Growth of mycobacteria on carbon monoxide and methanol

Several mycobacterial strains, such as Mycobacterium flavescens, Mycobacterium gastri, Mycobacterium neoaurum, Mycobacterium parafortuitum, Mycobacterium peregrinum, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium tuberculosis, and Mycobacterium vaccae, were found to grow on carbon monoxide (CO) as the sole source of carbon and energy. These bacteria, except for M. tuberculosis, also utilized methanol as the sole carbon and energy source. A CO dehydrogenase (CO-DH) assay, staining by activity of CO-DH, and Western blot analysis using an antibody raised against CO-DH of Mycobacterium sp. strain JC1 (formerly Acinetobacter sp. strain JC1 [J. W. Cho, H. S. Yim, and Y. M. Kim, Kor. J. Microbiol. 23:1-8, 1985]) revealed that CO-DH is present in extracts of the bacteria prepared from cells grown on CO. Ribulose bisphosphate carboxylase/oxygenase (RubisCO) activity was also detected in extracts prepared from all cells, except M. tuberculosis, grown on CO. The mycobacteria grown on methanol, except for M. gastri, which showed hexulose phosphate synthase activity, did not exhibit activities of classic methanol dehydrogenase, hydroxypyruvate reductase, or hexulose phosphate synthase but exhibited N,N-dimethyl-4-nitrosoaniline-dependent methanol dehydrogenase and RuBisCO activities. Cells grown on methanol were also found to have dihydroxyacetone synthase. Double immunodiffusion revealed that the antigenic sites of CO-DHs, RuBisCOs, and dihydroxyacetone synthases in all mycobacteria tested are identical with those of the Mycobacterium sp. strain JC1 enzymes.     

Components derived from Pelargonium stimulate macrophage killing of Mycobacterium species

Aims:  To determine the capacity of extracts of Pelargonium reniforme and Pelargonium sidoides , plants of the Geraniaceae family, to stimulate the uptake and killing of mycobacteria by murine macrophages and to identify the constituents that are responsible.
Methods and results:  Bioassay-guided fractionation of aqueous P. reniforme extracts yielded five chemically distinct structures with the capacity to increase the rate of intracellular killing by macrophages. These were: gallic acid, methyl gallate, myricetin and quercitin-3- O -β- d -glucoside, in addition to the previously unrecognized constituent 1- O -(2-(4-methoxyphenyl)ethyl-6- O -galloyl-glucopyranoside. Kinetics of intracellular accumulation of Mycobacterium tuberculosis and Mycobacterium fortuitum by macrophages were indistinguishable; pure preparations of the four previously known plant constituents stimulated macrophage killing, but not uptake, of M. tuberculosis and M. fortuitum equally well.
Conclusions:  A number of distinct molecular species are present in the medicinal plant P. reniforme that stimulate the killing of the intracellular pathogen M. tuberculosis.
Significance and Impact of the Study:  These observations support the view that Pelargonium extracts may have utility in the treatment of tuberculosis.     

Antigenic glycolipids of Mycobacterium fortuitum based on trehalose acylated with 2-methyloctadec-2-enoic acid

Three characteristic glycolipids were identified in extracts of 10 representative strains of Mycobacterium fortuitum. The two most polar major lipids were shown to be antigens with strong reactions against homologous antiserum and sera raised against Mycobacterium tuberculosis H37Rv. Both of these lipids were shown to be acyl trehaloses, containing substantial amounts of 2-methyloctadec-2-enoic acid in addition to straight-chain and monounsaturated acids. These lipids correspond to 'mycoside F', lipids previously identified by infra-red spectroscopy in extracts of Myco. fortuitum.     

