Sensing of mycobacterial arabinogalactan by galectin-9 exacerbates mycobacterial infection.

Mycobacterial arabinogalactan (AG) is an essential cell wall component of mycobacteria and a frequent structural and bio-synthetical target for anti-tuberculosis (TB) drug development. Here, we report that mycobacterial AG is recognized by galectin-9 and exacerbates mycobacterial infection. Administration of AG-specific aptamers inhibits cellular infiltration caused by Mycobacterium tuberculosis (Mtb) or Mycobacterium bovis BCG, and moderately increases survival of Mtb-infected mice or Mycobacterium marinum-infected zebrafish. AG interacts with carbohydrate recognition domain (CRD) 2 of galectin-9 with high affinity, and galectin-9 associates with transforming growth factor ß-activated kinase 1 (TAK1) via CRD2 to trigger subsequent activation of extracellular signal-regulated kinase (ERK) as well as induction of the expression of matrix metalloproteinases (MMPs). Moreover, deletion of galectin-9 or inhibition of MMPs blocks AG-induced pathological impairments in the lung, and the AG-galectin-9 axis aggravates the process of Mtb infection in mice. These results demonstrate that AG is an important virulence factor of mycobacteria and galectin-9 is a novel receptor for Mtb and other mycobacteria, paving the way for the development of novel effective TB immune modulators.

Clinical value of ELISA-MPT64 for the diagnosis of tuberculous pleurisy.

Tuberculous pleurisy is one of the common extrapulmonary tuberculosis diseases. However, the diagnosis of tuberculous pleurisy still lacks a useful and effective tool, mainly due to paucity of Mycobacterium tuberculosis organisms in pleural effusion. Previous studies have confirmed that the MPT64 protein is highly specific and is secreted only by M. tuberculosis (MTB) complex. Therefore, in this study, we developed ELISA based on recombinantly expressed MPT64 in combination with rabbit polyclonal antibodies. The ELISA-MPT64 method was validated using MTB strains and tested against clinical samples. Nested PCR, Löwenstein-Jensen (L-J) culture and smear microscopy were employed as the comparative tools for assessing the performance of the assay. Our results demonstrate that the newly established ELISA-MPT64 technique is a rapid and useful tool for the diagnosis of tuberculous pleurisy.

Perspective on sequence evolution of microsatellite locus (CCG)n in Rv0050 gene from Mycobacterium tuberculosis.

BACKGROUND: The mycobacterial genome is inclined to polymerase slippage and a high mutation rate in microsatellite regions due to high GC content and absence of a mismatch repair system. However, the exact molecular mechanisms underlying microsatellite variation have not been fully elucidated. Here, we investigated mutation events in the hyper-variable trinucleotide microsatellite locus MML0050 located in the Rv0050 gene of W-Beijing and non-W-Beijing Mycobacterium tuberculosis strains in order to gain insight into the genomic structure and activity of repeated regions. RESULTS: Size analysis indicated the presence of five alleles that differed in length by three base pairs. Moreover, nucleotide gains occurred more frequently than loses in this trinucleotide microsatellite. Mutation frequency was not completely related with the total length, though the relative frequency in the longest allele was remarkably higher than that in the shortest. Sequence analysis was able to detect seven alleles and revealed that point mutations enhanced the level of locus variation. Introduction of an interruptive motif correlated with the total allele length and genetic lineage, rather than the length of the longest stretch of perfect repeats. Finally, the level of locus variation was drastically different between the two genetic lineages. CONCLUSION: The Rv0050 locus encodes the bifunctional penicillin-binding protein ponA1 and is essential to mycobacterial survival. Our investigations of this particularly dynamic genomic region provide insights into the overall mode of microsatellite evolution. Specifically, replication slippage was implicated in the mutational process of this microsatellite and a sequence-based genetic analysis was necessary to determine that point mutation events acted to maintain microsatellite size integrity while providing genomic diversity.

