The bcp gene in the bcp-recA-vimA-vimE-vimF operon is important in oxidative stress resistance in Porphyromonas gingivalis W83

The ability of Porphyromonas gingivalis to overcome oxidative stress in the inflammatory environment of the periodontal pocket is critical for its survival. We have previously demonstrated that the recA locus, which carries the bacterioferritin co-migratory protein (bcp) gene and has a unique genetic architecture, plays a role in virulence regulation and oxidative stress resistance in P. gingivalis. To further characterize the bcp gene, which was confirmed to be part of the bcp-recA-vimA-vimE-vimF operon, we created a P. gingivalis bcp-defective isogenic mutant (FLL302) by allelic exchange. Compared with the wild-type, FLL302 had a similar growth rate, black pigmentation, β-hemolysis and UV sensitivity. Although there was no change in the distribution of gingipain activity, there was a 30% reduction in both Arg-X and Lys-X activities in the mutant strain compared with the wild-type. When exposed to 0.25 mm hydrogen peroxide, P. gingivalis FLL302 was more sensitive than the wild-type. In addition, the cloned P. gingivalis bcp gene increased resistance to 0.25 mm hydrogen peroxide in a bcp-defective Escherichia coli mutant. The mutant also demonstrated decreased aerotolerance when compared with the wild-type. Porphyromonas gingivalis FLL302 and the wild-type strain had similar virulence profiles in a mouse model of virulence. These observations suggest that the bcp gene may play a role in oxidative stress resistance but has a decreased functional significance in the pathogenic potential of P. gingivalis.     

变形链球菌gcp基因敲除菌株表达谱基因芯片

背景：前期研究中经证实变形链球菌内部存在单磷酸鸟苷环二聚体信号通路,构建了变形链球菌gcp基因敲除菌株。 目的：比较变形链球菌野生菌种和gcp基因突变菌株基因表达的差异情况,筛选与生物膜相关的基因,进入后续研究。 方法：提取两种细菌的总RNA,反转录后分别用cy3和cy5染色。与基因芯片杂交后,扫描结果,进行数据分析,获取差异基因信息,对筛选的基因进行Real-Time PCR验证。 结果与结论：差异基因主要与糖代谢、生物膜形成有关,选择了2个基因进行验证,PCR结果与芯片结果相符合。变形链球菌gcp基因敲除后,突变菌株ahpC基因表达上调,磷酸转移酶系统基因表达下调,说明这2个基因与c-di-GMP信号通路的下游途径相关。     

空肠弯曲菌AhpC蛋白的克隆表达及抗原性分析

目的构建空肠弯曲菌烷基过氧化氢还原酶（ahpC）基因的原核表达系统,克隆表达AhpC蛋白,用Western blot以及ELISA方法检测表达蛋白的抗原性,为筛选空肠弯曲菌感染后特异性抗体检测试剂的制备奠定基础。方法用PCR方法从中国分离菌株ICDCCJ07001的染色体DNA中扩增出ahpC基因,将目的基因插入表达载体pGEX-4T-1后转化入大肠杆菌E.coliBL21（DE3）,IPTG诱导重组质粒pGEX-ahpC的表达。SDS-PAGE分析表达产物,采用GST标签亲和层析柱纯化表达蛋白。应用兔免疫血清、临床感染者血清以及健康无感染者血清,利用Western blot以及ELISA的方法分析AhpC蛋白的抗原性并初步评价其在ELISA检测方法中的应用。结果 PCR扩增及双酶切鉴定证实重组质粒构建成功。重组表达蛋白经飞行质谱鉴定为空肠弯曲菌AhpC蛋白,Western blot及ELISA检测结果显示AhpC具有特异抗原性。结论成功构建了ahpC重组表达载体pGEX-4T-1/ahpC,并在大肠杆菌中高效表达。空肠弯曲菌AhpC蛋白具有抗原性,可以作为空肠弯曲菌感染后血清抗体检测的特异抗原。     

Does cross-reactivity between mycobacterium avium paratuberculosis and human intestinal antigens characterize Crohn's disease?

BACKGROUND & AIMS: Most Crohn's disease (CD) patients show seroreactivity against Mycobacterium avium paratuberculosis (MAP), suggesting a pathogenic role for this organism. Our aim was to seek amino acid similarities between MAP and intestinal proteins that, through molecular mimicry, could serve as targets for cross-reactive immunity in CD. METHODS: Fifty-three peptides comprising 23 sets of MAP/human intestinal peptidyl mimics chosen for maximal homology were constructed and tested for immunologic cross-reactivity by enzyme-linked immunosorbent assay in 50 patients with CD, 50 with ulcerative colitis, and 38 healthy controls. RESULTS: Antibody reactivity was present in only 7 of 23 peptide sets. MAP/self-reactivity in at least 1 of the 7 reactive sets was present in 21 (42%) CD patients but was virtually absent in the controls. Significant double-reactivity was found against MAP glycosyl transferase d (gsd)(230-244)/human gastrointestinal glutathione peroxidase (GPg)(111-125) homologues in 15 of 50 (30%) CD patients; MAP alkylohydroperoxidase C (ahpC)(20-34)/human tumor overexpressed protein (TOG)(637-651) double-reactivity was present in 10 (20%) CD patients, but in none of the controls. Inhibition studies confirmed that simultaneous reactivity to mimics was caused by cross-reactivity. Three-dimensional modeling predicts GPg(111-125) will be exposed in a solvent-accessible surface region of the protein compatible with antibody recognition. Antibody affinity was greater for the MAP mimics than for the self-sequences, suggesting that reactivity to the mycobacterial sequences precedes that against self-sequences. CONCLUSIONS: We describe MAP/self-mimics as targets of cross-reactive antibody responses characterizing patients with CD. Our findings indicate gastrointestinal glutathione peroxidase as a novel autoantigen in CD.     

