DAHP synthase from Mycobacterium tuberculosis H37Rv: Cloning, expression, and purification of functional enzyme

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains the leading cause of mortality due to a bacterial pathogen. According to the 2004 Global TB Control Report of the World Health Organization, there are 300,000 new cases per year of multi-drug resistant strains (MDR-TB), defined as resistant to isoniazid and rifampicin, and 79% of MDR-TB cases are now super strains, resistant to at least three of the four main drugs used to treat TB. Thus there is a need for the development of effective new agents to treat TB. The shikimate pathway is an attractive target for the development of antimycobacterial agents because it has been shown to be essential for the viability of M. tuberculosis, but absent from mammals. The M. tuberculosis aroG-encoded 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (mtDAHPS) catalyzes the first committed step in this pathway. Here we describe the PCR amplification, cloning, and sequencing of aroG structural gene from M. tuberculosis H37Rv. The expression of recombinant mtDAHPS protein in the soluble form was obtained in Escherichia coli Rosetta-gami (DE3) host cells without IPTG induction. An approximately threefold purification protocol yielded homogeneous enzyme with a specific activity value of 0.47 U mg-1 under the experimental conditions used. Gel filtration chromatography results demonstrate that recombinant mtDAHPS is a pentamer in solution. The availability of homogeneous mtDAHPS will allow structural and kinetics studies to be performed aiming at antitubercular agents development. © 2004 Elsevier Inc. All rights reserved.

Plantas do cerrado brasileiro com atividade contra Mycobacterium fortuitum

Mycobacterium fortuitum is a rapidly-growing species of bacteria, ubiquitous in the environment and related to important human mycobacterioses. It has been isolated from blood, abscesses, the endocardium and surgical and traumatic wounds. This mycobacterium is hard to treat, being recognized in the literature as resistant even to the drugs used in the treatment of tuberculosis. The objective of this study was to screen extracts prepared from plants of the Brazilian cerrado (extended savanna-like belt) with known activity against M. fortuitum, employing the Microplate Alamar Blue Assay (MABA) as the analytical method. Out of 26 extracts tested against M. fortuitum, the nonpolar extract of Quassia amara (in methylene dichloride) gave the best result (MIC 62.5μg/ mL), followed by the nonpolar extracts of Syngonanthus macrolepsis, Davilla elliptica and Turnera ulmifolia, with equal MICs of 125μg/ml. The polar extracts (in ethanol and methanol) obtained from the same plants were considered inactive, since the MIC values determined were above 500μg/mL and not significantly different from those of extracts from other plants, without known activity.

Use of skin graft punch graft type for the healing of leg ulcers in treated Hansen's disease patients

Hansen's disease is an infectious illness caused by Mycobacterium leprae. It affects preferentially the skin and the peripheral nervous system leading to incapacities, such as leg ulcers, which happens due to the direct action of the bacillus on the organs or its indirect action on the peripheral nervous system. Leg ulcers can occur by two physiopathologic processes. There are many treatments for general leg ulcers, which include the ones caused by Hansen's disease sequels. Among them, surgical treatment shows to be effective when using skin graft, which can be performed by several techniques. Considering the low number of techniques known for treating leg ulcers in Hansen's disease sequels, the aims of this work were to standardize alternative techniques, to detect the main bacteria found in ulcer secretion cultures, to analyze the patients profile and the ulcers, to describe the histophatologies found, and to correlate these data with those of literature from all over the world. Skin graft punch type was carried out and analyzed; males had a mean age of 59.4 years old and females, 54.2 years old. Patients were 73.6% male and 26.3% female. Lepromatous type was present in 89.4% patients and tuberculoid type was seen in 10.5% of them. Associated systemic diseases were observed in 26.3% patients. Mean time of ulcers evolution was 11.6 years in male and 12.8 years in women. The average diameter of ulcers in the pre-treatment period was 8.5 X 9.5 cm in male and 10.2 X 6.8 cm in women. After the graft, their average diameters were 3.2 X 2.7 cm in male and 5.1 X 5.6 cm in women. Statistical analysis showed that there was no significant correlation between the ulcer diameter and its reduction or not in the post-surgery period (p=0.269732). The mean age of patients whose ulcers diameter did not change or reduced by only 20% was 63.5 years. Using the Spearman's coefficient, it was possible to observe that there was no significant correlation between the patients' age and the ulcers diameter reduction after the skin graft (p=0.222531). Evolution time of ulcers that did not present any satisfactory result in the post-surgery period was 12.1 years. The Spearman's coefficient showed that there was no significant correlation between the ulcers evolution time and the ulcers diameter reduction in the post-surgery period (p=0.191655). Cultures presented 50% of cases with Pseudomonas aeruginosa. Statistical analysis showed there is no correlation between the bacterial types found and the ulcer evolution in the post-surgery period (p=0.697531). The average of the ulcers diameter reduction was 42.4%, and in 26.3% of the patients the lesions disappeared after the skin graft.

