derivatives, LTS63Y and LTdelta110/112, generated by immunizing through two different mucosal routes. Intragastric (IG) immunization with Helicobacter pylori urease alone resulted in poor systemic IgG and IgA responses and no detectable local secretory IgA, but IG co-immunization with urease and LTdelta110/112 induced high titers of urease-specific local secretory IgA and systemic IgG and IgA, comparable to those induced by wild-type LT. LTS63Y showed far lower adjuvant activity towards urease than LTdelta110/112 in IG immunization, but was more active than LTdelta110/112 in inducing immune responses to urease by intranasal (IN) immunization. LTdelta110/112 predominantly enhanced the induction of urease-specific IgG1 levels following IG immunization, whereas LTS63Y induced high levels of IgG1, IgG2a and IgG2b following IN immunization. In addition, quantitative H. pylori culture of stomach tissue following challenge with H. pylori demonstrated a 90-95% reduction (p < 0.0002) in bacterial burden in mice immunized intranasally with urease using either mutant LT as an adjuvant. These results indicate that the mechanism(s) underlying the adjuvant activities of mutant LTs towards coadmnistered H. pylori urease may differ between the IN and IG mucosal immunization routes. (+info)
Local pH elevation mediated by the intrabacterial urease of Helicobacter pylori cocultured with gastric cells.
(62/970)
Helicobacter pylori resists gastric acidity by modulating the proton-gated urea channel UreI, allowing for pH(out)-dependent regulation of urea access to intrabacterial urease. We employed pH- and Ca(2+)-sensitive fluorescent dyes and confocal microscopy to determine the location, rate, and magnitude of pH changes in an H. pylori-AGS cell coculture model, comparing wild-type bacteria with nonpolar ureI-deletion strains (ureI-ve). Addition of urea at pH 5.5 to the coculture resulted first in elevation of bacterial periplasmic pH, followed by an increase of medium pH and then pH in AGS cells. No change in periplasmic pH occurred in ureI-deletion mutants, which also induced a slower increase in the pH of the medium. Pretreatment of the mutant bacteria with the detergent C(12)E(8) before adding urea resulted in rapid elevation of bacterial cytoplasmic pH and medium pH. UreI-dependent NH(3) generation by intrabacterial urease buffers the bacterial periplasm, enabling acid resistance at the low urea concentrations found in gastric juice. Perfusion of AGS cells with urea-containing medium from coculture at pH 5.5 did not elevate pH(in) or [Ca(2+)](in), unless the conditioned medium was first neutralized to elevate the NH(3)/NH(4)(+) ratio. Therefore, cellular effects of intrabacterial ammonia generation under acidic conditions are indirect and not through a type IV secretory complex. The pH(in) and [Ca(2+)](in) elevation that causes the NH(3)/NH(4)(+) ratio to increase after neutralization of infected gastric juice may contribute to the gastritis seen with H. pylori infection. (+info)
Urease activity may contribute to the ability of Actinobacillus pleuropneumoniae to establish infection.
(63/970)
The contribution of urease activity to the pathogenesis of Actinobacillus pleuropneumoniae was investigated using 2 different urease-negative transposon mutants of the virulent serotype 1 strain, CM5 Nalr. One mutant, cbiK::Tn10, is deficient in the uptake of nickel, a cofactor required for urease activity. The other mutant, ureG::Tn10, is unable to produce active urease due to mutation of the urease accessory gene, ureG. In aerosol challenge experiments, pigs developed acute pleuropneumonia following exposure to high doses (10(6) cfu/mL) of the parental strain, CM5 Nalr, and to the cbiK::Tn10 mutant. When low dose (10(3) cfu/mL) challenges were used, neither urease-negative mutant was able to establish infection, whereas the parental strain was able to colonize and cause lesions consistent with acute pleuropneumonia in 8 of the 20 pigs challenged. These findings suggest that urease activity may be needed for A. pleuropneumoniae to establish infection in the respiratory tract of pigs. (+info)
Enhanced reduction of Helicobacter pylori load in precolonized mice treated with combined famotidine and urease-binding polysaccharides.
(64/970)
The present study investigated the effect of a model urease-binding polysaccharide in combination with a histamine H(2) receptor antagonist on Helicobacter pylori colonization in vivo. Euthymic hairless mice were treated daily with dextran sulfate via drinking water and/or famotidine via intragastric gavage starting at 1 week postchallenge with a CagA(+) VacA(+) (type 1) strain of H. pylori. Treatment of precolonized mice for 2 weeks with dextran sulfate combined with famotidine yielded a group mean bacterial load (per 100 mg of gastric tissue) of log(10) 1.04 CFU, which was significantly lower than those of the famotidine (log(10) 3.35 CFU, P < 0.01) and dextran sulfate (log(10) 2.45 CFU, P < 0.05) monotherapy groups and the infected nontreated group (log(10) 3.64 CFU, P < 0.01). Eradication was achieved after 2 weeks of treatment in 50% or more of the test mice using drug combinations (1 or 2 weeks of famotidine plus 2 weeks of dextran sulfate) versus none in the monotherapy and positive control groups. The enhanced activity of the drug combination may be related to the daily pattern of transient acid suppression by famotidine inducing periodic bacterial convergence to superficial mucus sites penetrated by dextran sulfate from the lumen. Increased urease-dextran sulfate avidity was observed in vitro in the presence of famotidine and may partly account for the enhanced activity. With potential utility in abbreviating treatment time and eradication of antibiotic-resistant strains, the use of urease-targeted polysaccharides concurrently with a gastric acid inhibitor warrants consideration as an additional component of the standard multidrug chemotherapy of H. pylori infection. (+info)