Identification and evaluation of a new nucleic acid amplification test target for specific detection of Mycobacterium tuberculosis.

BACKGROUND: Accurate and early diagnosis of tuberculosis (TB) is of major importance in the management and control of TB. Because the conventional bacteriological diagnosis of TB has several limitations, nucleic acid amplification (NAA) tests have emerged as promising alternatives. A potential problem with NAA tests is that some strains lack a target, which may be the one of main reasons for the much lower and highly variable accuracy in diagnosis. A possible solution may be to use more valid and applicable targets to increase detection accuracy. METHODS: In this paper, we designed a two-step program to obtain NAA test targets. Inter-simple sequence repeats (ISSR) based on oligonucleotide (GTG)(5) were first constructed to genotype Mycobacterium strains to obtain Mycobacterium tuberculosis (MTB)-specific fragment. Second, sequence characterized amplified region (SCAR) markers were developed from these species-specific sequences to identify MTB. Some 312 Mycobacterium strains were used to evaluate the efficacy of the SCAR markers, IS6110 element [specific identification of Mycobacterium tuberculosis complex (MTC)] and 16SrRNA gene (specific identification of Mycobacterium) amplification, together with traditional bacteriology testing was used as a control. RESULTS: MTB-specific sequences located in a gene coding for Rv1508c, as a new NAA test target, were obtained using ISSR-PCR genotyping. Based on these sequences, the SCAR primer pairs MISP1 and MISP2 were designed. All 312 strains from Mycobacterium accurately produced the genus-specific 16SrRNA amplicon. 271 MTB strains and M. africanum were positive. However, all nontuberculous mycobacteria (NTM) strains and 1 MTB strain named 1143 were negative in both SCAR and IS6110 PCR amplification. M. bovis, bacille Calmette-Guérin (BCG) were IS6110-PCR positive, while SCAR-PCR was negative. Strain 1143 was defined as M. arupense with 99% identity by 16SrRNA gene sequencing identification, despite being diagnosed as MTB using traditional testing. CONCLUSIONS: SCAR markers developed with this two-step program can be used as a new NAA test target to correctly detect MTB.

[Identification of the Mycobacterium tuberculosis complex by detecting recombinant secretory protein MPT64].

OBJECTIVE: To identify the Mycobacterium tuberculosis complex by detecting the secretory protein MPT64. METHODS: The gene mpt64 was amplified by polymerase chain reaction (PCR) from the genome of Mycobacterium tuberculosis H(37)Rv strain and cloned into expression vector. Immune sera from rabbits by recombinant proteins MPT64, were used to make enzyme-labeled antibodies and coated antibodies. A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was established to detect the secretory protein MPT64 of the culture supernatants in Mycobacterium strains. Results of ELISA were compared to those of the gene mpt64 amplified by PCR. RESULTS: A recombinant vector was constructed. The minimum detectable concentration of MPT64 was 0.01 mg/L. A total of 27 reference strains and 170 clinical isolate strains were evaluated. PCR for Mycobacterium tuberculosis reference strain, Mycobacterium bovis reference strain and Mycobacterium africanum reference strain was positive, but that for other reference stains was negative, consistent with the results of ELISA. In the 170 clinical isolate stains, the positive result of PCR and ELISA was 98.2% (111/113) and 97.3% (110/113) respectively, while the specificity of PCR and ELISA was both 100%, no positive result in non-tuberculosis mycobacterium strains. CONCLUSION: Identification of the Mycobacterium tuberculosis complex by detecting secretory protein MPT64 is rapid, sensitive, and specific, which can be used routinely in clinical laboratories.

Trehalose-6-phosphate Phosphatase from Mycobacterium tuberculosis induces humoral and cellular immune responses.

