Crystallographic and pre-steady-state kinetics studies on binding of NADH to wild-type and isoniazid-resistant enoyl-ACP(CoA) reductase enzymes from Mycobacterium tuberculosis

An understanding of isoniazid (INH) drug resistance mechanism in Mycobacterium tuberculosis should provide significant insight for the development of newer anti-tubercular agents able to control INH-resistant tuberculosis (TB). The inhA-encoded 2-trans enoyl-acyl carrier protein reductase enzyme (InhA) has been shown through biochemical and genetic studies to be the primary target for INH. In agreement with these results, mutations in the inhA structural gene have been found in INH-resistant clinical isolates of M. tuberculosis, the causative agent of TB. In addition, the InhA mutants were shown to have higher dissociation constant values for NADH and lower values for the apparent first-order rate constant for INH inactivation as compared to wild-type InhA. Here, in trying to identify structural changes between wild-type and INH-resistant InhA enzymes, we have solved the crystal structures of wild-type and of S94A, I47T and I21V InhA proteins in complex with NADH to resolutions of, respectively, 2.3 angstrom, 2.2 angstrom, 2.0 angstrom, and 1.9 angstrom. The more prominent structural differences are located in, and appear to indirectly affect, the dinucleotide binding loop structure. Moreover, studies on pre-steady-state kinetics of NADH binding have been carried out. The results showed that the limiting rate constant values for NADH dissociation from the InhA-NADH binary complexes (k(off)) were eleven, five, and tenfold higher for, respectively, I21V, I47T and S94A INH-resistant mutants of InhA as compared to INH-sensitive wildtype InhA. Accordingly, these results are proposed to be able to account for the reduction in affinity for NADH for the INH-resistant InhA enzymes. (c) 2006 Elsevier Ltd. All rights reserved.

Crystallographic studies on the binding of isonicotinyl-NAD adduct to wild-type and isoniazid resistant 2-trans-enoyl-ACP (CoA) reductase from Mycobacterium tuberculosis

The resumption of tuberculosis led to an increased need to understand the molecular mechanisms of drug action and drug resistance, which should provide significant insight into the development of newer compounds. Isoniazid (INH), the most prescribed drug to treat TB, inhibits an NADH-dependent enoyl-acyl carrier protein reductase (InhA) that provides precursors of mycolic acids, which are components of the mycobacterial cell wall. InhA is the major target of the mode of action of isoniazid. INH is a pro-drug that needs activation to form the inhibitory INH-NAD adduct. Missense mutations in the inhA structural gene have been identified in clinical isolates of Mycobacterium tuberculosis resistant to INH. To understand the mechanism of resistance to INH, we have solved the structure of two InhA mutants (121V and S94A), identified in INH-resistant clinical isolates, and compare them to INH-sensitive WT InhA structure in complex with the INH-NAD adduct. We also solved the structure of unliganded INH-resistant S94A protein, which is the first report on apo form of InhA. The salient features of these structures are discussed and should provide structural information to improve our understanding of the mechanism of action of, and resistance to, INH in M. tuberculosis. The unliganded structure of InhA allows identification of conformational changes upon ligand binding and should help structure-based drug design of more potent antimycobacterial agents. (c) 2007 Elsevier B.V. All rights reserved.

pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis clinical isolates from the southeast region of Brazil

Objectives: To investigate the presence of mutations in the pncA gene in 31 pyrazinamide-resistant Mycobacterium tuberculosis and 5 susceptible strains. MICs and pyrazinamidase (PZase) activity were also determined.Methods: All 36 M. tuberculosis clinical isolates were genotyped by mycobacterial interspersed repetitive units (MIRUs) and most were also typed by spoligotyping. The MIC value necessary to inhibit 99% of the resistant mycobacterial isolates was determined by microplate Alamar Blue assay (MABA) and by Lowenstein-Jensen assay (LJA). The PZase activity was measured by pyrazinamide deamination to pyrazinoic acid and ammonia, and the entire pncA sequence including the 410 by upstream from the start codon was determined by DNA sequencing of purified PCR products.Results: of the 31 isolates resistant to pyrazinamide, 26 (83.9%) showed at least one mutation in the pncA gene or in its putative regulatory region: Among the 22 different mutations detected in the pncA gene and in its regulatory region, 9 (40.9%) mutations (consisting of six substitutions, two insertions and one deletion) have not been described in previous studies. Three pyrazinamide-resistant isolates, confirmed by MIC varying from 800 to 1600 mg/L, carried the wild-type pncA sequence and retained PZase activity.Conclusions: These results contribute to the knowledge of the molecular mechanism of pyrazinamide resistance in Brazil and also expand the profile of pncA mutations worldwide. The MABA was successfully used to determine the MICs of pyrazinamide.

