The promoter of Rv0560c is induced by salicylate and structurally-related compounds in Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a major global health threat. During infection, bacteria are believed to encounter adverse conditions such as iron depletion. Mycobacteria synthesize iron-sequestering mycobactins, which are essential for survival in the host, via the intermediate salicylate. Salicylate is a ubiquitous compound which is known to induce a mild antibiotic resistance phenotype. In M. tuberculosis salicylate highly induces the expression of Rv0560c, a putative methyltransferase. We identified and characterized the promoter and regulatory elements of Rv0560c. P(Rv0560c) activity was highly inducible by salicylate in a dose-dependent manner. The induction kinetics of P(Rv0560c) were slow, taking several days to reach maximal activity, which was sustained over several weeks. Promoter activity could also be induced by compounds structurally related to salicylate, such as aspirin or para-aminosalicylic acid, but not by benzoate, indicating that induction is specific to a structural motif. The -10 and -35 promoter elements were identified and residues involved in regulation of promoter activity were identified in close proximity to an inverted repeat spanning the -35 promoter element. We conclude that Rv0560c expression is controlled by a yet unknown repressor via a highly-inducible promoter.     

Salicylate uniquely induces a 27-kDa protein in tubercle bacillus

Salicylate was found to uniquely induce a 27-kDa protein in Mycobacterium tuberculosis complex organisms but not in Mycobacterium smegmatis or Escherichia coli. The structural analogue antitubercular para-amino-salicylate also induced the 27-kDa protein but to a somewhat lower level than salicylate. Other structural analogues such as benzoic acid and acetyl salicylic acid (aspirin) did not induce the 27-kDa protein. Western blot analysis indicated that the 27-kDa protein was localized mainly in the cytoplasm. The 27-kDa protein was not expressed at different growth phases in the absence of salicylate. The 27-kDa protein was identified as a putative benzoquinone methyltransferase (Rv0560c), which has several homologues in the M. tuberculosis genome. The cloned 27-kDa gene was found to express constitutively in E. coli, M. smegmatis and BCG with or without salicylate.     

Molecular cloning, overexpression and biochemical characterization of hypothetical beta-lactamases of Mycobacterium tuberculosis H37Rv

Aim:  Molecular cloning, overexpression and biochemical characterization of the genes from the Mycobacterium tuberculosis H37Rv genome having hypothetical β-lactamases activity.
Methods and Results:  Analysis of the M. tuberculosis H37Rv genome revealed that Rv 2068c , Rv 0406 c and Rv 3677 c gene products were predicted to exhibit β-lactamases activity. All the three genes were cloned in pET28a vector and overexpressed in C41 (DE3) Escherichia coli cells. The His-tagged recombinant proteins were confirmed by immunoblotting and were shown to have β-lactamase activity by the hydrolysis of nitrocefin and other β-lactams. Catalytic parameters for all the recombinant proteins were derived followed by the enzyme inhibition studies. Antibiotic susceptibility studies using the recombinant strains showed an increased resistance against different classes of β-lactam antibiotics.
Conclusion:  The study revealed the possibility of more than one gene in M. tuberculosis , encoding proteins having β-lactamase or β-lactamase-like activity, giving wide spectrum of resistance against β-lactams.
Significance and Impact of the Study:  Systematic study of hypothetical β-lactamases of M. tuberculosis and related species and their correlation with β-lactam and inhibitor susceptibility profile might be useful in developing new antibiotic regime for the treatment of tuberculosis caused by multiple drug resistant (MDR) strains.     

Rv3303c of Mycobacterium tuberculosis protects tubercle bacilli against oxidative stress in vivo and contributes to virulence in mice

Ability of Mycobacterium tuberculosis to survive under oxidative stress in vivo is an important aspect of pathogenesis. Rv3303c gene from M. tuberculosis encodes an NAD(P)H quinone reductase. These enzymes have been shown to manage oxidative stress in other pathogenic bacteria. We have hypothesized that Rv3303c protein will remove reactive oxygen species released by the host and hence reduce oxidative stress to M. tuberculosis. rv3303c was PCR cloned and the purified recombinant enzyme reduced superoxide generator menadione. Antisense and sense RNA constructs of rv3303c were electroporated in M. tuberculosis H37Rv. The transformants were characterized by difference in expression of specific mRNA and protein. Antisense transformants were markedly reduced in virulence as compared to sense transformants as judged by several parameters such as weight and survival of infected mice, growth in vivo, colonization and histopathology of lungs. In the presence of menadione, the sense transformant was more resistant to killing in vitro than the antisense transformant. It may be concluded that the rv3303c gene contributes to virulence of M. tuberculosis in vivo and this might be mediated in part by increased resistance to reactive oxygen intermediates thereby enhancing intracellular growth and colonization.     

