Differential expression of NF-κB in mycobacteria infected THP-1 affects apoptosis

The present study was conducted to see the role of NF-κB in virulent (Mycobacterium tuberculosis H37Rv) and avirulent (M. tuberculosis H37Ra) mycobacterial infection in THP-1 cells. To inactivate NF-κB, pCMV-IκBαM dn containing THP-1 cell line was generated which showed marked increase in apoptosis with M. tuberculosis H37Rv and M. tuberculosis H37Ra. Infected THP-1-IκBαM dn cells showed decrease in mitochondrial membrane potential, cytochrome c release, activation of caspase-3 and enhanced TNF-α production. Increase in apoptosis of infected THP-1-IκBαM dn cells resulted in inhibition of intracellular mycobacterial growth. Differential NF-κB activation potential was observed with M. tuberculosis H37Rv and M. tuberculosis H37Ra. Both the strains activated NF-κB after 4 h in THP-1 cells however after 48 h only M. tuberculosis H37Rv activated NF-κB which lead to up-regulation of bcl-2 family anti-apoptotic member, bfl-1/A1. Our results indicated that NF-κB activation may be a determinant factor for the success of virulent mycobacteria within macrophages.     

Deciphering kas operon locus in Mycobacterium aurum and genesis of a recombinant strain for rational-based drug screening

Aims: To generate a recombinant Mycobacterium aurum strain for screening of antimycobacterial compounds affecting fatty acid synthase type II (FAS-II) elongation pathway. Methods and Results: kas operon locus was delineated in M. aurum, a fast growing nonpathogenic strain. Cloning and sequencing all the genes of the operon showed similar organization and sequence similarities with Mycobacterium tuberculosis (H37Rv) orthologues. Further, we cloned the upstream region of M. tuberculosis kas operon in fusion with lacZ reporter gene and put it in M. aurum. Recombinant M. aurum strain showed continued expression of reporter gene throughout the growth while an increased expression of the reporter gene was noticed only after treatment with FAS-II pathway inhibitors. Swapping of the regulatory sequence aborts the increased reporter gene expression after same antibiotic treatments. Conclusions: kas operon genes are similarly organized in M. tuberculosis and M. aurum. H37Rv kas operon promoter upregulates the reporter gene expression in M. aurum only upon treatment with drugs inhibiting FAS-II pathway. Significance and Impact of the Study: It would serve as a good second-line screen for characterization of compounds showing antimycobacterial activity in a first-line screen. With the simplicity of β-galactosidase enzyme assay the system can be easily adapted in high-throughput mode.     

Mammalian heterotrimeric G-protein-like proteins in mycobacteria: implications for cell signalling and survival in eukaryotic host cells

Mammalian heterotrimeric GTP-binding proteins (G proteins) are involved in transmembrane signalling that couples a number of receptors to effectors mediating various physiological processes in mammalian cells. We demonstrate that bacterial proteins such as a Ras-like protein from Pseudomonas aeruginosa or a 65 kDa protein from Mycobacterium smegmatis can form complexes with human or yeast nucleoside diphosphate kinase (Ndk) to modulate their nucleoside triphosphate synthesizing specificity to GTP or UTP. In addition, we demonstrate that bacteria such as M. smegmatis or Mycobacterium tuberculosis harbour proteins that cross react with antibodies against the α-, β- or the γ-subunits of heterotrimeric G proteins. Such antibodies also alter the GTP synthesizing ability of specific membrane fractions isolated from glycerol gradients of such cells, suggesting that a membrane-associated Ndk–G-protein homologue complex is responsible for part of GTP synthesis in these bacteria. Indeed, purified Ndk from human erythrocytes and M. tuberculosis showed extensive complex formation with the purified mammalian α and β G-protein subunits and allowed specific GTP synthesis, suggesting that such complexes may participate in transmembrane signalling in the eukaryotic host. We have purified the α-, β- and γ-subunit homologues from M. tuberculosis and we present their internal amino acid sequences as well as their putative homologies with mammalian subunits and the localization of their genes on the M. tuberculosis genome. Using oligonucleotide probes from the conserved regions of the α- and γ-subunit of M. tuberculosis G-protein homologue, we demonstrate hybridization of these probes with the genomic digest of M. tuberculosis H37Rv but not with that of M. smegmatis, suggesting that M. smegmatis might lack the genes present in M. tuberculosis H37Rv. Interestingly, the avirulent strain H37Ra showed weak hybridization with these two probes, suggesting that these genes might have been deleted in the avirulent strain or are present in limited copy numbers as opposed to those in the virulent strain H37Rv.     