INCIDENCE OF MYCOBACTERIUM-TUBERCULOSIS AND OTHER MYCOBACTERIA ON PULMONARY INFECTIONS IN ARARAQUARA - SP, 1993

The incidence of tuberculosis and other infections by mycobacteria was analyzed in 559 patients admitted to the Tisiology Section of the Special Health Care Unit of Araraquara (SESA). Mycobacteria were isolated from 78 individuals out of this total. Among these patients, 15 were also HIV positive. The occurrence of isolated species was: M. tuberculosis: 69 patients; M. avium-intracellulare: 5 patients; M. fortuitum: 2 patients; M. chelonae: 1 patient; and M. simiae 1 patient. The latter was for the first time isolated from humans in Brazil. In most cases, non tubercular mycobacteria (NTM) were found in the HIV positive patients.

Screening and characterization of mutations in isoniazid-resistant Mycobacterium tuberculosis isolates obtained in Brazil

We investigated mutations in the genes katG, inhA (regulatory and structural regions), and kasA and the oxyR-ahpC intergenic region of 97 isoniazid (INH)-resistant and 60 INH-susceptible Mycobacterium tuberculosis isolates obtained in two states in Brazil: São Paulo and Parana. PCR-single-strand conformational polymorphism (PCR-SSCP) was evaluated for screening mutations in regions of prevalence, including codons 315 and 463 of katG, the regulatory region and codons 16 and 94 of inhA, kasA, and the oxyR-ahpC intergenic region. DNA sequencing of PCR amplicons was performed for all isolates with altered PCR-SSCP profiles. Mutations in katG were found in 83 (85.6%) of the 97 INH-resistant isolates, including mutations in codon 315 that occurred in 60 (61.9%) of the INH-resistant isolates and 23 previously unreported katG mutations. Mutations in the inhA promoter region occurred in 25 (25.8%) of the INH-resistant isolates; 6.2% of the isolates had inhA structural gene mutations, and 10.3% had mutations in the oxyR-ahpC intergenic region (one, nucleotide -48, previously unreported). Polymorphisms in the kasA gene occurred in both INH-resistant and INH-susceptible isolates. The most frequent polymorphism encoded a G(269)A substitution. Although KatG(315) substitutions are predominant, novel mutations also appear to be responsible for INH resistance in the two states in Brazil. Since ca. 90.7% of the INH-resistant isolates had mutations identified by SSCP electrophoresis, this method may be a useful genotypic screen for INH resistance.

Immune modulation induced by tuberculosis DNA vaccine protects non-obese diabetic mice from diabetes progression

We have described previously the prophylactic and therapeutic effect of a DNA vaccine encoding the Mycobacterium leprae 65 kDa heat shock protein (DNA-HSP65) in experimental murine tuberculosis. However, the high homology of this protein to the corresponding mammalian 60 kDa heat shock protein (Hsp60), together with the CpG motifs in the plasmid vector, could trigger or exacerbate the development of autoimmune diseases. The non-obese diabetic (NOD) mouse develops insulin-dependent diabetes mellitus (IDDM) spontaneously as a consequence of an autoimmune process that leads to destruction of the insulin-producing beta cells of the pancreas. IDDM is characterized by increased T helper 1 (Th1) cell responses toward several autoantigens, including Hsp60, glutamic acid decarboxylase and insulin. In the present study, we evaluated the potential of DNA-HSP65 injection to modulate diabetes in NOD mice. Our results show that DNA-HSP65 or DNA empty vector had no diabetogenic effect and actually protected NOD mice against the development of severe diabetes. However, this effect was more pronounced in DNA-HSP65-injected mice. The protective effect of DNA-HSP65 injection was associated with a clear shift in the cellular infiltration pattern in the pancreas. This change included reduction of CD4(+) and CD8(+) T cells infiltration, appearance of CD25(+) cells influx and an increased staining for interleukin (IL)-10 in the islets. These results show that DNA-HSP65 can protect NOD mice against diabetes and can therefore be considered in the development of new immunotherapeutic strategies.