Reduced expression of nuclear cyclic adenosine 5'-monophosphate response element-binding proteins and IFN-gamma promoter function in disease due to an intracellular pathogen

Mycobacterium tuberculosis-induced IFN-gamma protein and mRNA expression have been shown to be reduced in tuberculosis patients, compared with healthy tuberculin reactors. To determine whether this decrease was associated with reduced activity of the IFN-gamma promoter, we first studied binding of nuclear proteins to the radiolabeled proximal IFN-gamma promoter (-71 to -40 bp), using EMSAs with nuclear extracts of freshly isolated peripheral blood T cells. Nuclear extracts of T cells from most tuberculosis patients showed markedly reduced expression of proteins that bind to the proximal IFN-gamma promoter, compared with findings in nuclear extracts of T cells from healthy tuberculin reactors. These DNA-binding complexes contained CREB proteins, based on competitive EMSAs, supershift assays, and Western blotting with an anti-CREB Ab. Transient transfection of PBLs with a luciferase reporter construct under the control of the IFN-gamma promoter revealed reduced IFN-gamma promoter activity in tuberculosis patients. Transient transfection of Jurkat cells with a dominant-negative CREB repressor plasmid reduced IFN-gamma promoter activity. These data suggest that reduced expression of CREB nuclear proteins in tuberculosis patients results in decreased IFN-gamma promoter activity and reduced IFN-gamma production.     

痰涂片标本用于结核分枝杆菌耐药性检测的研究

目的 研究抗酸染色结核分枝杆菌(简称结核杆菌)阳性痰涂片标本直接用于耐药性检测的方法。方法 对18株临床分离培养的结核杆菌用利福平进行药敏试验。分别提取菌株DNA和与之对应的痰涂片标本的菌体DNA,用聚合酶链反应(PcR)扩增ropB基因后进行固相杂交和核酸测序检测结核杆菌的耐药性。结果 18株结核杆菌中有12株对利福平耐药。经PCR扩增的ropB片段与探针杂交后,敏感菌株未发现rpoB基因的突变,自耐药菌株提取的DNA中rpoB突变体的检出率为100％(12／12),痰涂片提取DNA的检出率为91．7％(11／12)。所有耐药菌株DNA与痰涂片DNA核酸测序结果相吻合,都有rpoB基因核心区域碱基突变。结论 抗酸染色痰涂片阳性标本可直接用于检测结核杆菌利福平耐药基因rpoB突变体,是一种值得临床实验室推广使用的耐药菌诊断方法。     

Antimycobacterial evaluation of Peruvian plants

We present the results of an antimycobacterial screening of 270 Peruvian plant samples representing 216 species from 171 genera in 63 families. Dichloromethane extracts were tested at a concentration of 50 microg/ml for inhibition of Mycobacterium tuberculosis in radiometric culture. Slightly more than half of the samples tested showed inhibition of M. tuberculosis at this concentration.     

Application of an enzyme immunoassay for detecting antibodies in sera of Macaca fascicularis naturally exposed to Mycobacterium tuberculosis.

An enzyme immunoassay (EIA) was developed for detecting mycobacterial antibodies in the sera of 22 Macaca fascicularis following a natural outbreak of tuberculosis. EIAs were conducted using four antigens (lysozyme, triton, or deoxycholate extracts of Mycobacterium tuberculosis or a purified protein derivative) and two conjugates (protein A or antihuman). Mycobacterial antibodies were detected in two of two culture-positive monkeys, in nine of ten tuberculin test-suspect monkeys (culture-negative), and in five of ten tuberculin test-negative monkeys (culture-negative). Results indicate EIA may be of practical value in detecting monkeys exposed to M. tuberculosis.     