Partial physical and functional map of aMycobacterium aurum carotenogenesis operon

The genes controlling the biosynthesis of the carotenes inMycobacterium aurum were clustered in a 10.83-kb segment. Fragments generated by endonuclease digestions of the segment were cloned into a pHLD69 shuttle vector. The plasmids so constructed were used to transform a colorless (albino)M. aurum mutant (strain A₁₁), a brick-red mutant accumulating large amounts of lycopene (strain NgR9), the buff-coloredMycobacterium smegmatis MC²-155, and the buffcoloredMycobacterium tuberculosis H37Ra. From the endonuclease digestion patterns and the phenotypes of the transformed strains, the partial physical and functional maps of a carotenogenesis operon were established. This investigation also showed that the genes controlling the conversion of lycopene into the xanthophylls were not located in the 10.83-kb segment.     

Transformation of distinct mycobacterial species by shuttle vectors derived from theMycobacterium fortuitum pAL5000 plasmid

Mycobacterium tuberculosis H37Ra,M. smegmatisATCC 607,M. smegmatis MC²155,M. aurum A +,M. aurum A11, and one representative strain ofM. flavescens were transformed by electroporation with plasmid pMY10 and cosmid pDC100. Plasmid pMY 10 contained the origin of replication of pAL5000, the origin of replication of pBR322, a kanamycin resistance gene, and the origin of transfer of the Inc plasmid RK2; the cosmid pDC100 contained the pHC79 SS cosmid, the origin of replication of pAL5000, and a kanamycin resistance gene. The efficiency of transformation varied with the recipient cells used and was in decreasing order: 7×10⁵ forM. smegmatis MC²155, 6×10³ forM. tuberculosis H37Ra, 10³ forM. aurum, 50 forM. smegmatis ATCC 607, and 5 forM. flavescens. A rapid protocol for plasmid extraction from mycobacteria was developed.The satisfactory transformation of the nonvirulentM. tuberculosis strain H37Ra was of interest for future studies on cloning of virulence genes, while the satisfactory transformation ofM. aurum was of interest for future studies on the genetics of drug resistance because these bacteria are sensitive to drugs specifically used in the treatment of tuberculosis and leprosy. However, neither vector was stably maintained inM. smegmatis, indicating that further investigations are still necessary to resolve this difficulty.     

Variation among Genome Sequences of H37Rv Strains of Mycobacterium tuberculosis from Multiple Laboratories