Effect of environmental mycobacteria (Mycobacterium avium) on immunity induced by a DNA vaccine (DNAhsp65) against tuberculosis

The efficacy of BCG vaccine (attenuated Mycobacterium bovis) against pulmonary tuberculosis varies enormously among different populations. The prevailing hypothesis attributes this variation to interactions between the vaccine and mycobacteria common in the environment. Studies have revealed that most protective antigens expressed by the antituberculous vaccine are conserved in M. avium, supporting the hypothesis that exposure to environmental mycobacteria generates a cross-reactive immune response that interferes with BCG efficacy. In this study we investigated the effect of a prior exposure to heat-killed M. avium on the immune response and the protective efficacy induced by a genetic vaccine pVAXhsp65 (hsp65 gene from M. leprae inserted in pVAX vector) against experimental tuberculosis. To evaluate the effect on the immune response, female BALB/c mice were initially injected with distinct doses (0.08×106, 4×106, and 200×10 6) of heat-killed M. avium by subcutaneous route. Three weeks later, the animals were immunized with 3 doses of DNAhsp65 by intramuscular route (100μg/15 days apart). Control groups received only M. avium, vaccine (pVAXhsp65), vector (pVAX) or saline solution. Cytokine production and antibody levels were determined by ELISA. To evaluate the effect on the protective efficacy, animals were initially sensitized with 200×106 heat-killed CFU of M. avium by subcutaneous route and then immunized with 3 doses of pVAXhsp65 (100μg/15 days apart) by intramuscular route. Control groups were injected with saline, pVAX (4 doses), pVAXhsp65 (4 doses), M. avium or M. avium plus pVAX (3 doses). Fifteen days after last DNA dose, the animals were infected with 1×104 viable CFU of H37Rv M. tuberculosis by intratracheal route. Thirty days after challenge, the animals were sacrificed and the bacterial burden was determined by counting the number of CFU in the lungs. Lung histological sections were also analyzed. Splenic cells from primed animals produced more IL-5 but less IFN-gamma than non-primed ones. Also, prior contact with M. avium determined higher production of IgG1 and IgG2a anti-hsp65 antibodies in comparison to control groups. However, this higher immune response did not decrease the bacterial burden in the lungs. In addition, prior sensitization with M. avium decreased the parenchyma preservation observed in the group immunized only with pVaxhsp65. These results indicate that environmental mycobacteria can interfere with immunity and protective efficacy induced by DNAhsp65.

Immune response to Mycobacterium bovis-an5 infection in genetically selected mice (selection IV-A)

Mice genetically selected for high (H) and low (L) antibody production (Selection IV-A) were used as murine experimental model. The aim of the present work was to evaluate the macrophagic activity and to characterize the immune response in Mycobacterium bovis-AN5 infected mice (3×10 7 bacteria). The response profile previously observed in such strains was not similar to that obtained during M. bovis infection; however, it corroborated works carried out using Selection I, which is very similar to Selection IV-A regarding infection by M. tuberculosis and Bacillus Calmette-Guérin (BCG). Considering bacterial recovery, LIV-A mice showed higher control of the infectious process in the lungs than in the spleen, whereas HIV-A mice presented more resistance in the spleen. With respect to macrophagic activity, hydrogen peroxide (H2O 2) was probably not involved in the infection control since there was an inhibition in the production of this metabolite. Nitric oxide (NO) and TNF-α production seemed to be important in the control of bacterial replication and varied according to the strain, period and organ. Evaluation of the antibody production indicated that the multi-specific effect commonly observed in these strains was not the same in the response to M. bovis. Antibody concentrations were higher in LIV-A than in HIV-A mice at the beginning of the infection, being similar afterwards. Such data were compared with delayed-type hypersensitivity (DTH), which was more intense in HIV-A than in LIV-A mice, indicating that antibody production is independent of the capability to trigger DTH reactions and that cellular and humoral responses to M. bovis antigens show a polygenic control and an independent quantitative genetic regulation. Differences were observed among organs and metabolites, suggesting that different mechanisms play an important role in this infection in natural heterogeneous populations, indicating that NO, TNF-α and Th1 cytokines are involved in the infection control.

