Rv2686c-Rv2687c-Rv2688c, an ABC fluoroquinolone efflux pump in Mycobacterium tuberculosis

The Mycobacterium tuberculosis Rv2686c-Rv2687c-Rv2688c operon, encoding an ABC transporter, conferred resistance to ciprofloxacin and, to a lesser extent, norfloxacin, moxifloxacin, and sparfloxacin to Mycobacterium smegmatis. The resistance level decreased in the presence of the efflux pump inhibitors reserpine, carbonyl cyanide m-chlorophenylhydrazone, and verapamil. Energy-dependent efflux of ciprofloxacin from M. smegmatis cells containing the Rv2686c-Rv2687c-Rv2688c operon was observed.     

结核分枝杆菌抗原Rv2654c、Rv1985c和Rv3868的克隆表达及血清诊断学初步评价

目的 评价结核分枝杆菌蛋白抗原Rv2654c、Rv1985c和Rv3868的血清诊断价值和潜在应用前景。方法 以结核分枝杆菌H37Rv标准株全基因组DNA为模板扩增得到Rv2654c、Rv1985c和Rv3868基因的完整序列, 并与表达载体pET-32a构建重组质粒。原核表达Rv2654c、Rv1985c和Rv3868蛋白, 利用亲和层析的方法进行纯化。采用棋盘滴定的方法, 确定各抗原的酶联免疫吸附试验(ELISA)最佳反应条件, 并应用190份血清进行血清IgG抗体检测, 结合受试者工作特征曲线(ROC)对其诊断效能进行分析和评价。通过ROC计算各抗原组合的诊断效能, 确定最佳组合方案。结果 成功构建了重组蛋白, 对重组后的片段进行测序, 经BLAST比对与目的基因完全一致, 且能够稳定表达。对经纯化后的3种蛋白进行ELISA检测, 经统计学分析, Rv2654c、Rv1985c和Rv3868抗原的诊断效能分别达到73.16%、56.84%和71.05%。结合ROC得到最佳的抗原组合方案为Rv2654c+Rv3868, 其敏感性、特异性和诊断效能分别达到78.95%、72.63%和75.79%。结论 结核分枝杆菌重组抗原Rv2654c、Rv1985c和Rv3868具有作为结核病诊断抗原的潜力, 可以作为结核病免疫学快速诊断的候选蛋白。抗原组合Rv2654c+Rv3868具有较高的诊断效能, 有较好的潜在应用价值。     

结核分枝杆菌Rv1884c和Rv0867c基因的克隆表达及其促生长作用的研究

目的 构建结核分枝杆菌Rv1884c和Rv0867c基因的原核表达质粒,获得结核分枝杆菌Rvl884c和Rv0867c基因的表达蛋白,并初步研究其促生长作用.方法 制备结核分枝杆菌基因组DNA,采用PCR技术扩增目的 基因片段;将2个片段分别克隆入克隆载体pGEx-4T-1和pUC19,再分别克隆入原核表达载体pGEX-4T-1和pPRO-EXHT,经序列测定证实正确后,再经异丙基硫代-β-D半乳糖苷(IPTG)诱导表达GST标记的Rv1884c融合蛋白和His标记的Rv0867c融合蛋白;用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析重组蛋白的相对分子质量大小及表达形式.结果 成功扩增出了结核分枝杆菌Rv1884c和Rv0867c基因,构建了具有正确基因序列的质粒载体pGEX-4T-1-Rv1884c和pPRO-EXHT-Rv0867c,转化人大肠杆菌DH5α中经诱导产生高水平的表达产物.经SDS分析,在相对分子质量为45 000和80 000处出现新生蛋白带,凝胶薄层扫描检测表达量分别约占菌体蛋白的18.3%和23.7%.用GSTrap FF亲和层析柱和Ni2+-NTA纯化柱进行蛋白纯化,并研究这两种蛋白对藤黄微球菌、BCG和结核分枝杆菌H37Rv的促生长作用.结论 成功克隆了结核分枝杆菌Rv1884c和Rv0867c基因并得到了其大肠杆菌表达产物,为进一步研究Rv1884c和Rv0867c基因蛋白的活性及其功能,以及研究结核分枝杆菌快速促生长作用奠定了基础.     