The publication of the complete genome sequence for Mycobacterium tuberculosis H37Rv in 1998 has had a great impact on the research community. Nonetheless, it is suspected that genetic differences have arisen in stocks of H37Rv that are maintained in different laboratories. In order to assess the consistency of the genome sequences among H37Rv strains in use and the extent to which they have diverged from the original strain sequenced, we carried out whole-genome sequencing on six strains of H37Rv from different laboratories. Polymorphisms at 73 sites were observed, which were shared among the lab strains, though 72 of these were also shared with H37Ra and are likely to be due to sequencing errors in the original H37Rv reference sequence. An updated H37Rv genome sequence should be valuable to the tuberculosis research community as well as the broader microbial research community. In addition, several polymorphisms unique to individual strains and several shared polymorphisms were identified and shown to be consistent with the known provenance of these strains. Aside from nucleotide substitutions and insertion/deletions, multiple IS6110 transposition events were observed, supporting the theory that they play a significant role in plasticity of the M. tuberculosis genome. This genome-wide catalog of genetic differences can help explain any phenotypic differences that might be found, including a frameshift mutation in the mycocerosic acid synthase gene which causes two of the strains to be deficient in biosynthesis of the surface glycolipid phthiocerol dimycocerosate (PDIM). The resequencing of these six lab strains represents a fortuitous “in vitro evolution” experiment that demonstrates how the M. tuberculosis genome continues to evolve even in a controlled environment.Publication of the whole genome sequence of the H37Rv strain of Mycobacterium tuberculosis by Stewart Cole and colleagues in 1998 provided a breakthrough in tuberculosis (TB) research (8), leading to insights into the biology, metabolism, and evolution of this infectious pathogen. Large protein families related to fatty acid and polyketide biosynthesis, regulation (e.g., sigma factors and two-component sensor systems), drug efflux pumps and transporters, and the PE_PGRS proteins (a large duplicated family unique to the M. tuberculosis group of mycobacteria) were identified. In addition, transposons, prophage-like elements, and other repetitive and/or mobile genetic elements were identified (18). This genomic information has played an essential role in interpreting gene expression studies, modeling persistence, and identifying essential proteins as putative targets for drug discovery. However, to date the functions of only half of the genes (1,756/4,066) have been determined or predicted, and the rest remain annotated as “hypothetical proteins” (6).The H37Rv strain was initially selected for sequencing because it is a widely used laboratory strain that has retained its virulence. H37Rv was initially derived from a clinical isolate, H37, obtained from a patient with pulmonary tuberculosis in 1905. H37Rv falls in the T clade (5) and single-nucleotide polymorphism (SNP) cluster group SCG-6b (12). The virulence of H37Rv can be demonstrated in a number of animal models. For example, SCID mice infected with H37Rv typically have a mean time to death of 30 to 35 days, depending on the dose and route of inoculation (13).An avirulent strain, H37Ra, was also derived from H37 by culturing on solid egg medium and selecting for resistance to lysis (42). The strain was found not to cause disease in guinea pigs (43) or in mice (27). It has a colony morphology (smooth) different from that of H37Rv (rough) and several other phenotypic differences (14, 29). The H37Rv (ATCC 25618) and H37Ra (ATCC 25177) strains are maintained at the Trudeau Institute in New York (3), although unfortunately, the original H37 clinical isolate has been lost. Strain ATCC 27294 (TMC 102) is also frequently used as a representative of H37Rv in studies and treated equivalently in the literature. ATCC 25618 and ATCC 27294 were both isolated from the same patient in different years, and both are fully drug susceptible.The complete genome of H37Ra has been sequenced by Zheng et al. (48), who found 272 polymorphisms compared to the genome sequence determined by Cole et al. (8) for H37Rv. However, a subset of the polymorphic sites were found to match CDC1551, and upon resequencing of 85 such sites in H37Rv, 79 were determined to be errors in the H37Rv reference sequence. In addition, H37Ra has insertions of IS6110 at two novel sites and a loss of one, compared to the 16 sites in H37Rv. The 130 genuine H37Ra-specific polymorphisms found were divided into those in coding regions, those in upstream regulatory regions, and those in noncoding, nonregulatory intergenic regions in order to assess potential relevance to virulence. Polymorphisms in the promoter regions of sigC, nrdH (glutaredoxin-like electron transporter), and pabB (para-amino benzoate synthase), as well as nonsynonymous substitutions in mazG (regulator of stringent response), phoP (two-component sensor regulating biosynthesis of cell surface lipid antigens), pks12 (polyketide synthase involved in biosynthesis of mycoketides), and nrp (nonribosomal peptide synthetase potentially involved in phthiocerol dimycocerosate [PDIM] biosynthesis), were highlighted as possible causes of the loss of virulence. H37Ra does not synthesize a number of cell surface antigens, including sulfolipid-1, trehalose mycolates, and PDIM (7). The roles of mutations in phoP and sigM, both of which regulate expression of genes involved in biosynthesis of cell surface antigens, have been subsequently investigated, though neither seems to be singularly responsible for the avirulence of H37Ra (17, 35). Multiple mutations in PPE and PE_PGRS genes are also observed in H37Ra, and there has been speculation about the role of these genes in virulence (39). However, the RvD2 region (an 8-kb region present in H37Ra but deleted in H37Rv, including an IS6110 insertion element, mmpL14, and several hypothetical genes) is known not to be responsible for differences in virulence (25).Because of its importance as a model strain used in laboratory studies, it is essential to determine how consistent different stocks of H37Rv in different laboratories are with the reference genome sequence and with each other. Different stocks could accumulate independent polymorphisms over time, and such inconsistencies could potentially make results of studies obtained with H37Rv cultures from different labs difficult to compare, particularly if they affect virulence, drug tolerance, metabolism, cell wall constitution, etc. Furthermore, sequencing errors in the original genome sequence are possible. In order to evaluate differences among currently used variants of H37Rv, we resequenced the complete genomes of six extant H37Rv strains (two samples of ATCC 25618 and four of ATCC 27294) using Illumina sequencing technology. We compared differences among them and differences from the reference sequences for H37Rv and H37Ra available from GenBank. The results of this study identify a common set of 73 polymorphisms shared among all six sequenced strains relative to the H37Rv reference strain. Most (72) of these are shared with H37Ra and likely correspond to sequencing errors in the original H37Rv genome sequence. However, there are several sites where additional polymorphisms are shared among a subset of strains, and several strains have a small number of unique polymorphisms. Furthermore, examination of insertion sites of the IS6110 transposable element reveals several changes that have occurred among these strains. These results illustrate the ongoing evolution of this strain and divergence from the sequenced reference strain of H37Rv and highlight the importance of understanding the genetic differences unique to the stock used in each laboratory.     