牛分支杆菌与肺结核分支杆菌基因组的比较

通过比较基因组学的方法研究发现,牛分支杆菌与肺结核杆菌基因组的同源性为99．95％,但在牛分枝杆菌基因组中有11个缺失区,大小从1kb到12．7kb,遗传信息的缺失引起牛分枝杆菌的基因组减小;牛分枝杆菌与肺结核分枝杆菌H37Rv间存在着2437个单核苷酸多态性（SNPs）,与肺结核分枝杆菌CDC1551间存在着2423个单核苷酸多态性（SNPs）,牛分支杆菌与肺结核分枝杆菌在编码细胞壁和分泌蛋白上变异程度也是巨大的。研究结果揭示了牛分支杆菌与肺结核分枝杆菌的遗传关系,为研究分支杆菌疫苗和诊断试剂提供理论依据,对牛肺结核病的防治有着非常重要的意义。     

Recognition of novel glycolipid antigens from smooth variants of Mycobacterium tuberculosis

A major polar and three minor slightly less polar glycolipids were identified in extracts of two smooth (Canetti) strains of Mycobacterium tuberculosis. Immunostaining on thin-layer chromatograms and enzyme-linked immunosorbent assay (ELISA) of purified lipids demonstrated that the major and the two most polar of the minor glycolipids are potent antigens, reacting with homologous antisera and also with that raised against the type strain (H37Rv).     

The Mycobacterium bovis homologous protein of the Mycobacterium tuberculosis serine/threonine protein kinase Mbk (PknD) is truncated

We previously identified a 70-kDa serine/threonine protein kinase (MbK or PknD) from Mycobacterium tuberculosis Erdman containing a transmembrane domain and bearing a 270-amino acid N-terminal kinase domain. With the use of a polyclonal serum, Mbk has now been identified by Western blotting in protein extracts from M. tuberculosis and confirmed to be localised in the envelope. An identical mbk gene has been found by sequencing different M. tuberculosis and M. africanum strains. Surprisingly, in two virulent M. bovis strains and four different strains of M. bovis BCG, an additional adenine after position 829 of the open reading frame was found that produces a frame shift resulting in a predicted truncated, presumably free cytoplasmic protein, encoding only the N-terminal 30-kDa Mbk kinase domain. This sequence polymorphism has been confirmed by Western blot analysis of M. bovis BCG protein extracts.     

猫爪草提取物对结核分枝杆菌临床分离株的可能作用靶标

利用双向电泳技术, 对猫爪草提取物作用前后的结核分枝杆菌临床分离株的全细胞蛋白表达图谱进行差异比较和分析, 发现其中22个蛋白质斑点的浓度具有差异,利用基质辅助激光解吸/电离飞行时间质谱技术, 对其中4个表达明显下调和1个明显上调的蛋白质斑点进行分析鉴定, 获得5个明确的肽质量指纹图谱.通过数据库检索, 确定这5个蛋白质分别为S-腺苷甲硫氨酸合成酶、吲哚-3-甘油磷酸合酶、烯酰-CoA水合酶、琥珀酰辅酶A合成酶和60 kD的分子伴侣2.其中前4个分子是首次报道参与结核分枝杆菌的重要生理活动.该结果有助于了解猫爪草提取物对结核分枝杆菌生理的影响, 为进一步确定中药猫爪草提取物对结核分枝杆菌的作用靶标和机理提供了基础.     

Protein tyrosine phosphorylation in Mycobacterium tuberculosis

Abstract Crude cell extracts from three strains of Mycobacterium tuberculosis were analyzed for the presence of proteins possessing phosphorylated tyrosine residues. A protein migrating at approximately 55 kDa was detected using an antiphosphotyrosine monoclonal antibody. In addition, less predominant bands were observed between 50 kDa and 60 kDa. That M. tuberculosis contains specific tyrosine phosphorylated proteins implies that M. tuberculosis has tyrosine kinase activity. Examination of other, non-pathogenic mycobacterium species yielded no major antiphosphotyrosine reactive proteins. This suggests that the antiphosphotyrosine reactive protein is specific to M. tuberculosis strains. These results provide evidence that M. tuberculosis contains an antiphosphotyrosine reactive protein.     