Trehalose-6-phosphate phosphatase is an enzyme strictly essential for the growth of mycobacteria. Subcellular fractionation of Mycobacterium tuberculosis and M. bovis bacillus Calmette-Guérin (BCG) located the trehalose-6-phosphate phosphatase in the cell wall and membrane fractions. Trehalose-6-phosphate phosphatase induced an increased Th1-type immune response in mice, characterized by an elevated level of interferon-gamma in antigen-stimulated splenocyte culture and a strong IgG2a antibody response. The trehalose-6-phosphate phosphatase was recognized by the sera of tuberculosis patients and BCG-vaccinated donors. The mycobacterial trehalose-6-phosphate phosphatase is an immunodominant antigen, and it may be a candidate for vaccine development for the control of tuberculosis.

Rv2629 Overexpression Delays Mycobacterium smegmatis and Mycobacteria tuberculosis Entry into Log-Phase and Increases Pathogenicity of Mycobacterium smegmatis in Mice.

Objective: The aim of the present study was to explore the potential biological role of Rv2629 in Mycobacterium smegmatis and Mycobacterium tuberculosis.Methods: Recombinant wild type and mutant Rv2629 strains were constructed. Rv2629 expression was evaluated by real-time PCR and western blot. Microarray and interaction network analyses were used to identify the gene interactions associated with wild type and mutant Rv2629. Bacterial growth was assessed in Balb/c mice infected with wild type and mutant Rv2629 strains using CFU assay and histological analysis of the organs. Results: Overexpression of Rv2629 could delay the entry of the Mycobacterium tuberculosis cells into the log-phase, while Rv2629 decreased the number of ribosomes and the expression of uridylate kinase in Mycobacterium smegmatis. The Gene Ontology (GO) and pathway analysis indicated that 122 genes correlated with wild type Rv2629, whereas the Rv2629 mutation led to decrease in the ribosome production, oxidative phosphorylation, and virulence in Mycobacterium tuberculosis. Overexpression of Rv2629 slightly enhanced the drug resistance of Mycobacterium smegmatis to antibiotics, and increased its survival and pathogenicity in Balb/c mice. Conclusion: It is suggested that Rv2629 is involved in the survival of the clinical drug-resistant strain via bacterial growth repression and bacterial persistence induction.

Design, synthesis, and in vitro biological evaluation of novel 6-methyl-7-substituted-7-deaza purine nucleoside analogs as anti-influenza A agents.

Among many subtypes of influenza A viruses, influenza A(H1N1) and A(H3N2) subtypes are currently circulating among humans (WHO report 2014-15). Therapeutically, the emergence of viral resistance to currently available drugs (adamantanes and neuraminidase inhibitors) has heightened alarms for developing novel drugs that could address diverse targets in the viral replication cycle in order to improve treatment outcomes. To this regard, the design and synthesis of nucleoside analog inhibitors as potential anti-influenza A agents is a very active field of research nowadays. In this study, we designed and synthesized a series of hitherto unknown 6-methyl-7-substituted-7-deaza purine nucleoside analogs, and evaluated for their biological activities against influenza A virus strains, H1N1 and H3N2. From the viral inhibition assay, we identified some effective compounds, among which, compounds 5x (IC50 = 5.88 µM and 6.95 µM for H1N1 and H3N2, respectively) and 5z (IC50 = 3.95 µM and 3.61 µM for H1N1 and H3N2, respectively) demonstrated potent anti-influenza A activity. On the basis of selectivity index, we conceive that compound 5x may serve as a chemical probe of interest for further lead optimization studies with a general aim of developing novel and effective anti-influenza A virus agents.

Regulation of aroP expression by tyrR gene in Escherichia coli.