Immune regulatory effect of pHSP65 DNA therapy in pulmonary tuberculosis: activation of CD8(+) cells, interferon-gamma recovery and reduction of lung injury

A DNA vaccine based on the heat-shock protein 65 Mycobacterium leprae gene (pHSP65) presented a prophylactic and therapeutic effect in an experimental model of tuberculosis. In this paper, we addressed the question of which protective mechanisms are activated in Mycobacterium tuberculosis-infected mice after immune therapy with pHSP65. We evaluated activation of the cellular immune response in the lungs of infected mice 30 days after infection (initiation of immune therapy) and in those of uninfected mice. After 70 days (end of immune therapy), the immune responses of infected untreated mice, infected pHSP65-treated mice and infected pCDNA3-treated mice were also evaluated. Our results show that the most significant effect of pHSP65 was the stimulation of CD8(+) lung cell activation, interferon-gamma recovery and reduction of lung injury. There was also partial restoration of the production of tumour necrosis factor-alpha. Treatment with pcDNA3 vector also induced an immune stimulatory effect. However, only infected pHSP65-treated mice were able to produce significant levels of interferon-gamma and to restrict the growth of bacilli.

Effects of the magnesium and chloride ions and shikimate on the structure of shikimate kinase from Mycobacterium tuberculosis

Bacteria, fungi and plants can convert carbohydrate and phosphoenolpyruvate into chorismate, which is the precursor of various aromatic compounds. The seven enzymes of the shikimate pathway are responsible for this conversion. Shikimate kinase (SK) is the fifth enzyme in this pathway and converts shikimate to shikimate-3-phosphate. In this work, the conformational changes that occur on binding of shikimate, magnesium and chloride ions to SK from Mycobacterium tuberculosis (MtSK) are described. It was observed that both ions and shikimate influence the conformation of residues of the active site of MtSK. Magnesium influences the conformation of the shikimate hydroxyl groups and the position of the side chains of some of the residues of the active site. Chloride seems to influence the affinity of ADP and its position in the active site and the opening length of the LID domain. Shikimate binding causes a closing of the LID domain and also seems to influence the crystallographic packing of SK. The results shown here could be useful for understanding the catalytic mechanism of SK and the role of ions in the activity of this protein.

Single dose of a vaccine based on DNA encoding mycobacterial hsp65 protein plus TDM-loaded PLGA microspheres protects mice against a virulent strain of Mycobacterium tuberculosis

The high incidence of tuberculosis around the world and the inability of BCG to protect certain populations clearly indicate that an improved vaccine against tuberculosis is needed. A single antigen, the mycobacterial heat shock protein hsp65, is sufficient to protect BALB/c mice against challenge infection when administered as DNA vaccine in a three-dose-based schedule. In order to simplify the vaccination schedule, we coencapsulated hsp65-DNA and trehalose dimicolate (TDM) into biodegradable poly(DL-lactide-co-glycolide) (PLGA) microspheres. BALB/c mice immunized with a single dose of DNA-hsp65/TDM-1oaded microspheres produced high levels of IgG2a subtype antibody and high amounts of IFN-gamma in the supernatant of spleen cell cultures. DNA-hsp65/TDM-loaded microspheres were also able to induce high IFN-gamma production in bulk lung cells from challenged mice and confer protection as effective as that attained after three doses of naked DNA administration. This new formulation also allowed a ten-fold reduction in the DNA dose when compared to naked DNA. Thus, this combination of DNA vaccine and adjuvants with immunomodulatory and carrier properties holds the potential for an improved vaccine against tuberculosis.