Comparative cytotoxic and anti-tuberculosis activity of Aplysina caissara marine sponge crude extracts

Three crude extracts of Aplysina caissara, a marine sponge endemic to Brazil, were tested against a hepatoma cell line and Mycobacterium tuberculosis. The results demonstrate that all extracts are toxic and capable of inhibiting cellular growth. Additionally, the extracts produced morphological aberrations and inhibited cell attachment to culture substrates. These effects were dose/time dependent. Our results also suggest that reactive oxygen species (ROS) production is not involved in the cytotoxic processes levied by the extracts employed in this study and that active metabolites are likely to be present in the polar fractions of the crude extracts. Finally, our results indicate that all three extracts exhibit a moderate anti-tuberculosis capacity, and that the removal of an extract's lipid fraction appears to diminish this activity.     

Detection of antituberculous activity in plant extracts

Ten of 408 ethanolic extracts of plants inhibited growth of Mycobacterium tuberculosis H37Rv at dilutions of 1 in 160 to 1280 and a high proportion of the other extracts inhibited growth at lower dilutions. Some of the plants, after purification, may be therapeutically useful. In view of the environmental threat to many plant species, screening of such plants for potential therapeutic properties, including antituberculous and other antimicrobial activities, are urgently required before they are lost forever.     

Inhibition of mycobacterial arylamine N-acetyltransferase contributes to anti-mycobacterial activity of Warburgia salutaris

In this study, we show that extracts and a purified compound of Warburgia salutaris exhibit anti-mycobacterial activity against Mycobacterium tuberculosis H37Rv and Mycobacterium bovis BCG Pasteur. The extracts did not inhibit growth of Escherichia coli and were not toxic to cultured mammalian macrophage cells at the concentrations at which anti-mycobacterial activity was observed. The extract and pure compound inhibited pure recombinant arylamine N-acetyltransferase (NAT), an enzyme involved in mycobacterial cell wall lipid synthesis. Moreover, neither extract nor pure compound inhibited growth of a strain of M. bovis BCG in which nat has been deleted suggesting that NAT may indeed be a target within the mycobacterial cell. The purified compound is a novel drimane sesquiterpenoid lactone, 11alpha-hydroxycinnamosmolide. These studies show that W. salutaris is a useful source of anti-tubercular compounds for further analysis and supports the hypothesis of a link between NAT inhibition and anti-mycobacterial activity.     

一种新型抗结核先导化合物S28 对结核分枝杆菌蛋白质组表达的影响

目的 探讨从化合物库中高通量筛选得到的、可有效抑制结核分枝杆菌生长和繁殖的新型活性化合物S28 的作用机制及其可能的作用靶点。方法 采用双向电泳技术, 比较分析活性化合物作用于结核分枝杆菌H37Ra 前、后的全细胞蛋白表达差异。结果 13 个蛋白质斑点表达下调, 对其中6 个改变明显的蛋白质斑点进行基质辅助激光解吸/ 电离飞行时间质谱分析, 成功测定2 个蛋白质斑点。数据库检索分析确定这2 个差异蛋白点分别为延长因子Tu 和短链脱氢酶, 是参与蛋白质翻译和氧化呼吸、能量代谢等生理过程的重要蛋白。结论 为 进一步深入探索新型抗结核活性化合物的作用机制和可能的靶点提供研究基础和方向。     

Molecular study of the T cell repertoire in family contacts and patients with leprosy

The specificity of lymphocyte proliferative responses of 22 family contacts and 7 patients with leprosy were analyzed using Ag fractions from soluble extracts of Mycobacterium leprae and Mycobacterium tuberculosis. Fractions 10-100 kDa m.w. from each extract were separated by SDS-polyacrylamide gel electrophoresis, electroblotted to nitrocellulose membrane and solubilized for use in lymphocyte culture. The main immunogenic fractions for both contacts and patients had m.w. of 12,000 to 22,000, 35,000 to 40,000, and 65,000. Determinants which were either distinct or shared by the two extracts were active in each of the immunogenic fractions. Lymphocyte proliferation following stimulation with separated Ag was found also in five subjects who failed to respond to the whole soluble extracts. Stimulatory synthetic peptides were identified for the 65 kDa protein Ag. This technique has permitted the screening of the T cell immune repertoire for the identification of the immunodominant Ag which merit further purification and molecular characterization.     