Characterization of three glycosyltransferases involved in the biosynthesis of the phenolic glycolipid antigens from the Mycobacterium tuberculosis complex

Mycobacterium tuberculosis and Mycobacterium leprae, the two main mycobacterial pathogens in humans, produce highly specific long chain beta-diols, the dimycocerosates of phthiocerol, and structurally related phenolic glycolipid (PGL) antigens, which are important virulence factors. In addition, M. tuberculosis also secretes glycosylated p-hydroxybenzoic acid methyl esters (p-HBAD) that contain the same carbohydrate moiety as the species-specific PGL of M. tuberculosis (PGL-tb). The genes involved in the biosynthesis of these compounds in M. tuberculosis are grouped on a 70-kilobase chromosomal fragment containing three genes encoding putative glycosyltransferases: Rv2957, Rv2958c, and Rv2962c. To determine the functions of these genes, three recombinant M. tuberculosis strains, in which these genes were individually inactivated, were constructed and biochemically characterized. Our results demonstrated that (i) the biosynthesis of PGL-tb and p-HBAD involves common enzymatic steps, (ii) the Rv2957, Rv2958c, and Rv2962c genes are involved in the formation of the glycosyl moiety of the two classes of molecules, and (iii) the product of Rv2962c catalyzes the transfer of a rhamnosyl residue onto p-hydroxybenzoic acid ethyl ester or phenolphthiocerol dimycocerosates, whereas the products of Rv2958c and Rv2957 add a second rhamnosyl unit and a fucosyl residue to form the species-specific triglycosyl appendage of PGL-tb and p-HBAD. The recombinant strains produced provide the tools to study the role of the carbohydrate domain of PGL-tb and p-HBAD in M. tuberculosis pathogenesis.     

两株超级广泛耐药结核分枝杆菌全基因组比较分析

【目的】结合现有数据,通过对两株临床超级广泛耐药的结核分枝杆菌全基因组的测序和分析,发现其型别相关的突变位点,解释发生广泛耐药的基因组突变机制。【方法】利用Solexa第二代测序技术对两株广泛耐药结核分枝杆菌(FJ05194和GuangZ0019)进行全基因组测序分析。以H37Rv为参考序列得到两株广泛耐药菌株的单核苷酸多态性(SNPs),构建系统发育树鉴定菌株型别,判断突变位点中型别相关和非型别相关的SNPs。定位SNPs所在的基因组区域,对型别相关的突变基因进行KEGG通路的富集分析,对非型别相关的突变基因和间隔区判断是否与耐药相关。【结果】两株广泛耐药菌株分别属于Lineage2和Lineage4型别,两菌株在碱基替换方面存在差异性,Lineage2型别相关的基因功能富集于ABC转运蛋白和核苷酸切除修复的通路。耐药方面,发现了已知的耐药相关基因的突变(rpoB、katG、rpsl、gyrA、gyrB、embB和ethA等),但卷曲霉素和卡那霉素相关的rrs、tlyA和eis启动子区域未发生突变,不足以解释其耐药性的产生。与最新报道的候选耐药基因比较,发现了卷曲霉素和卡那霉素相关的突变(Rv1393c、Rv0265c和narX等)和外排泵相关的pstB、Rv2333c和Rv2687c突变。【结论】结核分枝杆菌Lineage2型别相关的SNPs中含有影响结核分枝杆菌突变率和耐药性的突变。对于两株超级广泛耐药的结核菌,已知的激活药物或药靶相关的单耐药基因突变集合不能完全解释其广泛耐药性,还涉及新候选结核耐药基因、外排泵和补偿等其他潜在机制的相关基因突变。     

Expression of the naphthoate synthase gene in Mycobacterium tuberculosis in a self-generated oxygen depleted liquid culture system

Mycobacterium tuberculosis has been classified for decades as a strict aerobic species. Whole genome sequencing of the type culture strain H37Rv has revealed the presence of a full set of genes allowing for anaerobic metabolism. Naphthoate synthase (menB) is a key enzyme required for the synthesis of menaquinone, which plays a crucial role in anaerobic electron transport, ultimately resulting in the formation of energy generating intermediates. Interrupting the synthesis of this enzyme will interfere with the production of menaquinone and therefore this enzyme is a potential drug target. This study serves to investigate the role of naphtoate synthase in the survival of M. tuberculosis H37Rv when incubated under oxygen limiting conditions of unagitated liquid culture over 15 weeks. M.?tuberculosis H37Rv was grown in Middlebrook 7H9 media. The tubes were kept undisturbed at 37?°C for up to 15 weeks. At selected time points, aliquots of cells were removed and frozen. RNA was simultaneously extracted from all aliquots. The RNA was converted to cDNA for Real-Time PCR on the ABI 7000 SDS. Gene expression was normalized against 16S RNA quantities at each time point. A systematic increase in the expression of the menB gene product was observed over the incubation period with a 4.3-fold increase seen at week 6 (P?相似文献   