A point mutation in the two-component regulator PhoP-PhoR accounts for the absence of polyketide-derived acyltrehaloses but not that of phthiocerol dimycocerosates in Mycobacterium tuberculosis H37Ra

Similarities between Mycobacterium tuberculosis phoP-phoR mutants and the attenuated laboratory strain M. tuberculosis H37Ra in terms of morphological and cytochemical properties, lipid content, gene expression and virulence attenuation prompted us to analyze the functionality of this two-component regulator in the latter strain. Sequence analysis revealed a base substitution resulting in a one-amino-acid change in the likely DNA-binding region of PhoP in H37Ra relative to H37Rv. Using gel-shift assays, we show that this mutation abrogates the ability of the H37Ra PhoP protein to bind to a 40-bp segment of its own promoter. Consistent with this result, the phoP gene from H37Rv but not that from H37Ra was able to restore the synthesis of sulfolipids, diacyltrehaloses and polyacyltrehaloses in an isogenic phoP-phoR knock-out mutant of M. tuberculosis Moreover, complementation of H37Ra with phoP from H37Rv fully restored sulfolipid, diacyltrehalose and polyacyltrehalose synthesis, clearly indicating that the lack of production of these lipids in H37Ra is solely due to the point mutation in phoP. Using a pks2-3/4 knock-out mutant of M. tuberculosis H37Rv, evidence is further provided that the above-mentioned polyketide-derived acyltrehaloses do not significantly contribute to the virulence of the tubercle bacillus in a mouse model of infection. Reasons for the attenuation of H37Ra thus most likely stand in other virulence factors, many of which are expected to belong to the PhoP regulon and another of which, unrelated to PhoP, appears to be the lack of production of phthiocerol dimycocerosates in this strain.     

利用抑制消减杂交技术研究结核分支杆菌强毒株H37Rv和弱毒株H37Ra的基因差异

为了解结核病的致病分子机理和筛选结核病致病菌的毒力基因,利用抑制消减杂交（SSH）技术分析了结核分支杆菌强毒株H37Rv和弱毒株H37Ra间的基因组DNA间差异。通过Southern杂交验证及序列分析得到仅在强毒株H37Rv基因组中有的DNA片段8个,其中一个编码已知的毒力因子mce蛋白,1个编码PE家族蛋白,1个编码purC合成酶,和4个潜在蛋白,另一个为非编码区片段。其中有2个基因经PCR方法已证实在强毒株H37Rv和临床分离的强毒株中存在,而在H37Ra和临床弱毒株中无;仅在弱毒株H37Ra基因组中的DNA片段3个,其中2个为新基因片段,已被GenBank收录。     

差显技术分析结核杆菌H37Rv与H37Ra差异表达的基因

采用差异显示技术比较了结核分支杆菌强毒株H37Rv和弱毒株H37Ra在体外培养条件下的基因表达差异。通过20种引物组合进行mRNA差异显示,克隆到了两菌株间的20余个差异表达基因,经序列分析及杂交鉴定发现其中2个基因仅在H37Rv中表达。其中Rv1894c基因编码的可能是H37Rv中的一个新蛋白。而在H37Ra的基因组中含有这2个基因的编码序列,但均未检测到基因的表达。     

ESX-1 dependent impairment of autophagic flux by Mycobacterium tuberculosis in human dendritic cells

