Characterization of the putative tryptophan synthase β-subunit from Mycobacterium tuberculosis

The increasing emergence of drug-resistant tuberculosis （TB） poses a serious threat to the control of this disease. It is in urgent need to develop new TB drugs. Tryptophan biosynthetic pathway plays an important role in the growth and replication of Mycobacterium tuberculosis （Mtb）. The β-subunit of tryptophan synthase （TrpB） catalyzes the last step of the tryptophan biosynthetic pathway, and it might be a potential target for TB drug design. In this study, we overexpressed, purified, and characterized the putative TrpB-encoding gene Rv1612 in Mtb H37Rv. Results showed that Mtb His-TrpB optimal enzymatic activity is at pH 7.8 with 0.15 M Na^＋ or 0.18 M Mg^2＋ at 37℃. Structure analysis indicated that Mtb TrpB exhibited a typical β/α barrel structure. The amino acid residues believed to interact with the enzyme cofactor pyridoxal-5＇-phosphate were predicted by homology modeling and structure alignment. The role of these residues in catalytic activity of the Mtb His-TrpB was confirmed by site-directed mutagenesis. These results provided reassuring structural information for drug design based on TrpB.     

Is Mycobacterium bovis in the environment important for the persistence of bovine tuberculosis?

Mycobacterium bovis is the causative agent of bovine tuberculosis (bTB) in cattle and wildlife. Direct aerosol contact is thought to be the primary route of infection between conspecifics, whereas indirect transmission via an environmental reservoir of M. bovis is generally perceived not to be a significant source for infection. Here, we report on the application of molecular technology (PCR) to quantify the prevalence of M. bovis in the environment and to explore its epidemiological significance. We show that the detectability of viable M. bovis at badger setts and latrines is strongly linked to the frequency of M. bovis excretion by infected badgers, and that putative M. bovis in the environment is prevalent on a large proportion of endemic cattle farms in Britain. These results raise important questions about the role of an environmental reservoir in bTB persistence.     

Analytical Performance of the Roche LightCycler? Mycobacterium Detection Kit for the Diagnosis of Clinically Important Mycobacterial Species

Background

The LightCycler® Mycobacterium Detection Kit based on real-time PCR technology for the detection of Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium kansasii was recently developed. This study evaluated its analytical sensitivity, specificity and reproducibility.

Methodology/Principal Findings

Plasmid standards were prepared and used to determine the limit of detection. The assay was also performed against organisms other than mycobacteria, other mycobacterial strains and interfering substances to exclude cross-reactivity and interference. Reference standards were prepared and tested to assess the assay''s reproducibility. All PCR assays were performed using the LightCycler® 2.0 Instrument. The detection limit for M. tuberculosis was 28 copies per microlitre. Neither cross-reactivity nor interference occurred with non-mycobacterial organisms and substances tested. Overall reproducibility for consecutive measurements, run-to-run, lot-to-lot, day-to-day and laboratory-to-laboratory achieved a coefficient of variance of less than two percent.

Significance

The LightCycler® Mycobacterium Detection kit has shown to be a robust and accurate assay with the potential to be used as a rapid TB diagnostic test.     

Leprosy – clues about the biochemistry of Mycobacterium leprae and its host-dependency from the genome

Deletions and the appearance of pseudogenes in pathways of carbon source utilisation and energy metabolism best explain the host-dependency and failure to culture Mycobacterium leprae axenically. From the genome sequence it is possible to predict that acetate and galactose cannot be used as carbon sources, while pyruvate can only be catabolised. Glycerol, glucose, and fatty acids could be used for glycolysis, the pentose cycle and -oxidation which are complete. Retrospective functional genomics – interpreting work before the completion of the genome project – supports the failure of M. leprae to use acetate as well as another prediction that metabolic flux from pyruvate to acetyl-CoA would be very low. However, the loss of a second icd gene (compared with M. tuberculosis), predicted to encode isocitrate dehydrogenase, did not diminish the specific activity of the enzyme. The genes for respiratory pathways are extremely limited, being present for oxidative phosphorylation as a result of electron transport only using FADH as an electron donor. In contrast, all the major biosynthetic pathways are complete except that M. leprae is a natural methionine auxotroph: this is predicted not to be attenuating, or explain host-dependency since methionine would be present in rich culture media.     