tyrR gene encodes a global regulatory protein (TyrR), which plays an important role in the transcriptional regulation of eight transcription units (including tyrR gene itself) whose protein products catalyze key steps in aromatic amino acid biosynthesis and/or transport. The aroP gene encodes an integral membrane protein (AroP) that transports aromatic amino acids through the cell membrane. The transcription of aroP was reported to be repressed by TyrR. In this work, aroP(p) (aroP gene carrying its own promoter), aroP (aroP gene without promoter) and tyrR genes were amplified by PCR from genomic DNA of E. coli K12 and introduced into E. coli WT5. The expression of aroP and tyrR were detected and the activities of AroP and TyrR were determined. The introduction of either aroP(p) or aroP elevated the strain's transport activity by 1.40 or 1.46-fold respectively. Transformant carrying tyrR gene showed an ATPase activity 1.69-fold compared with the control. When the genes were linked in tandem and co-expressed in a plasmid, the relative AroP transport activity of the strain harboring aroP(p) -tyrR (0.95) was significantly lower than that of aroP-tyrR (1.31). The results indicated that TyrR might be able to reduce the expression of aroP gene by binding with the aroP promoter region in E.coli.

[Knockout of tyrR gene in Escherichia coli and its effects on the phenylalanine biosynthesis].

TyrR is a global regulation protein encoded by tyrR gene which controls several transcriptional units involving biosynthesis and transportation of aromatic amino acids in Escherichia coli. In this work, the tyrR gene was knocked out by a double-cross homologous recombination. The tyrR- mutant was verified by structural identification by PCR and sequencing, and functional demonstration using lacZ reporter gene. In tyrR- mutant, the activities of two key enzymes in the phenylalanine biosynthesis pathway, whose expression was controlled by TyrR, DAHPS and AT, had been shown to elevate by a 1.08-fold and 2.70-fold compared with the parent strain, respectively. The yield of phenylalanine biosynthesis in the mutant was 1.59 times higher than that of wild type strain. The repression on the transcription of aroP, encoding an aromatic amino acid permease, was eliminated, resulting in a 70.2% increase of the aromatic amino acid transportation in tyrR- mutant strain.

A novel B-cell epitope identified within Mycobacterium tuberculosis CFP10/ESAT-6 protein.

BACKGROUND: The 10-kDa culture filtrate protein (CFP10) and 6-kDa early-secreted target antigen (ESAT-6) play important roles in mycobacterial virulence and pathogenesis through a 1:1 complex formation (CFP10/ESAT-6 protein, CE protein), which have been used in discriminating TB patients from BCG-vaccinated individuals. The B-cell epitopes of CFP10 and ESAT-6 separately have been analyzed before, however, the epitopes of the CE protein are unclear and the precise epitope in the positions 40 to 62 of ESAT-6 is still unknown. METHODS: In the present study, we searched for the B-cell epitopes of CE protein by using phage-display library biopanning with the anti-CE polyclonal antibodies. The epitopes were identified by sequence alignment, binding affinity and specificity detection, generation of polyclonal mouse sera and detection of TB patient sera. RESULTS: One linear B-cell epitope (KWDAT) consistent with the 162(nd)-166(th) sequence of CE and the 57(th)-61(st) sequence of ESAT-6 protein was selected and identified. Significantly higher titers of E5 peptide-binding antibodies were found in the sera of TB patients compared with those of healthy individuals. CONCLUSION: There was a B-cell epitope for CE and ESAT-6 protein in the position 40 to 62 of ESAT-6. E5 peptide may be useful in the serodiagnosis of tuberculosis, which need to be further confirmed by more sera samples.

Evaluation of a novel biphasic culture medium for recovery of mycobacteria: a multi-center study.