Emerging evidence points to an important role of autophagy in the immune response mediated by dendritic cells (DC) against Mycobacterium tuberculosis (Mtb). Since current vaccination based on Bacillus Calmette-Guerin (BCG) is unable to stop the tuberculosis epidemic, a deeper comprehension of the alterations induced by Mtb in DC is essential for setting new vaccine strategies. Here, we compared the capacity of virulent (H37Rv) and avirulent (H37Ra) Mtb strains as well as BCG to modulate autophagy in human primary DC. We found that Mtb H37Rv impairs autophagy at the step of autophagosome-lysosome fusion. In contrast, neither Mtb H37Ra nor BCG strains were able to hamper autophagosome maturation. Both these attenuated strains have a functional inhibition of the 6kD early secreted antigenic target ESAT-6, an effector protein of the ESAT-6 Secretion System-1(ESX-1)/type VII secretion system. Notably, the ability to inhibit autophagy was fully restored in recombinant BCG and Mtb H37Ra strains in which ESAT-6 secretion was re-established by genetic complementation using either the ESX-1 region from Mtb (BCG::ESX-1) or the PhoP gene (Mtb H37Ra::PhoP), a regulator of ESAT-6 secretion. Importantly, the autophagic block induced by Mtb was overcome by rapamycin treatment leading to an increased interleukin-12 expression and, in turn, to an enhanced capacity to expand a Th1-oriented response. Collectively, our study demonstrated that Mtb alters the autophagic machinery through the ESX-1 system, and thereby opens new exciting perspectives to better understand the relationship between Mtb virulence and its ability to escape the DC-mediated immune response.     

Aptamer inhibits <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> (H37Rv) invasion of macrophage

There is an urgent need to develop new anti-tuberculosis drugs due to the rising tendency in tuberculosis (TB) around the world. It is known that Mycobacterium tuberculosis (M. tuberculosis) generally infects mammalian host via aerosol route. The pathogenic process has been fully studied that it can initially invade alveolar macrophage, then established stable residence within those phagocytic cells, suggesting that one of the possible ways to prevent this pathogen is to inhibit its invasion and growth in the macrophage. Aptamers from SELEX (Systematic Evolution of Ligands by Exponential Enrichment) have been used to rival virulent M. tuberculosis (H37Rv) in our previous work, and the materials to which aptamers bound were proved to be some outer membrane proteins of H37Rv. In the present study, the interaction between M. tuberculosis and macrophage in the presence of aptamers was investigated in more details. The results suggested that the selective aptamers significantly inhibited H37Rv invasion of macrophage in vitro, and the effect correspond to the binding affinity of these aptamers to H37Rv. The values of equilibrium dissociation constant (Kd) was calculated by flow cytometry, all in the nanomolar range, showed much higher affinity to H37Rv than M. bovis Bacillus Guerin (BCG). Moreover, the aptamer-treated H37Rv can stimulate IFN-γ, IL-15 and IL-17 secretion of macrophages compared with H37Rv (no treated). In summary, our data indicated that the NK2 aptamer not only acted as anti-tuberculosis agent by inhibiting virulent M. tuberculosis (H37Rv) invasion of macrophage, but also might be used as molecular probe for exploring the interaction between the outer membrane of M. tuberculosis and macrophage.     

Mitogen-activated protein kinases mediate <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>–induced CD44 surface expression in monocytes

CD44, an adhesion molecule, has been reported to be a binding site for Mycobacterium tuberculosis (M. tuberculosis) in macrophages and it also mediates mycobacterial phagocytosis, macrophage recruitment and protective immunity against pulmonary tuberculosis in vivo. However, the signalling pathways that are involved in M. tuberculosis–induced CD44 surface expression in monocytic cells are currently unknown. Exposure of THP-1 human monocytes to M. tuberculosis H37Rv and H37Ra induced distinct, time-dependent, phosphorylation of mitogen-activated protein kinase kinase-1, extracellular signal regulated kinase 1/2, mitogen-activated protein kinase kinase 3/6, p38 mitogen-activated protein kinase and c-jun N-terminal kinases. The strains also differed in their usage of CD14 and human leukocyte antigen-DR (HLA-DR) receptors in mediating mitogen-activated protein kinase activation. M. tuberculosis H37Rv strain induced lower CD44 surface expression and tumour necrosis factor-alpha levels, whereas H37Ra the reverse. Using highly specific inhibitors of mitogen-activated protein kinase kinase-1, p38 mitogen-activated protein kinase and c-jun N-terminal kinase, we report that inhibition of extracellular signal regulated kinase 1/2 and c-jun N-terminal kinases increases, but that inhibition of p38 mitogen-activated protein kinase decreases M. tuberculosis–induced CD44 surface expression in THP-1 human monocytes.     