Functional plasticity of a peroxidase allows evolution of diverse disulfide-reducing pathways

In Escherichia coli, the glutathione/glutaredoxin and thioredoxin pathways are essential for the reduction of cytoplasmic protein disulfide bonds, including those formed in the essential enzyme ribonucleotide reductase during its action on substrates. Double mutants lacking thioredoxin reductase (trxB) and glutathione reductase (gor) or glutathione biosynthesis (gshA) cannot grow. Growth of Deltagor DeltatrxB strains is restored by a mutant (ahpC*) of the peroxiredoxin AhpC, converting it to a disulfide reductase that generates reduced glutathione. Here, we show that ahpC* also restores growth to a DeltagshB DeltatrxB strain, which lacks glutathione and accumulates only its precursor gamma-glutamylcysteine (gamma-GC). It suppresses this strain by allowing accumulation of reduced gamma-GC, which can substitute for glutathione. Surprisingly, new ahpC suppressor mutations arose in a DeltagshA DeltatrxB strain lacking both glutathione and gamma-GC, a strain that ahpC* does not suppress. Some of these mutant AhpC proteins channel electrons into the disulfide-reducing pathways via either the thioredoxins or the glutaredoxins without, evidently, the intermediary of glutathione. Our results provide insights into the physiological functioning of the glutathione pathway and reveal surprising plasticity of a peroxidase because different mutant versions of AhpC can channel electrons into the disulfide-reducing pathways by at least four distinct routes. Despite the reductase activity of mutant AhpCs, these various suppressor strains exhibit an oxidizing cytoplasm and accumulate correctly folded disulfide-bonded proteins in their cytoplasm. Proteins most effectively oxidized vary between strains, potentially providing useful tools for expressing different disulfide-bonded proteins.     

变形链球菌gcp基因敲除菌株表达谱基因芯片

背景：前期研究中经证实变形链球菌内部存在单磷酸鸟苷环二聚体信号通路,构建了变形链球菌 gcp 基因敲除菌株。目的：比较变形链球菌野生菌种和 gcp 基因突变菌株基因表达的差异情况,筛选与生物膜相关的基因,进入后续研究。方法：提取两种细菌的总 RNA,反转录后分别用 cy3 和 cy5 染色。与基因芯片杂交后,扫描结果,进行数据分析,获取差异基因信息,对筛选的基因进行 Real-Time PCR 验证。结果与结论：差异基因主要与糖代谢、生物膜形成有关,选择了 2 个基因进行验证,PCR 结果与芯片结果相符合。变形链球菌 gcp 基因敲除后,突变菌株 ahpC 基因表达上调,磷酸转移酶系统基因表达下调,说明这 2个基因与 c-di-GMP 信号通路的下游途径相关。     

PCR-SSCP技术直接快速检测痰标本中结核分支杆菌rpoB,katG,ahpC基因突变的研究

目的：应用PCR－SSCP技术直接快速检测痰标本中结核分支杆菌rpoB,katG及ahpC基因突变,评价此方法用于结核分杆菌利福平（RFP）及异烟肼（INH）耐药性试验的意义。方法：以结核分支杆菌H37Rv为对照,30例耐RFP（单耐或多耐）、21例耐INH（单耐和多耐）的结核分枝杆菌临床分离株及10例敏感菌株;39例菌阳肺结核患者及30例非结核 性肺部疾病患者痰标本,采用PCR－SS－CP技术检测痰标本和菌株中的结核杆菌rpoB,katG,ahpC基因突变,并与药敏试验对照。结果：PCR－SSCP检测痰标本中结核分支杆菌rpoB,katG及ahpC基因突变的阳性率分别为79％（31／39）、46％（18／39）及5％（2／39）.结论：PCR－SSCP可望成为直接检测临床痰标本中结核分枝杆菌耐利福平及耐异烟肼的快速方法。     

Alkyl hydroperoxide peroxidase subunit C (ahpC) protects against organic peroxides but does not affect the virulence of Porphyromonas gingivalis W83

The cloned Porphyromonas gingivalis alkyl hydroperoxide reductase (ahpC) gene complemented an ahpC defect in Escherichia coli. To study the role of ahpC in protecting against oxidative stress in P. gingivalis a 1.8 kb fragment containing the ahpC gene was amplified from the chromosome of P. gingivalis W83. This gene was insertionally inactivated using the ermF-ermAM antibiotic resistance cassette and used to create a ahpC-deficient mutant by allelic exchange. One mutant strain, designated FLL141, demonstrated no change in the growth rate, black pigmentation, beta-hemolysis or level of proteolytic activity compared to the parent strain. Although P. gingivalis FLL141 was more sensitive to hydrogen peroxide than the parent strain, there was no change in its virulence potential in the mouse model compared to the wild-type strain. These findings suggest that the ahpC gene plays a role in peroxide resistance in P. gingivalis but does not contribute significantly to virulence.