结核分枝杆菌与巨噬细胞相互作用的研究进展

结核分枝杆菌（Mycobacterium tuberculosis,MTB）是一种典型的胞内致病菌,巨噬细胞是MTB在体内的主要宿主细胞。巨噬细胞具有强大的吞噬功能,在机体固有免疫和适应性免疫中均发挥着重要作用,可有效保护宿主免受结核分枝杆菌的感染。MTB在与宿主巨噬细胞的长期相互作用过程中,逐渐形成多种逃避杀灭的有效策略,得以在宿主体内存活并增殖。该文从巨噬细胞抗MTB感染及MTB逃避巨噬细胞杀灭两个方面综述国内外的研究进展。     

Mycobacterium tuberculosis possesses a functional enzyme for the synthesis of vitamin C, L-gulono-1,4-lactone dehydrogenase

The last step of the biosynthesis of L-ascorbic acid (vitamin C) in plants and animals is catalyzed by L-gulono-1,4-lactone oxidoreductases, which use both L-gulono-1,4-lactone and L-galactono-1,4-lactone as substrates. L-gulono-1,4-lactone oxidase is missing in scurvy-prone, vitamin C-deficient animals, such as humans and guinea pigs, which are also highly susceptible to tuberculosis. A blast search using the rat L-gulono-1,4-lactone oxidase sequence revealed the presence of closely related orthologs in a limited number of bacterial species, including several pathogens of human lungs, such as Mycobacterium tuberculosis, Pseudomonas aeruginosa, Burkholderia cepacia and Bacillus anthracis. The genome of M. tuberculosis, the etiologic agent of tuberculosis, encodes a protein (Rv1771) that shows 32% identity with the rat L-gulono-1,4-lactone oxidase protein. The Rv1771 gene was cloned and expressed in Escherichia coli, and the corresponding protein was affinity-purified and characterized. The FAD-binding motif-containing Rv1771 protein is a metalloenzyme that oxidizes L-gulono-1,4-lactone (Km 5.5 mm) but not L-galactono-1,4-lactone. The enzyme has a dehydrogenase activity and can use both cytochrome c (Km 4.7 microm) and phenazine methosulfate as exogenous electron acceptors. Molecular oxygen does not serve as a substrate for the Rv1771 protein. Dehydrogenase activity was measured in cellular extracts of a Mycobacterium bovis BCG strain. In conclusion, M. tuberculosis produces a novel, highly specific L-gulono-1,4-lactone dehydrogenase (Rv1771) and has the capacity to synthesize vitamin C.     

Application of an easy and reliable method for sulfolipid-I detection in the study of its distribution in Mycobacterium tuberculosis strains

Sulfolipid-I (SL-I) is a specific Mycobacterium tuberculosis glycolipid that has been involved in the mechanisms of tuberculoid infection. Until now, a limited number of M. tuberculosis strains have been studied to ascertain their SL-I content, mainly due to the laborious techniques of purification used: DEAE-cellulose column chromatography (DEAE) or extensive solvent extractions. We designed a two-dimensional thin layer chromatographic (2D-TLC) system which allows the easy and reliable detection of SL-I in small amounts of M. tuberculosis-free glycolipid extracts without previous purification. A characteristic SL-I signal was clearly identified by a differential cresyl violet metachromatic stain. Seven clinical isolates, M. tuberculosis H(37)Ra, H(37)Rv and Canetti strains were tested by DEAE and the 2D-TLC system. Identical results were found using both methodologies. The 2D-TLC methodology devised could be applied to a large number of strains to ascertain easily the distribution of SL-I in the strains of M. tuberculosis species.