结核分枝杆菌Rv3872基因的克隆、表达和纯化

目的原核表达并纯化结核分枝杆菌Rv3872蛋白。方法聚合酶链反应（PCR）技术从结核分枝杆菌标准株H37Rv全基因组中扩增出的表达Rv3872基因序列，将其克隆到pMD18-T载体后，测序分析。亚克隆该目的基因到pET28a表达载体，最终转化至大肠杆菌BL21（DE3），获得正确的阳性克隆子后大量表达重组Rv3872蛋白，再经纯化、定量后，通过Western blot分析其抗原性。结果扩增Rv3872基因经序列测定与GenBank公布的序列完全一致，表达蛋白经SDS-PAGE分析，在约15000kD处有表达条带，纯化后的重组蛋白占总蛋白的90％以上。免疫印迹分析显示重组的Rv3872蛋白具有抗原性。结论成功表达结核分枝杆菌的Rv3872蛋白，为结核病血清学诊断候选抗原筛选和开发应用打下基础。     

结核分枝杆菌Rv1009结构域多肽的免疫学特性研究

目的 研究结核分枝杆菌Rv1009结构域多肽的免疫学特性.方法 用原核表达的Rv1009结构域多肽免疫BALB/c小鼠3次.每次间隔2周.用ELISA法检测免疫小鼠血清中特异性抗体滴度.分离免疫小鼠的脾淋巴细胞,体外用抗原再刺激后,用MTT比色法检测免疫小鼠脾淋巴细胞的增殖指数.ELISA方法检测淋巴细胞悬液中γ干扰素(IFN-γ)、白细胞介素(IL)-10和IL-12的产生水平.另一部分免疫的小鼠经尾静脉感染MTB毒株H37Rv,4周后,计数脾脏细菌负荷数.结果 Rv1009结构域多肽免疫小鼠血清特异性抗体滴度为1:12 800.淋巴细胞增殖指数为2.40±0.18,明显高于生理盐水对照组的0.90±0.21.ELISA方法检测Rv1009结构域多肽免疫组IFN-γ、IL-10和IL-12水平为(1432±30)ng/L、(503±11)ng/L和(311±11)ng/L,显著高于生理盐水对照组的(256±20)ng/L、(76±6)ng/L和(56±8)ng/L(P<0.01).与生理盐水免疫组(细菌负荷6.64±0.13)相比较,Rv1009结构域多肽免疫组小鼠,对攻击感染后抗MTB在脾脏中增殖有显著作用(细菌负荷为4.86±0.14,P<0.05),但不及BCG免疫组的3.81±0.16.结论 Rv1009结构域多肽有可能作为新型结核疫苗的候选组分.     

结核分枝杆菌H37Rv株菌体抗原及分泌抗原分析

目的 对比分析人型结核枝杆菌（结核菌）H37Rv株固体培养菌、液体培养菌及分泌蛋白中抗原成分的差异，寻找结核菌的主要免疫抗原。方法 应用SDS-聚丙烯酰胺凝胶电泳技术、免疫印迹技术及单克隆抗体技术分析人型结核菌H37Rv株固体培养、液体培养及其分泌蛋白中的抗原成分。结果 固体培养菌与液体培养菌具有基本一致的多肽抗原成分，其主要成分分子量为79000、64000、54000、50000、28000～38000、23000等，而分泌蛋白差异稍大，其主要成分分子量为66000、63000～65000、55000、42000、32000～34000、24000、16000等。单克隆抗体进一步证明66000、55000、16000为分泌蛋白。多克隆抗体免疫印迹分析表明，菌体主要免疫原有79000、54000、38000～50000、28000蛋白成分，而分泌蛋白主要免疫原为分子量66000、55000～64000、30000～34000、24000、16000成分。结论 人型结核菌H37Rv株菌体蛋白和分泌蛋白具有不同的主要免疫原，将有助于认识H37Rv株的总体蛋白组成及寻找与结核病相关的特异性抗原成分。     

Accumulation of five fluoroquinolones by Mycobacterium tuberculosis H37Rv.

The accumulation of ciprofloxacin, norfloxacin, moxifloxacin, levofloxacin and ofloxacin by Mycobacterium tuberculosis H37Rv was determined with a modified fluorescence method. The time to achieve a steady-state concentration (SSC) of each agent in M. tuberculosis was 60-240 s. Moxifloxacin was accumulated to the lowest concentration and ciprofloxacin to the highest. However, ciprofloxacin took longer to achieve an SSC than the other four agents; levofloxacin reached steady state in the shortest time. Larger fluoroquinolones accumulated to the lowest concentration and more slowly. Although all five agents had low hydrophobicity values (P(app) < or =0.11), those with the lowest values accumulated to the higher concentrations.     