Evaluation of the microscopic observation drug susceptibility assay for detection of Mycobacterium tuberculosis resistance to pyrazinamide

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Palladium(II) Complexes with Thiosemicarbazones. Syntheses, Characterization and Cytotoxicity against Breast Cancer Cells and Anti-Mycobacterium tuberculosis Activity

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Implication of the RDRio Mycobacterium tuberculosis sublineage in multidrug resistant tuberculosis in Portugal

Multidrug and extensively drug resistant Mycobacterium tuberculosis are a threat to tuberculosis control programs. Genotyping methods, such as spoligotyping and MIRU-VNTR typing (Mycobacterial Interspersed Repetitive Units), are useful in monitoring potentially epidemic strains and estimating strain phylogenetic lineages and/or genotypic families. M. tuberculosis Latin American Mediterranean (LAM) family is a major worldwide contributor to tuberculosis (TB). LAM specific molecular markers, Ag85C(103) single nucleotide polymorphism (SNP) and RDRio long-sequence polymorphism (LSP), were used to characterize spoligotype signatures from 859 patient isolates from Portugal. LAM strains were found responsible for 57.7% of all tuberculosis cases. Strains with the RDRio deletion (referred to as RDRio) were estimated to represent 1/3 of all the strains and over 60% of the multidrug resistant (MDR) strains. The major spoligotype signature SIT20 belonging to the LAM1 RDRio sublineage, represented close to 1/5th of all the strains, over 20% of which were MDR. Analysis of published datasets according to stipulated 12 loci MIRU-VNTR RDRio signatures revealed that 96.3% (129/134) of MDR and extensively drug resistant (XDR) clusters were RDRio. This is the first report associating the LAM RDRio sublineage with MDR. These results are an important contribution to the monitoring of these strains with heightened transmission for future endeavors to arrest MDR-TB and XDR-TB. (c) 2012 Elsevier B.V. All rights reserved.

Tuberculosis diagnosis after bleach processing for early stage tuberculosis laboratory capacity building

The diagnosis of tuberculosis is seriously hampered in the absence of standard biosafety laboratory facilities for specimen concentration and Mycobacterium tuberculosis culture. Within a laboratory twinning arrangement, heat-fixed direct smear and sediment from 74 bleach-processed and 20 non-processed specimens from Cumura Hospital, Guinea-Bissau, were sent to Lisbon for molecular evaluation of rifampicin resistance. Sequence analysis of a 369 base-pair ppoB locus detected 3.2% (3/94) resistant specimens. To our knowledge, this represents the first report on the molecular analysis of M. tuberculosis from bleach-processed sputum, an alternative to current diagnostic practice in low-resource settings.

Design of novel iron compounds as potential therapeutic agents against tuberculosis

In the search for new therapeutic tools against tuberculosis two novel iron complexes, [Fe(L-H)3], with 3-aminoquinoxaline-2-carbonitrile N(1),N(4)-dioxide derivatives (L) as ligands, were synthesized, characterized by a combination of techniques, and in vitro evaluated. Results were compared with those previously reported for two analogous iron complexes of other ligands of the same family of quinoxaline derivatives. In addition, the complexes were studied by cyclic voltammetry and EPR spectroscopy. Cyclic voltammograms of the iron compounds showed several cathodic processes which were attributed to the reduction of the metal center (Fe(III)/Fe(II)) and the coordinated ligand. EPR signals were characteristic of magnetically isolated high-spin Fe(III) in a rhombic environment and arise from transitions between m(s) = +/- 1/2 (geff-9) or m(s) = +/- 3/2 (g(eff)similar to 4.3) states. Mossbauer experiments showed hyperfine parameters that are typical of high-spin Fe(III) ions in a not too distorted environment. The novel complexes showed in vitro growth inhibitory activity on Mycobacterium tuberculosis H(37)Rv (ATCC 27294), together with very low unspecific cytotoxicity on eukaryotic cells (cultured murine cell line J774). Both complexes showed higher inhibitory effects on M. tuberculosis than the "second-line" therapeutic drugs. (C) 2010 Elsevier B.V. All rights reserved.