Co-induction of methyltransferase Rv0560c by naphthoquinones and fibric acids suggests attenuation of isoprenoid quinone action in Mycobacterium tuberculosis

The superoxide generator menadione was previously demonstrated as an inducer of growth stage dependent protein patterns in Mycobacterium tuberculosis. The present study refines this observation by characterizing a novel 27-kDa protein that had not been observed in previous studies relying on younger cultures. A very similar response, based on two-dimensional gel electrophoretic analyses, was induced by the closely related naphthoquinone plumbagin. The 27-kDa protein was also induced by the pro-oxidant peroxisome proliferator gemfibrozil and to a lesser extent by the structurally related compounds fenofibrate and clofibrate. N-terminal sequence data of proteolytic fragments from the 27-kDa protein demonstrated its identity with protein Rv0560c, previously demonstrated to be inducible by salicylate, which also possesses peroxisome proliferating properties. Protein Rv0560c bears three conserved motifs characteristic of S-adenosylmethionine-dependent methyltransferases. Further sequence similarities suggest a function in the bio syn thesis of isoprenoid compounds, e.g., tocopherol, ubiquinone, and sterols. Such involvement is supported by the recognized yet unexplained widespread interference of menadione, salicylate, and fibrates with the isoprenoid quinones ubiquinone, menaquinone, and vitamin K. Induction of Rv0560c by fibrates, salicylate, and naphthoquinones is thus suggested to be caused by action on the plasma membrane, reminiscent of cytochrome P450BM-3 induction by fibrates in Bacillus megaterium, which catalyzes the hydroxylation of fatty acids and thus modulates membrane properties.     

结核杆菌活动期高表达基因Rv1776c的克隆及其在耻垢分枝杆菌中的表达

目的 构建表达结核分枝杆菌Rv1776c基因的重组耻垢分支杆菌,并鉴定该基因在重组耻垢分支杆菌中的活性。方法 采用PCR技术克隆结核分枝杆菌Rv1776c基因,构建大肠埃希菌‒分支杆菌穿梭表达质粒pMV-Rv1776c,通过酶切和测序鉴定其正确性,用电穿孔法将重组质粒转染到耻垢分支杆菌mc2155中。以SDS-PAGE及Western blot检测证实Rv1776c蛋白在重组耻垢分支杆菌内的表达。结果 重组耻垢分支杆菌构建成功,生长曲线说明重组质粒不会影响耻垢分支杆菌的体外生长;SDS-PAGE及Western blot检测证实Rv1776c在耻垢分枝杆菌内表达出相对分子量约56 kD的Rv1776c蛋白。结论 成功构建了Rv1776c基因的穿梭质粒pMV-Rv1776c,且该质粒在耻垢分枝杆菌内具有生物活性,为进一步研究其表达产物的功能提供基础。     

Large Mg(2+)-dependent currents are associated with the increased expression of ALR1 in Saccharomyces cerevisiae

Mycobacteria adapt to a decrease in oxygen tension by entry into a non-replicative persistent phase. It was shown earlier that the two-component system, DevR-DevS, was induced in Mycobacterium tuberculosis and Mycobacterium bovis BCG cultures during hypoxia, suggesting that it may play a regulatory role in their adaptation to oxygen limitation. The presence of a homologous genetic system in Mycobacterium smegmatis was predicted by scanning its unfinished genome sequence with devR and devS genes of M. tuberculosis. Rv3134c, which is cotranscribed with devR-devS in M. tuberculosis, was also present in M. smegmatis at a similar location upstream from devR. The expression of all three genes was induced at the RNA and protein levels in M. smegmatis cultures grown under microaerobic and anaerobic conditions. The M. smegmatis genome also contained the hspX gene, encoding chaperone alpha-crystallin, Acr, that was induced during hypoxia. The similarity in sequences and hypoxia-responsive behaviour of devR-devS, Rv3134c and hspX genes in M. smegmatis and M. tuberculosis suggests that the molecular mechanisms involved in the dormancy response are likely conserved in these two species. M. smegmatis could therefore serve as a useful model for the delineation of the hypoxia response in general and DevR-DevS regulated pathways in particular.     