Mycobacterium tuberculosis Rv3802c Encodes a Phospholipase/Thioesterase and Is Inhibited by the Antimycobacterial Agent Tetrahydrolipstatin

The cell wall of M. tuberculosis is central to its success as a pathogen. Mycolic acids are key components of this cell wall. The genes involved in joining the α and mero mycolates are located in a cluster, beginning with Rv3799c and extending at least until Rv3804c. The role of each enzyme encoded by these five genes is fairly well understood, except for Rv3802c. Rv3802 is one of seven putative cutinases encoded by the genome of M. tuberculosis. In phytopathogens, cutinases hydrolyze the waxy layer of plants, cutin. In a strictly mammalian pathogen, such as M. tuberculosis, it is likely that these proteins perform a different function. Of the seven, we chose to focus on Rv3802c because of its location in a mycolic acid synthesis gene cluster, its putative essentiality, its ubiquitous presence in actinomycetes, and its conservation in the minimal genome of Mycobacterium leprae. We expressed Rv3802 in Escherichia coli and purified the enzymatically active form. We probed its activities and inhibitors characterizing those relevant to its possible role in mycolic acid biosynthesis. In addition to its reported phospholipase A activity, Rv3802 has significant thioesterase activity, and it is inhibited by tetrahydrolipstatin (THL). THL is a described anti-tuberculous compound with an unknown mechanism, but it reportedly targets cell wall synthesis. Taken together, these data circumstantially support a role for Rv3802 in mycolic acid synthesis and, as the cell wall is integral to M. tuberculosis pathogenesis, identification of a novel cell wall enzyme and its inhibition has therapeutic and diagnostic implications.     

Contribution of the Rv2333c efflux pump (the Stp protein) from Mycobacterium tuberculosis to intrinsic antibiotic resistance in Mycobacterium bovis BCG

OBJECTIVES: To characterize the efflux pump encoded by the gene Rv2333c from Mycobacterium tuberculosis, and assess its contribution to intrinsic antibiotic resistance using Mycobacterium bovis BCG as a model organism. METHODS: Firstly, the Rv2333c gene was expressed from a multicopy plasmid in M. bovis BCG. Secondly, the gene was inactivated in the chromosome of M. bovis BCG. Antibiotic susceptibility tests and tetracycline uptake/efflux experiments were carried out with the strains mentioned above. RESULTS: When the Rv2333c gene was inactivated in the M. bovis BCG chromosome, there was a decrease in the MIC values of spectinomycin and tetracycline, and an increase in [3H]tetracycline accumulation. When the Rv2333c gene was cloned into a multicopy plasmid, there was an increase in the MIC values of spectinomycin and tetracycline, and a decrease in [3H]tetracycline accumulation. These results indicate that both antibiotics are substrates of the Rv2333c efflux pump, which has been named Stp, for Spectinomycin Tetracycline efflux Pump. CONCLUSIONS: The Rv2333c efflux pump (Stp protein) of M. tuberculosis contributes to intrinsic spectinomycin and tetracycline resistance.     

Characterization of spontaneous, In vitro-selected, rifampin-resistant mutants of Mycobacterium tuberculosis strain H37Rv

Resistance to rifampin in Mycobacterium tuberculosis results from mutations in the gene coding for the beta subunit of RNA polymerase (rpoB). At least 95% of rifampin-resistant isolates have mutations in rpoB, and the mutations are clustered in a small region. About 40 distinct point mutations and in-frame insertions and deletions in rpoB have been identified, but point mutations in two codons, those coding for Ser(531) and His(526), are seen in about 70% of rifampin-resistant clinical isolates, with Ser(531)-to-Leu (TCG-to-TGG) mutations being by far the most common. To explore this phenomenon, we isolated independent, spontaneous, rifampin-resistant mutant versions of well-characterized M. tuberculosis laboratory strain H37Rv by plating 100 separate cultures, derived from a single low-density inoculum, onto rifampin-containing medium. Rifampin-resistant mutants were obtained from 64 of these cultures. Although we anticipated that the various point mutations would occur with approximately equal frequencies, sequencing the rpoB gene from one colony per plate revealed that 39 (60.9%) were Ser(531) to Leu. We conclude that, for unknown reasons, the associated rpoB mutation occurs at a substantially higher rate than other rpoB mutations. This higher mutation rate may contribute to the high percentage of this mutation seen in clinical isolates.     