2-N-Arylthiazole inhibitors of Mycobacterium tuberculosis

To develop agents for the treatment of infections caused by Mycobacterium tuberculosis, a novel phenotypic screen was undertaken that identified a series of 2-N-aryl thiazole-based inhibitors of intracellular Mycobacterium tuberculosis. Analogs were optimized to improve potency against an attenuated BSL2 H37Ra laboratory strain cultivated in human macrophage cells in vitro. The insertion of a carboxylic acid functionality resulted in compounds that retained potency and greatly improved microsomal stability. However, the strong potency trends we observed in the attenuated H37Ra strain were inconsistent with the potency observed for virulent strains in vitro and in vivo.     

结核分枝杆菌H37有毒株与无毒株的代谢相关基因pabB和lpdA启动子活性分析

【目的】通过分析结核分枝杆菌无毒株H37Ra的全基因组序列,并与H37Rv基因组序列比较,发现pabB和lpdA预测的启动子区发生了突变。我们利用报告基因,确认启动子突变与其基因转录水平的关系,探索结核分枝杆菌H37Ra毒力丧失的内在原因。【方法】利用生物信息学方法预测这两对基因的启动子区,采用PCR技术克隆这两对基因的启动子,与分枝杆菌启动子探针载体pMC210相连,DNA测序证实连接片段正确后,用电穿孔法将重组质粒转化至耻垢分枝杆菌mc2155。利用Quantitative Real-Time RT-PCR检测报告基因lacZ转录水平的差异,进一步验证这两对基因启动子的突变对相应基因转录水平的影响。【结果】Quantitative Real Time PCR检测结果显示H37RapabB启动子活性是H37Rv pabB启动子活性的6倍(p0.05),而H37Rv lpdA启动子的活性是H37RalpdA启动子的2倍(p0.05)。【结论】pabB,lpdA的启动子在H37Ra中的突变对其启动子的活性产生了影响,其中lpdA启动子的突变可能与结核分枝杆菌H37Ra的毒力丧失有关。     

Mycobacterial growth and ultrastructure in mouse L-929 fibroblasts and bone marrow-derived macrophages: Evidence that infected fibroblasts secrete mediators capable of modulating bacterial growth in macrophages

The intracellular growth kinetics ofMycobacterium avium and H37Rv (virulent) and H37Ra (avirulent) strains ofMycobacterium tuberculosis were compared by use of both the professional (mouse bone marrow-derived macrophages, BMMØ) and nonprofessional (mouse L-929 fibroblast cell line) phagocytes. The results obtained showed that all the mycobacterial strains grew more actively in fibroblasts than in BMMØ. This difference was paralleled by lesser acid phosphatase (AcP) labeling of noninfected fibroblasts and the observation that upon infection both the proportion of AcP-positive cells and AcP content were higher in BMMØ than in L-cells during the 7 days of infection. In parallel experiments, intracellular growth ofM. tuberculosis H37Rv andM. avium was compared inside BMMØ from both theBcg ^s (C57BL/6) andBcg ^r (DBA-2) mice, which were matured and differentiated with either an L-cell-conditioned medium (LCM) obtained from control, noninfected L-929 cells, or a LCM obtained withM. tuberculosis-orM. avium-infected L-cells. Upon mycobacterial infection, fibroblasts were able to secrete mediators that stimulated the BMMØ to better control the infection by pathogenic mycobacteria. These results are discussed in terms of the mycobacteria-fibroblast interactions and their eventual role in the immune modulation of the host's response to invading mycobacteria.     

MTB-PCDB: Mycobacterium tuberculosis proteome comparison database

The Mycobacterium tuberculosis Proteome Comparison Database (MTB-PCDB) is an online database providing integrated access to proteome sequence comparison data for five strains of Mycobacterium tuberculosis (H37Rv, H37Ra, CDC 1551, F11 and KZN 1435) sequenced completely so far. MTB-PCDB currently hosts 40252 protein sequence comparison data obtained through inter-strain proteome comparison of five different strains of MTB. 2373 proteins were found to be identical in all 5 strains using MTB H(37)Rv as reference strain. To enable wide use of this data, MTB-PCDB provides a set of tools for searching, browsing, analyzing and downloading the data. By bringing together, M. tuberculosis proteome comparison among virulent & avirulent strains and also drug susceptible & drug resistance strains MTB-PCDB provides a unique discovery platform for comparative proteomics among these strains which may give insights into the discovery & development of TB drugs, vaccines and biomarkers. AVAILABILITY: The database is available for free at http://www.bicjbtdrc-mgims.in/MTB-PCDB/     

