Protein-protein interaction of HP137 with histidine kinase HP244 does not contribute to flagellar regulation in Helicobacter pylori

Flagellar motility is essential for the ability of Helicobacter pylori to colonize the gastric mucosa. Expression of the flagella is controlled by a complex regulatory cascade involving the two-component system FlgR-HP244, the sigma factors sigma54 and sigma28 and the anti-sigma28 factor FlgM. The protein-protein interaction map of H. pylori, which is based on a high-throughput two-hybrid screen (Rain et al., 2001. Nature 409, 211-215) indicated a protein-protein interaction between the gene product of ORF hp137 and both the histidine kinase HP244 and the flagellar hook protein HP908. We hypothesized that HP137 might be involved in a feedback regulatory mechanism controlling the activity of histidine kinase HP244. Here we demonstrate that HP137 does not participate in the regulation of flagellar gene expression, neither in H. pylori nor in the closely related bacterium Campylobacter jejuni.     

Sequential inactivation of rdxA (HP0954) and frxA (HP0642) nitroreductase genes causes moderate and high-level metronidazole resistance in Helicobacter pylori

Helicobacter pylori is a human-pathogenic bacterial species that is subdivided geographically, with different genotypes predominating in different parts of the world. Here we test and extend an earlier conclusion that metronidazole (Mtz) resistance is due to mutation in rdxA (HP0954), which encodes a nitroreductase that converts Mtz from prodrug to bactericidal agent. We found that (i) rdxA genes PCR amplified from 50 representative Mtz(r) strains from previously unstudied populations in Asia, South Africa, Europe, and the Americas could, in each case, transform Mtz(s) H. pylori to Mtz(r); (ii) Mtz(r) mutant derivatives of a cultured Mtz(s) strain resulted from mutation in rdxA; and (iii) transformation of Mtz(s) strains with rdxA-null alleles usually resulted in moderate level Mtz resistance (16 microg/ml). However, resistance to higher Mtz levels was common among clinical isolates, a result that implicates at least one additional gene. Expression in Escherichia coli of frxA (HP0642; flavin oxidoreductase), an rdxA paralog, made this normally resistant species Mtz(s), and frxA inactivation enhanced Mtz resistance in rdxA-deficient cells but had little effect on the Mtz susceptibility of rdxA(+) cells. Strains carrying frxA-null and rdxA-null alleles could mutate to even higher resistance, a result implicating one or more additional genes in residual Mtz susceptibility and hyperresistance. We conclude that most Mtz resistance in H. pylori depends on rdxA inactivation, that mutations in frxA can enhance resistance, and that genes that confer Mtz resistance without rdxA inactivation are rare or nonexistent in H. pylori populations.     

Use of HP selective medium to detect Helicobacter pylori associated with other enteric bacteria in seawater and marine molluscs

AIMS: This project investigated the utility of HP selective medium to isolate H. pylori cells from seawater and from marine molluscs. METHODS AND RESULTS: Nested-PCR was performed to reveal the presence of Helicobacter genus. All samples were cultured in HP selective medium and 16 cultures were initially selected as putative Helicobacter. Helicobacter spp. DNA were detected in 9/16 cultures and three of them had 99-100% homology to H. pylori based on 16S RNA gene sequence. Helicobacter pylori isolation was unsuccessful. On the basis of 16S RNA gene sequences the contaminating organisms were shown to be Proteus mirabilis and Vibrio cholerae. CONCLUSIONS: These results indicate the coexistence of three predominant bacterial genera in the cultures and that HP selective medium can grow other enteric bacteria besides Helicobacter. Additional assays will improve the HP selective medium formulation for marine samples avoiding P. mirabilis and V. cholerae interferents. SIGNIFICANCE AND IMPACT OF THE STUDY: This work shows the effectiveness of the selective HP medium for the Helicobacter culture from marine samples.     

幽门螺杆菌HP0762蛋白的原核表达纯化及多克隆抗体制备

目的:表达和纯化幽门螺杆菌HP0762蛋白,并制备该蛋白的多克隆抗体。方法:从幽门螺杆菌SS1中经PCR扩增得到了hp0762基因,将其克隆至含有6×His编码序列的原核表达载体pET-28a(+)中,再将重组质粒转化大肠杆菌BL21(DE3),在IPTG诱导下进行蛋白表达;用HiTrap Chelating HP亲和柱纯化重组蛋白,Western印迹进一步鉴定;以纯化后的蛋白为抗原免疫新西兰大耳白兔,制备该蛋白的多克隆抗体;用ELISA和Western印迹检测抗血清。结果:目的蛋白在大肠杆菌BL21(DE3)中获得了可溶性表达,纯化后纯度可达90%以上;制备了针对HP0762重组蛋白的抗血清,抗体ELISA效价为1:256000,Western印迹分析表明该抗体能特异性识别内源性HP0762。结论:完成了HP0762蛋白的原核高效表达与纯化,并制备了其高效价的多克隆抗体,为进一步对其进行疫苗研制与基因功能研究奠定了基础。     