On the evolution of ‘Indian Bison type’ strains of Mycobacterium avium subspecies paratuberculosis

Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease (JD) in animals, has also been linked with Crohn's disease in human beings. Lack of indigenous diagnostics and vaccine hampered control of JD in India. Designing effective control strategies require thorough understanding of the etiological agent at phenotypic and molecular levels. On the basis of cultural phenotypes and IS1311 PCR-REA typing, MAP strains have been genotyped as ‘Cattle type’, ‘Sheep type’ and ‘Bison type’. Information exists on genetic differences and comparative evolution of ‘Cattle type’ and ‘Sheep type’ strains after divergence from M. avium; however, emphasis has been little on ‘Bison type’ strains. Recently, a new ‘Indian Bison type’ genotype has been reported as principal strain infecting different animal species and human beings in India. The study analyzed few genetic markers to have inferences on the molecular evolution of native MAP isolates belonging to ‘Bison type’ genotype. Results pointed towards recent evolution of ‘Bison type’ genotype.     

Is Mycobacterium tuberculosis stressed out? A critical assessment of the genetic evidence

Mycobacterium tuberculosis is an obligate human intracellular pathogen which remains a major killer worldwide. A remarkable feature of M. tuberculosis infection is the ability of the pathogen to persist within the host for decades despite an impressive onslaught of stresses. In this review we seek to outline the host-inflicted stresses experienced by M. tuberculosis, the bacterial strategies used to withstand these stresses, and how this information should guide our efforts to combat this global pathogen.     

Inhibition of the β-carbonic anhydrases from Mycobacterium tuberculosis with C-cinnamoyl glycosides: Identification of the first inhibitor with anti-mycobacterial activity

A small series of C-cinnamoyl glycoside containing the phenol moiety was tested for the inhibition of the three Mycobacterium tuberculosis β-carbonic anhydrases (CAs, EC 4.2.1.1) with activities in the low micromolar range detected. The compounds were also tested for the inhibition of growth of M. tuberculosis H₃₇Rv strain, leading to the identification of (E)-1-(2′,3′,4′,6′-tetra-O-acetyl-β-d-glucopyranosyl)-4-(3-hydroxyphenyl)but-3-en-2-one (1) as the first carbonic anhydrase inhibitor with anti-tubercular activity.     

The dimeric structure of the Cpn60.2 chaperonin of Mycobacterium tuberculosis at 2.8 Å reveals possible modes of function

Mycobacterium tuberculosis expresses two proteins (Cpn60.1 and Cpn60.2) that belong to the chaperonin (Cpn) family of heat shock proteins. Studies have shown that the two proteins have different functional roles in the bacterial life cycle and that Cpn60.2 is essential for cell viability and may be involved in M. tuberculosis pathogenicity. Cpn60.2 does not form a tetradecameric double ring, which is typical of other Cpns. We have determined the crystal structure of recombinant Cpn60.2 to 2.8 Å resolution by molecular replacement; the asymmetric unit (AU) contains a dimer, which is consistent with size-exclusion high-performance liquid chromatography and dynamic light-scattering measurements of the soluble recombinant protein. However, we suggest that the actual Cpn60.2 dimer may be different from that identified within the AU on the basis of surface contact stability, solvation free-energy gain, and functional aspects. Unlike the dimer found in the AU, which is formed through apical domain interactions, the dimeric form we propose here provides a free apical domain that is required for normal chaperone activity and may be involved in M. tuberculosis association with macrophages and arthrosclerosis plaque formation. Here we describe in detail the structural aspects that lead to Cpn60.2 dimer formation and prevent the formation of heptameric rings and tetradecameric double rings.     