BACKGROUND: Mycobacterial culture and identification provide a definitive diagnosis of TB. Culture on Löwenstein-Jensen (L-J) medium is invariably delayed because of the slow growth of M. tuberculosis on L-J slants. Automated liquid culture systems are expensive. A low-cost culturing medium capable of rapidly indicating the presence of mycobacteria is needed. The aim of this study was to develop and evaluate a novel biphasic culture medium for the recovery of mycobacteria from clinical sputum specimens from suspected pulmonary tuberculosis patients. METHODS AND FINDINGS: The biphasic medium consisted of 7 ml units of L-J slant medium, 3 ml units of liquid culture medium, growth indicator and a mixture of antimicrobial agents. The decontamination sediments of sputum specimens were incubated in the biphasic culture medium at 37°C. Mycobacterial growth was determined based on the appearance of red granule sediments and the examination using acid-fast bacilli (AFB). The clinical sputum specimens were cultured in the biphasic medium, on L-J slants and in the Bactec MGIT 960 culture system. Among smear-positive specimens, the mycobacteria recovery rate of the biphasic medium was higher than that of the L-J slants (P<0.001) and similar to that of MGIT 960 (P>0.05). Among smear-negative specimens, the mycobacterial recovery rate of the biphasic medium was higher than that of L-J slants (P<0.001) and lower than that of MGIT 960 (P<0.05). The median times to detection of mycobacteria were 14 days, 20 days and 30 days for cultures grown in MGIT, in biphasic medium, on L-J slants for smear negative specimens, respectively (P<0.001). CONCLUSIONS: The biphasic culture medium developed in this study is low-cost and suitable for mycobacterial recovery. It does not require any expensive detection instrumentation, decreases the time required for detection of M. tuberculosis complex, and increases the detection rate of M. tuberculosis complex.

Relationship between polymorphism of DC-SIGN (CD209) gene and the susceptibility to pulmonary tuberculosis in an eastern Chinese population.

Dendritic cell-specific intracellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is an important receptor for Mycobacterium tuberculosis on human dendritic cells. Previous studies have shown that the variation, especially the -871A/G and -336A/G in DC-SIGN promoter influenced the susceptibility to tuberculosis. We therefore investigated whether polymorphisms in the DC-SIGN gene were associated with susceptibility to tuberculosis in an eastern Chinese population. A total of 237 culture-positive pulmonary tuberculosis case patients and 244 controls were genotyped for -871A/G and -336A/G by pyrosequencing. Our results suggested that the 2 promoter variants of DC-SIGN gene were not associated with susceptibility to tuberculosis in Chinese. Further analysis showed that the allele -336G was associated with a protective effect against fever in pulmonary tuberculosis patients, but not against cavity formation. In addition, we compared the allelic frequencies of -871A/G and -336A/G in African, Caucasian, and Asian groups. The results showed that the tw forms of allelic frequencies detected Chinese individuals in our study were similar to the reported frequencies in other Asian populations but differed significantly from those in the African and Caucasian groups studied.

A multi-center study to evaluate the performance of phage amplified biologically assay for detecting TB in sputum in the pulmonary TB patients.

OBJECTIVE: To evaluate the performance of phage amplified biologically assay (PhaB) for detecting tuberculosis (TB) in sputum in the pulmonary tuberculosis (PTB) patients. METHODS: Shanghai Tuberculosis Key Laboratory of Shanghai Pulmonary Hospital participated in the project in collaboration with the laboratories of six hospitals and a total of 1660 eligible participants (1351 PTB patients and 309 non-TB patients) were included in the study. The sputum samples from the participants were detected by smear microscopy, PhaB, and Löwenstein-Jensen (L-J) culture method, respectively. RESULTS: The overall sensitivity of PhaB were higher than that of L-J culture and smear microscopy (p<0.05). The sensitivity of PhaB for detecting smear-negative specimens was obviously higher than that of L-J culture (p<0.05). Compared with L-J culture, the overall sensitivity, specificity, PPV, NPV, ACC and Kappa value of PhaB were 98.4 (95% Cl: 96.9-99.3), 71.6 (95% Cl: 68.4-74.6), 67.7, 98.7, 81.7% and 0.643, respectively. The detection median time of PhaB only needed 48 hours, which was significantly less than that (31 days) of L-J culture method. CONCLUSION: PhaB method is a rapid and sensitive method for detecting TB in sputum in PTB patients; especially for the diagnosis of smear-negative PTB, PhaB method is obviously more sensitive than L-J culture method.

Selection and application of peptide mimotopes of MPT64 protein in Mycobacterium tuberculosis.