Helicobacter pylori FlhB function: the FlhB C-terminal homologue HP1575 acts as a "spare part" to permit flagellar export when the HP0770 FlhBCC domain is deleted

In Helicobacter pylori 26695, a gene annotated HP1575 encodes a putative protein of unknown function which shows significant similarity to part of the C-terminal domain of the flagellar export protein FlhB. In Salmonella enterica, this part (FlhB(CC)) is proteolytically cleaved from the full-length FlhB, a processing event that is required for flagellar protein export and, thus, motility. The role of FlhB (HP0770) and its C-terminal homologue HP1575 was studied in H. pylori using a range of nonpolar deletion mutants defective in HP1575, HP0770, and the CC domain of HP0770 (HP0770(CC)). Deletion of HP0770 abolished swimming motility, whereas mutants carrying a deletion of either HP1575 or HP0770(CC) retained their ability to swim. An H. pylori strain containing deletions in both HP1575 and HP0770(CC) was nonmotile and did not produce flagella, suggesting that at least one of the two proteins had to be present for flagellar assembly to occur. Indeed, motility was restored when HP1575 was reintroduced into this strain immediately downstream of, but not fused to, the truncated HP0770 gene. Thus, HP1575 can functionally replace HP0770(CC) in this background. Like FlhB in S. enterica, HP0770 appeared to be proteolytically processed at a conserved NPTH processing site. However, mutation of the proline contained within the NPTH site of HP0770 did not affect motility and flagellar assembly, although it clearly interfered with processing when the protein was heterologously produced in Escherichia coli.     

Identification of genes associated with natural competence in Helicobacter pylori by transposon shuttle random mutagenesis.

To identify genes involved in DNA transformation, we generated 1500 insertion mutants of a Helicobacter pylori strain by transposon shuttle mutagenesis. All mutant strains were screened for their frequency of natural transformation. A total of 20 mutant strains were found to exhibit a significantly decreased transformation frequency. DNA sequencing revealed seven genetic loci, including the reported comB locus, HP0017 (a putative virB4 homologue) and five loci without database match (HP0015, HP1089, HP1326, HP1424, and HP1473) from the 20 mutants. Reknockout of HP1326 revealed no impairment in natural transformation, while the other 5 mutants showed the same defective in natural transformation. Mutation of HP0017 severely impaired natural transformation both chromosome and plasmid DNA. Slot blot analysis revealed that some noncompetent strains had decreased virB4 RNA expression levels compared with competent strains. Nineteen ORFs had decreased expression levels in virB4 knockout mutant by microarray. Therefore, our data indicate that HP0017 is a virB4 homologue and is essential in the natural competence of H. pylori. HP0015, HP1089, HP1424, and HP1473 genes could be also involved in natural transformation.     

Stable accumulation of sigma54 in Helicobacter pylori requires the novel protein HP0958

Several flagellar genes in Helicobacter pylori are dependent on sigma(54) (RpoN) for their expression. These genes encode components of the basal body, the hook protein, and a minor flagellin, FlaB. A protein-protein interaction map for H. pylori constructed from a high-throughput screen of a yeast two-hybrid assay (http://pim.hybrigenics.com/pimriderext/common/) revealed interactions between sigma(54) and the conserved hypothetical protein HP0958. To see if HP0958 influences sigma(54) function, the corresponding gene was disrupted with a kanamycin resistance gene (aphA3) in H. pylori ATCC 43504 and the resulting mutant was analyzed. The hp0958:aphA3 mutant was nonmotile and failed to produce flagella. Introduction of a functional copy of hp0958 into the genome of the hp0958:aphA3 mutant restored flagellar biogenesis and motility. The hp0958:aphA3 mutant was deficient in expressing two sigma(54)-dependent reporter genes, flaB'-'xylE and hp1120'-'xylE. Levels of sigma(54) in the hp0958 mutant were substantially lower than those in the parental strain, suggesting that the failure of the mutant to express the genes in the RpoN regulon and produce flagella was due to reduced sigma(54) levels. Expressing sigma(54) at high levels by putting rpoN under the control of the ureA promoter restored flagellar biogenesis and motility in the hp0958:aphA3 mutant. Turnover of sigma(54) was more rapid in the hp0958:aphA3 mutant than it was in the wild-type strain, suggesting that HP0958 supports wild-type sigma(54) levels in H. pylori by protecting it from proteolysis.