Mycobacterium tuberculosis Promotes Anti-apoptotic Activity of the Macrophage by PtpA Protein-dependent Dephosphorylation of Host GSK3α

Mycobacterium tuberculosis tyrosine phosphatase PtpA inhibits two key cellular events in macrophages required for the elimination of invading organisms, phagosome acidification, and maturation. Kinome analysis revealed multiple PtpA-dependent changes to the phosphorylation status of macrophage proteins upon M. tuberculosis infection. Among those proteins we show that PtpA dephosphorylates GSK3α on amino acid Tyr²⁷⁹, which leads to modulation of GSK3α anti-apoptotic activity, promoting pathogen survival early during infection.     

Standardization of in-house polymerase chain reaction for the identification of Mycobacterium tuberculosis at the reference Tropical Disease Hospital in the State of Goiás, Brazil

This study compares smear, growth in Lowenstein-Jensen medium, and in-house polymerase chain reaction (PCR) techniques for the detection of Mycobacterium tuberculosis. A total of 72 specimens from 72 patients with clinical symptoms of tuberculosis, including 70 sputum and two bronchial aspirate samples, were tested in parallel by smear, culture, and in-house PCR techniques. From these, 48 (66.6%) were negative by the 3 methods, 2 (2.8%) were smear positive and negative by culture and in-house PCR, 11 (15.3%) were both smear and culture negative, and in-house PCR positive, 7 (9.7%) were positive by the 3 methods, 2 (2.8%) were positive by smear and culture, and negative by PCR, 2 (2.8%) were positive by culture and PCR, but smear negative. After the resolution of discrepancies in PCR results, the sensitivity and specificity for in-house PCR technique to M. tuberculosis relative to the culture, were 81.8% and 81.9%, respectively. These results confirm that this method, in-house PCR, may be a sensitive and specific technique for M. tuberculosis detection, occurring in both positive and negative smear and negative cultures.     

Molecular characterization of Mycobacterium kansasii isolates in the State of São Paulo between 1995-1998

Mycobacterium kansasii is the most common cause of pulmonary nontuberculous mycobacteria infection and classical identification of this pathogen needs a time consuming phenotypic tests. Polymerase chain reaction-restriction fragment length polymorphism analysis (PRA) of the gene enconding for the 65 kDa heat shock (hsp65) protein offers an easy, rapid, and inexpensive procedure to identify and subtype M. kansasii isolates. In the present study, we performed a retrospective analysis of patients who had mycobacteria identified on the basis of phenotypic tests by means of a review of database at Mycobacteria Laboratory of the Instituto Adolfo Lutz in the period 1995-1998. A total of 9381 clinical isolates were analyzed of which 7777 (82.9%) were identified as M. tuberculosis complex and 1604 (17.1%) as nontuberculous mycobacteria. Of the 296 M. kansasii isolates, 189 (63.8%) isolates obtained from 119 patients were viable and were analyzed by PRA-hsp65. Hundred eight two (98.9%) were classified as M. kansasii type I. Two isolates were classified as type II and III and five isolates were characterized as other Mycobacterium species. Clinical isolates of M. kansasii in the state of Sao Paulo was almost exclusively subtype I regardless of HIV status.     

The role of the β5-α11 loop in the active-site dynamics of acylated penicillin-binding protein A from Mycobacterium tuberculosis

Penicillin-binding protein A (PBPA) is a class B penicillin-binding protein that is important for cell division in Mycobacterium tuberculosis. We have determined a second crystal structure of PBPA in apo form and compared it with an earlier structure of apoenzyme. Significant structural differences in the active site region are apparent, including increased ordering of a β-hairpin loop and a shift of the SxN active site motif such that it now occupies a position that appears catalytically competent. Using two assays, including one that uses the intrinsic fluorescence of a tryptophan residue, we have also measured the second-order acylation rate constants for the antibiotics imipenem, penicillin G, and ceftriaxone. Of these, imipenem, which has demonstrable anti-tubercular activity, shows the highest acylation efficiency. Crystal structures of PBPA in complex with the same antibiotics were also determined, and all show conformational differences in the β5-α11 loop near the active site, but these differ for each β-lactam and also for each of the two molecules in the crystallographic asymmetric unit. Overall, these data reveal the β5-α11 loop of PBPA as a flexible region that appears important for acylation and provide further evidence that penicillin-binding proteins in apo form can occupy different conformational states.