Proteome Analysis of Alkylhydroxide Peroxidase-Deficient Isogenic Mutant of Helicobacter pylori 26695

In order to investigate the antioxidant effect of alkylhydroxide peroxidase (ahpC) of Helicobacter pylori (H. pylori) 26695, an ahpC-deficient mutant (H. pylori 26695 ahpC::cat) was generated. ahpC-deficient mutant was grown slowly at lower pressure of oxygen (5% oxygen) compared to the H. pylori 26695. Whole cell proteins isolated form H. pylori 26695 and H. pylori 26695 ahpC::cat were analyzed by MALDI-TOF and tandem-MS. The expression of 15 proteins, including Ppa, HypB, GrpE, Elp, RecA, GroES, Mda66, RibE, NapA, GlnA, BioB, TrxB, Tsf, FumC and Icd, was more than doubled in H. pylori 26695 ahpC::cat. Production of 10 proteins such as UreG, FabE, Adk, Pnp, OorC, AtpA, AtpD, Nqq3, Pfr, and TagD decreased below 50% in H. pylori 26695 ahpC::cat compared to the H. pylori 26695. In microarray analysis, 9 genes including sul1, amiE, frxA, fecA, hyuA, and katA increased in transcription level in H. pylori 26695 ahpC::cat compared to H. pylori 26695. A total of 24 genes, including flaB, protein kinase C inhibitor, cag16, pabC, and sabA, reduced in transcription. 27 genes, including HP0889, showed common expression changes in ahpC, katA, and sodB-deficient mutations. As a result of this study, there were not many genes whose expression was commonly changed by the deletion of each of the three major antioxidant enzymes of H. pylori. These results showed the functions and regulation of the three antioxidant enzymes were different in H. pylori.

Role of alkyl hydroperoxide reductase subunit C (AhpC) as a peroxiredoxin in Leptospira interro-gans against oxidative stress / 中华微生物学和免疫学杂志

Objective To analyze the enzymatic properties of alkyl hydroperoxide reductase sub-unit C (AhpC) from Leptospira interrogans (L.interrogans) and to elucidate its physiological roles in host-pathogen interactions in macrophages during Leptospira infection. Methods A prokaryotic expression system for ahpC gene of L.interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai was established to ex-press the recombinant AhpC(rAhpC). After purified by Ni-NTA affinity chromatography,the enzymatic ac-tivity of the rAhpC and its role in protecting DNA from oxidation were analyzed. The importance of each cys-teine in its molecule was evaluated through site-directed mutation. L.interrogans strains were pretreated with or without Conoidin A, a covalent inhibitor of peroxiredoxin, and then were used to infect macrophages. Changes in oxidative status in leptospires and survival rates of L.interrogans strains were analyzed by fluores-cence-activated cell sorting and colony counting method. Results The rAhpC was successfully expressed in the established prokaryotic expression system. It had peroxiredoxin activity that was able to catalyze the re-duction of hydrogen peroxide. Its ability of reducing hydrogen peroxide depended on the thioredoxin/thiore-doxin reductase system. Cys47 (a peroxidatic cysteine) and Cys167 (a resolving cysteine) were critical to maintaining the enzymatic activity of AhpC. AhpC could protect DNA from hydrogen peroxide induced-oxida-tive damage. When L.interrogans strains were pretreated with Conoidin A,the oxidative status in leptospires was elevated and the survival of L.interrogans in macrophages was significantly reduced in a dose-dependent manner. Conclusion The AhpC of L.interrogans is a thioredoxin-dependent peroxiredoxin that plays an im-portant role in protecting L.interrogans against oxidative stress in macrophages.

Streptococcus mutans gcp gene knockout strains expression profile gene chip / 中国组织工程研究

BACKGROUND:Previous studies have confirmed the presence of bis-(3'-5')-cyclic dimeric guanosine monophosphate signaling pathway in Streptococcus mutans, which construct the streptococcus mutans gcp gene knockout strains. OBJECTIVE:To compare the gene expression differences between Streptococcus mutans wild strains and gcp mutant strains, and to screen the biofilm-related genes from them for the fol ow-up study. METHODS:The total RNA of two kinds of strains were extracted and stained with cy3 and cy5 respectively after reverse transcription. The gene chip was scanned after hybridization and the differential gene were obtained through the data analysis. The different expression genes were verified by real-time PCR. RESULTS AND CONCLUSION:Differential genes were mainly relative about glucose metabolism and biofilm formation. We selected two genes for real-time PCR verification. The PCR results were consistent with the microarray results. After Streptococcus mutans gcp gene knockout, the gene expressions of gcp mutant strains were upregulated and the gene expressions of phosphotransferase system were downregulated, this result suggested that two different genes were related with the c-di-GMP signal pathway downstream.

Cloning and expression of H.pylori ahpC in prokaryotic expression vector / 西安交通大学学报(医学版)

Objective To construct a prokaryotic expression system of ahpC gene of Helicobacter pylori. Methods The ahpC gene was amplified from Hp chromosomal DNA by PCR technique and cloned into the expression vector pET-30a. The recombinant vector pET30a-ahpC was identified by DNA sequencing and transformed to E.coli BL21 (DE3) for expression under induction by IPTG. The expression product was analyzed by SDS-PAGE. Results PCR product showed that ahpC gene consisted of 594bp. The gene fragment that was inserted into the recombinant vector was identified to GenBank for 99%. SDS-PAGE showed that the induced protein was expressed highly in the host bacterium. Conclusion A prokaryotic high-expression system for ahpC gene has been successfully constructed. It can highly express r-AhpC protein in E.coli.

Rapid Drug Susceptibility Testing for Isoniazid and Rifampicin by Reverse Hybridization Assay / 결핵

BACKGROUND: Development of rapid drug susceptibility testing provides the opportunity for rapid identification of individuals with drug resistant tubercle bacilli, allowing selection of appropriate therapeutic regimens. METHODS: A total of 502 drug resistant isolates were subjected to reverse blot hybridization assay to detect mutations within genes (rpoB, katG, inhA, and ahpC) associated with rifampicin (RMP) and isoniazid (INH) resistance. RESULTS: Among the 264 RMP resistant strains (RMPR) tested, the most prevalent mutation was the Ser531Leu seen in 121 strains (46%). The second common mutation occurred in 84 strains (32%) at codon 526. And 27 strains (10%) showed the mutation at codon 516. Among all 469 INH resistant strains (INHR), the katG mutation was responsible for INH. The inhA mutation was present in 88 strains (19%). In 11 isolates (2%), coexisting of the katG and inhA mutations were identified. Reverse hybridization assay successfully detected over 80% of INHR and over 92% of RMPR among Korean isolates. CONCLUSION: Reverse hybridization was useful for rapid detection of INHR and RMPR.