Antibody responses can be useful markers of tuberculosis (TB) infection, especially in the screening of extra-pulmonary TB. MPT64 is an important antigen in Mycobacterium tuberculosis (MTB) infection and is used in serological diagnosis. However, large variability in the diagnostic accuracy of MPT64 as a serological tool has limited its application. Phage-displayed random peptide libraries have emerged as a powerful technique to select peptides (epitopes) or mimotopes that may serve as surrogate diagnostic markers in serological tests. In the present study, this method was employed to identify mimotopes of the MPT64 protein of MTB by screening a linear heptapeptide library with rabbit antibodies raised against MPT64 protein. Two antigenic mimotopes (M2 and M6) resembling B-cell epitopes of MPT64 were identified that bound the affinity purified anti-MPT64 polyclonal antibodies and competed with MPT64 for antibody binding. From the results of sequence alignment and a structure modelling figure of MPT64, the sequence of the 2nd to 5th amino acids (DSML) of M2 was totally consistent with the sequence of the 224th to 227th amino acids of MPT64 and the peptide is located on the surface of the space structure of MPT64, suggesting that it might be a linear epitope of MPT64. The recognition of both phage-displayed and synthetic peptides of M2 by the anti-MPT64 polyclonal antibodies also supported this. Although no recurring sequence and no analogue to MPT64 of M6 were found for sequence alignment, the recognition of both phage-displayed and synthetic peptides of M6 by the anti-MPT64 polyclonal antibodies indicated that it might be a mimotope of a conformational epitope of MPT64. According to the results of the reactivity of human sera with synthetic M2 and M6 peptides and MPT64, M2 showed a significantly higher AUC and sensitivity than M6 and MPT64, especially for the sera from sputum-negative TB patients, suggesting that the M2 mimotope may be useful in serological diagnostic testing for TB.

Altered protein expression patterns of Mycobacterium tuberculosis induced by ATB107.

ATB107 is a potent inhibitor of indole-3-glycerol phosphate synthase (IGPS). It can effectively inhibit the growth of clinical isolates of drug-resistant Mycobacterium tuberculosis strains as well as M. tuberculosis H37Rv. To investigate the mechanism of ATB107 action in M. tuberculosis, two-dimensional gel electrophoresis coupled with MALDI-TOF-MS analysis (2-DE-MS) was performed to illustrate alterations in the protein expression profile in response to ATB107. Results show that ATB107 affected tryptophan biosynthesis by decreasing the expression of protein encoded by Rv3246c, the transcriptional regulatory protein of MtrA belonging to the MtrA-MtrB two-component regulatory system, in both drug-sensitive and drug-resistant virulent strains. ATB107 might present a stress condition similar to isoniazid (INH) or ethionamide for M. tuberculosis since the altered expression in response to ATB107 of some genes, such as Rv3140, Rv2243, and Rv2428, is consistent with INH or ethionamide treatment. After incubation with ATB107, the expression of 2 proteins encoded by Rv0685 and Rv2624c was down-regulated while that of protein encoded by Rv3140 was up-regulated in all M. tuberculosis strains used in this study. This may be the common response to tryptophan absence; however, relations to ATB107 are unknown and further evaluation is warranted.

The selection and application of ssDNA aptamers against MPT64 protein in Mycobacterium tuberculosis.