Multiple drug resistance of Mycobacterium tuberculosis

Mycobacterium tuberculosis acquires drug resistance by chromosomal mutation resulting in alterations of target molecules of drugs.     

Metabolic bifunctionality of Rv0812 couples folate and peptidoglycan biosynthesis in Mycobacterium tuberculosis

Comparative sequence analysis has enabled the annotation of millions of genes from organisms across the evolutionary tree. However, this approach has inherently biased the annotation of phylogenetically ubiquitous, rather than species-specific, functions. The ecologically unusual pathogen Mycobacterium tuberculosis (Mtb) has evolved in humans as its sole reservoir and emerged as the leading bacterial cause of death worldwide. However, the physiological factors that define Mtb’s pathogenicity are poorly understood. Here, we report the structure and function of a protein that is required for optimal in vitro fitness and bears homology to two distinct enzymes, Rv0812. Despite diversification of related orthologues into biochemically distinct enzyme families, rv0812 encodes a single active site with aminodeoxychorismate lyase and D–amino acid transaminase activities. The mutual exclusivity of substrate occupancy in this active site mediates coupling between nucleic acid and cell wall biosynthesis, prioritizing PABA over D-Ala/D-Glu biosynthesis. This bifunctionality reveals a novel, enzymatically encoded fail-safe mechanism that may help Mtb and other bacteria couple replication and division.     

结核分枝杆菌抗原Rv0173的克隆、表达与纯化

目的 在大肠杆菌中表达、纯化结核分枝杆菌Rv0173抗原,为研制新型结核病疫苗打下基础.方法 将结核杆菌抗原Rv0173的全长cDNA插入到原核表达载体pGES-4T-1中,构建成Rv0173重组质粒.将重组质粒转入大肠杆菌BL21后用IPTG进行诱导表达,通过SDS-PAGE和Western-blot鉴定重组表达蛋白.结果 获得了pGEX4T-Rv0173 重组子,Rv0173蛋白在BL21菌中获得表达,表达的蛋白条带大小约45KD,与预期结果相符.结论 成功地对结核杆菌免疫保护性抗原Rv0173进行了基因克隆与表达,为进一步研究其在结核病新型疫苗研制中的应用奠定了基础.     

结核分枝杆菌H_(37)Rv株菌体抗原及分泌抗原分析

目的对比分析人型结核枝杆菌(结核菌)H37Rv株固体培养菌、液体培养菌及分泌蛋白中抗原成分的差异,寻找结核菌的主要免疫抗原。方法应用SDS聚丙烯酰胺凝胶电泳技术、免疫印迹技术及单克隆抗体技术分析人型结核菌H37Rv株固体培养、液体培养及其分泌蛋白中的抗原成分。结果固体培养菌与液体培养菌具有基本一致的多肽抗原成分,其主要成分分子量为79000、64000、54000、50000、28000～38000、23000等,而分泌蛋白差异稍大,其主要成分分子量为66000、63000～65000、55000、42000、32000～34000、24000、16000等。单克隆抗体进一步证明66000、55000、16000为分泌蛋白。多克隆抗体免疫印迹分析表明,菌体主要免疫原有79000、54000、38000～50000、28000蛋白成分,而分泌蛋白主要免疫原为分子量66000、55000～64000、30000～34000、24000、16000成分。结论人型结核菌H37Rv株菌体蛋白和分泌蛋白具有不同的主要免疫原,将有助于认识HRv株的总体蛋白组成及寻找与结核病相关的特异性抗原成分。     

艾滋病患者淋巴结结核分枝杆菌培养及耐药情况分析

目的比较两种不同方法培养艾滋病(AIDS)患者淋巴结抗酸杆菌的阳性率,并探讨此类患者抗酸杆菌的耐药情况。方法选择该院53例AIDS患者作为研究对象,采集淋巴结,用仪器法(3D法)和L-J法进行抗酸杆菌培养及其耐药实验。结果53例AIDS患者淋巴结经两种方法培养及检测,共培养出24例阳性标本,阳性率45.28%,其中3D法培养阳性阳性率为45.28%(24/53),罗氏培养法阳性率为41.50%(22/53),两种方法阳性率比较差异无统计学意义(χ2=0.15,P>0.05)。药敏结果有9例出现耐药,耐药率为37.5%。结论 3D法与罗氏法相互结合可提高检出率,淋巴结抗酸杆菌的培养和药敏试验能为临床诊断AIDS/TB和指导用药提供重要依据。