Proteomic analysis of sensitive and multi drug resistant <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> strains

Multidrug-resistant tuberculosis (MDR-TB) is caused by bacteria that are resistant to the most effective anti TB drugs (Isoniazid and Rifampicin) with or without resistance to other drugs. Novel intervention strategies to eliminate this disease based on finding proteins can be used for designing new drugs or new and reliable kits for diagnosis. The aim of this study was to compare the protein profile of MDR-TB with sensitive isolates. Two-dimensional gel electrophoresis (2DE) along with mass spectrometry is a powerful and effective tool to identification and characterization of Mycobacterium tuberculosis. Two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was used for diagnosis and comparison of proteins. We identified 14 protein spots in MDR-TB isolates that 2DE analysis showed these spots absent in M. tuberculosis sensitive isolates (Rv1876, Rv0379, Rv0147, Rv2031c, Rv3597c, Rv1886c, MT0493, Rv0440, Rv3614c, Rv1626, Rv0443, Rv0475, Rv3057 and unknown protein. The results showed 22 protein spots which were up regulated (or expressed) by the MDR-TB isolates, (Rv1240, Rv3028c, Rv2971, Rv2114c, Rv3311, Rv3699, Rv1023, Rv1308, Rv3774, Rv0831c, Rv2890c, Rv1392, Rv0719, Rv0054, Rv3418c, Rv0462, Rv2215, Rv2986c, Rv3248c and Rv1908c)). Two up regulated protein spots were identified in sensitive isolate (Rv1133c and Rv0685). These data will provide valuable clues in further investigation for suitable TB rapid tests or drug targets against drug resistant and sensitive of M. tuberculosis.     

Molecular dissection of the biosynthetic relationship between phthiocerol and phthiodiolone dimycocerosates and their critical role in the virulence and permeability of Mycobacterium tuberculosis

Phthiocerol dimycocerosates and related compounds are important molecules in the biology of Mycobacterium tuberculosis, playing a key role in the permeability barrier and in pathogenicity. Both phthiocerol dimycocerosates, the major compounds, and phthiodiolone dimycocerosates, the minor constituents, are found in the cell envelope of M. tuberculosis, but their specific roles in the biology of the tubercle bacillus have not been established yet. According to the current model of their biosynthesis, phthiocerol is produced from phthiodiolone through a two-step process in which the keto group is first reduced and then methylated. We have previously identified the methyltransferase enzyme that is involved in this process, encoded by the gene Rv2952 in M. tuberculosis. In this study, we report the construction and biochemical analyses of an M. tuberculosis strain mutated in gene Rv2951c. This mutation prevents the formation of phthiocerol and phenolphthiocerol derivatives, but leads to the accumulation of phthiodiolone dimycocerosates and glycosylated phenolphthiodiolone dimycocerosates. These results provide the formal evidence that Rv2951c encodes the ketoreductase catalyzing the reduction of phthiodiolone and phenolphthiodiolone to yield phthiotriol and phenolphthiotriol, which are the substrates of the methyltransferase encoded by gene Rv2952. We also compared the resistance to SDS and replication in mice of the Rv2951c mutant, deficient in synthesis of phthiocerol dimycocerosates but producing phthiodiolone dimycocerosates, with those of a wild-type strain and a mutant without phthiocerol and phthiodiolone dimycocerosates. The results established the functional redundancy between phthiocerol and phthiodiolone dimycocerosates in both the protection of the mycobacterial cell and the pathogenicity of M. tuberculosis in mice.     

Deciphering the genetic bases of the structural diversity of phenolic glycolipids in strains of the Mycobacterium tuberculosis complex

Phenolic glycolipids (PGL) play a major role in the virulence of mycobacteria, notably in strains of the Mycobacterium tuberculosis complex and in Mycobacterium leprae. The structure of the carbohydrate domain of these compounds is highly variable, and the genetic bases for these variations remain unknown. We demonstrated that the monoglycosylated PGL formed by Mycobacterium bovis differs from the triglycosylated PGL synthesized by M. tuberculosis (PGL-tb) because of the following two genetic defects: a frameshift mutation within the gene Rv2958c, encoding a glycosyltransferase involved in the transfer of the second rhamnosyl residue of the PGL-tb, and a deletion of a region that encompasses two genes, which encode a GDP-D-mannose 4,6-dehydratase and a GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase/reductase, required for the formation of activated L-fucose. Expression of these three genes in M. bovis BCG allowed synthesis of PGL-tb in this recombinant strain. Additionally, we showed that all M. bovis, Mycobacterium microti, Mycobacterium pinnipedii, and some Mycobacterium africanum strains harbor the same frameshift mutation in their Rv2958c orthologs. Consistently, the structure of PGLs purified from M. africanum (harboring the Rv2958c mutation) and M. pinnipedii strains revealed that these compounds are monoglycosylated PGL. These findings explain the specificity of PGL-tb production by some strains of the M. tuberculosis complex and have important implications for our understanding of the evolution of this complex.