BACKGROUND: Tuberculosis (TB) remains a major health problem affecting millions of people worldwide. One-third of the world's population is infected with Mycobacterium tuberculosis, the etiologic agent of TB. A simple and rapid method to diagnose TB is urgently needed to be developed. The procedure of systematic evolution of ligands by exponential enrichment (SELEX) is a method in which single-stranded oligonucleotides (called aptamers) are selected from a wide variety of sequences, based on their interaction with a target molecule. Aptamers have been used in numerous investigations as therapeutic or diagnostic tools. METHODS: In this study, we apply a SELEX method to develop aptamers against MPT64 protein from M. tuberculosis. On this basis, a sandwich assay scheme with the complex of aptamer-MPT64 was designed and tested the feasibility of detecting M. tuberculosis by detecting MPT64 protein levels in the culture filtrates of 77 samples including M. tuberculosis and other Mycobacterium species. RESULTS: There was a highly significant difference (p<0.01) between group A (non-TB Mycobacterium, bacille Calmette-Guérin) and group B (M. tuberculosis, M. bovis), when they were diagnosed with the sandwich assay scheme based on aptamer-protein complex to detect MPT64 protein levels in the culture filtrates of samples. When the cut-off point was at the optical density value of 0.58 (95%=0.764-0.946; Z=6.130, p=0.0001), the sandwich assay scheme based on aptamer-protein complex had a high sensitivity (negative ration, 24/27, 86.3%) and specificity (positive ration, 46/52, 88.5%). CONCLUSIONS: Aptamer of MPT64 as a new detection tool, to a certain extent, is feasible to diagnose Mycobacterium tuberculosis.

Purification and characterization of a functionally active Mycobacterium tuberculosis prephenate dehydrogenase.

Tuberculosis (TB) remains to be a global health problem. New drugs are badly needed to drastically reduce treatment time and overcome some of the challenges with tuberculosis treatment, such as multi-drug resistant (MDR) strain infected patients or tuberculosis/HIV co-infected patients. The essentiality of mycobacterial aromatic amino acid biosynthesis pathways and their absence from human host indicate that the member enzymes of these pathways promising drug targets for therapeutic agents against pathogen mycobacteria. Prephenate dehydrogenase (PDH) is a key regulatory enzyme in tyrosine biosynthesis, catalyzing the NAD(+)-dependent conversion of prephenate to p-hydroxyphenylpyruvate, making it a potential drug target for antibiotics discovery. The recombinant PDH with an N-terminal His-tag (His-rMtPDH) was first purified in Escherichia coli, and using enterokinase rMtPDH was obtained by cleaving the N-terminal fusion partner. The effect of pH, temperature and the cation-Na(+) on purified enzyme activity was characterized. The N-terminal fusion partner was found to have little effect on the biochemical properties of PDH. We also provide in vitro evidence that Mycobacterium tuberculosis PDH does not possess any chorismate mutase (CM) activity, which suggests that, unlike many other enteric bacteria (where PDH exists as a fusion protein with CM), M. tuberculosis PDH is a monofunctional protein.

Purification and characterization of a functionally active Mycobacterium tuberculosis pyrroline-5-carboxylate reductase.

Pyrroline-5-carboxylate reductase (P5CR) plays an important role in the survival of Mycobacterium tuberculosis and is related to virulence of this pathogen. RT-PCR analysis indicated that proC, encoding P5CR, was expressed at the transcriptional level cultured in vitro. The His-rMtP5CR with an N-terminal His-tag (His-rMtP5CR) was firstly purified in Escherichia coli and rMtP5CR was obtained by removal of the N-terminal fusion partner using enterokinase. His-rMtP5CR had considerable beta-pleated sheet analyzed by circular dichroism spectroscopy. The effect of pH, temperature, cations, denaturants, and detergents on the purified enzyme activity and stability was characterized. The N-terminal fusion partner was found to have very little effect on the biochemical properties of P5CR.

Purification and characterization of Mycobacterium tuberculosis indole-3-glycerol phosphate synthase.

Indole-3-glycerol phosphate synthase (IGPS) plays an important role in the survival of Mycobacterium tuberculosis. The trpC gene, encoding IGPS, is essential for the growth of M. tuberculosis. It was expressed at the transcriptional level in cultured M. tuberculosis. The recombinant IGPS with an added His-tag was purified. The His-tag was found to have a small effect on the biochemical properties of IGPS. IGPS is a monofunctional enzyme in M. tuberculosis. Recombinant IGPS has considerable beta-pleated sheet and is relatively compact. The enzyme activity is significantly inhibited by denaturants and antibiotics, suggesting that IGPS may be a novel potential drug target of M. tuberculosis.