Synthesis and antitubercular activities of bis-glycosylated diamino alcohols

Conjugate addition of diamines to glycosyl olefinic esters 1a and 1b followed by reduction of resulting bis-glycosyl beta-amino esters (2-7 and 14-19) with lithium aluminium hydride led to the respective glycosyl amino alcohols (8-13 and 20-25) in moderate to good yields. All the compounds were evaluated for antitubercular activity against Mycobacterium tuberculosis H(37)Ra and H(37)Rv. Few of the compounds exhibited antitubercular activity with MIC as low as 6.25-3.12microg/mL in virulent and avirulent strains. Compound 13 was found to be active against MDR strain and showed mild protection in mice.     

Organization and copy number of initiator tRNA genes in slow- and fast-growing mycobacteria

We have previously reported the isolation and characterization of a functional initiator tRNA gene,metA, and a second initiator tRNA-like sequence,metB, fromMycobacterium tuberculosis. Here we describe the fine mapping of the initiator tRNA gene locus of the avirulent (H37Ra) and virulent (H37Rv) strains ofM. tuberculosis. The genomic blot analyses show that the 1.7 kb (harbouringmetE) and the 6.0 kb BamHI (harbouringmetA) fragments are linked. Further, sequencing of a portion of the 6.0kb fragment, in conjunction with the sequence of the 1.7 kb fragment confirmed the presence of an IS6110 element in the vicinity ofmetB. The IS element is flanked by inverted (28 bp, with 3 contiguous mismatches in the middle) and direct (3 bp) repeats considered to be the hallmarks of IS6110 integration sites. The organization of the initiator tRNA gene locus is identical in both the H37Ra and H37Rv strains and they carry a single copy of the functional initiator tRNA gene. Interestingly, the fast growingMycobacterium smegmatis also bears a single initiator tRNA gene. This finding is significant in view of the qualitative differences in total tRNA pools and the copy number of rRNA genes in the fast- and slow-growing mycobacteria. Finally, we discuss hypotheses related to the origin ofmetB inM. tuberculosis.     

Mitogen-activated protein kinases mediate the production of B-cell lymphoma 2 protein by <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> in Monocytes

Changes in the levels of antiapoptotic protein B-cell lymphoma 2 (Bcl-2) protein has been reported in murine and human tuberculosis. We investigated the role of mitogen-activated protein kinase pathways in the production of Bcl-2 protein in THP-1 human monocytes infected with Mycobacterium tuberculosis H37Rv and H37Ra. Analysis of phosphorylation profiles of mitogen-activated protein kinase kinase-1, extracellular-signal regulated kinase 1/2, mitogen-activated protein kinase kinase 3/6, and p38 mitogen-activated protein kinase; B-cell lymphoma 2 kinetics; and tumor necrosis factor-α (TNF-α) secretion levels showed variation between the two strains. Mycobacterium tuberculosis H37Rv induced higher Bcl-2 and lower TNF-α levels, whereas H37Ra the reverse. The strains also differed in their usage of CD14 and human leukocyte antigen-DR receptors in mediating extracellular-signal regulated kinase 1/2 and p38 mitogen-activated protein kinase activation. Mycobacterium tuberculosis H37Rv- and H37Ra-induced Bcl-2 production was reduced by specific inhibitors of mitogen-activated protein kinase kinase-1 (PD98059) and p38 (SB203580), but increased by nuclear factor κB (NF-κB) inhibitor (BAY 11-7082). TNF-α production by both strains was reduced in the presence of specific inhibitors of mitogen-activated protein kinase kinase-1 (PD98059), p38 (SB203580), and NF-κB (BAY 11-7082). Furthermore, inhibition of NF-κB was accompanied by an increase in strain-induced extracellular-signal regulated kinase 1/2 phosphorylation. Collectively, these results indicate for the first time that the production of Bcl-2 and TNF-α by M. tuberculosis H37Rv/H37Ra-infected THP-1 human monocytes is mediated through mitogen-activated protein kinases and NF-κB.