Tuberculose e o estudo molecular da sua epidemiologia

Systems that can distinguish epidemiologically-related Mycobacterium tuberculosis strains from unrelated ones are extremely valuable. Molecular biology techniques have allowed a great deal of information to be acquired about the infectious disease tuberculosis (TB) that was very hard or impossible to obtain by conventional epidemiology. A typing method based on bacterial DNA genome differences, known as RFLP (restriction fragment length polymorphism), is widely used to discriminate strains in the epidemiologic study of TB. However, RFLP is laborious and there is a tendency to replace it by other methods. Thus, other DNA sequences have been employed as epidemiological markers, as in Spoligotyping, a fast technique based on PCR followed by differential hybridization of amplified products. The polymorphism observed among different isolates is probably the product of strain-dependent recombination. MIRU (mycobacterial interspersed repetitive unit) typing is a reproducible and fast assay, involving the generation of genotypes based on the study of 12 loci containing VNTRs (variable-number tandem repeats) in strains of the M. tuberculosis complex. It compares strains from different geographic areas and allows the movement of individual lineages to be tracked, as in RFLP. This approach enables a greater number of isolates to be analyzed, leading to the identification of a larger number of foci of transmission within the population and thus to improved ways of slowing the progress of the disease.

The Mycobacterium tuberculosis Rv2540c DNA sequence encodes a bifunctional chorismate synthase

Background. The emergence of multi- and extensively-drug resistant Mycobacterium tuberculosis strains has created an urgent need for new agents to treat tuberculosis (TB). The enzymes of shikimate pathway are attractive targets to the development of antitubercular agents because it is essential for M. tuberculosis and is absent from humans. Chorismate synthase (CS) is the seventh enzyme of this route and catalyzes the NADH- and FMN-dependent synthesis of chorismate, a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Although the M. tuberculosis Rv2540c (aroF) sequence has been annotated to encode a chorismate synthase, there has been no report on its correct assignment and functional characterization of its protein product. Results. In the present work, we describe DNA amplification of aroF-encoded CS from M. tuberculosis (MtCS), molecular cloning, protein expression, and purification to homogeneity. N-terminal amino acid sequencing, mass spectrometry and gel filtration chromatography were employed to determine identity, subunit molecular weight and oligomeric state in solution of homogeneous recombinant MtCS. The bifunctionality of MtCS was determined by measurements of both chorismate synthase and NADH:FMN oxidoreductase activities. The flavin reductase activity was characterized, showing the existence of a complex between FMN ox and MtCS. FMNox and NADH equilibrium binding was measured. Primary deuterium, solvent and multiple kinetic isotope effects are described and suggest distinct steps for hydride and proton transfers, with the former being more rate-limiting. Conclusion. This is the first report showing that a bacterial CS is bifunctional. Primary deuterium kinetic isotope effects show that C4-proS hydrogen is being transferred during the reduction of FMNox by NADH and that hydride transfer contributes significantly to the rate-limiting step of FMN reduction reaction. Solvent kinetic isotope effects and proton inventory results indicate that proton transfer from solvent partially limits the rate of FMN reduction and that a single proton transfer gives rise to the observed solvent isotope effect. Multiple isotope effects suggest a stepwise mechanism for the reduction of FMNox. The results on enzyme kinetics described here provide evidence for the mode of action of MtCS and should thus pave the way for the rational design of antitubercular agents. © 2008 Ely et al; licensee BioMed Central Ltd.

Usefulness of Mycobacterium tuberculosis molecular typing in a tuberculosis low-endemic agro-industrial setting of Brazil

To highlight the transmission and major phylogenetic clades of Mycobacterium tuberculosis, a retrospective study was carried out at two health facilities in a small agro-industrial area in São Paulo, Brazil, that has a low tuberculosis incidence rate. IS6110-RFLP and spoligotyping were performed on the isolates, with the former revealing that 31.3% (35/112) of strains were clustered. Epidemiological links were found in 16 of the 35 clustered patients and were associated with transmission among patients living in public housing. Spoligotyping grouped 62.8% of the strains. The T genetic family predominated among the isolates. Of interest is that five strains had a pattern characteristic of African or Asian origin (ST535), and two others were of the rare localized type ST1888 (BRA, VEN). In addition, three new types-1889, 1890, and 1891-were identified. Spoligotyping showed that some ST may be circulating to or from Brazil, and RFLP revealed ongoing transmission in inadequately ventilated public-housing buildings. This may point to a failure in tuberculosis control policy.

The Rv1712 locus from Mycobacterium tuberculosis H37Rv codes for a functional CMP kinase that preferentially Phosphorylates dCMP

The Mycobacterium tuberculosis cmk gene, predicted to encode a CMP kinase (CMK), was cloned and expressed, and its product was purified to homogeneity. Steady-state kinetics confirmed that M. tuberculosis CMK is a monomer that preferentially phosphorylates CMP and dCMP by a sequential mechanism. A plausible role for CMK is discussed. Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Structural studies of Helicase NS3 variants from Hepatitis C virus genotype 3 in virological sustained responder and non-responder patients

Background. About 130 million people are infected with the hepatitis C virus (HCV) worldwide, but effective treatment options are not yet available. One of the most promising targets for antiviral therapy is nonstructural protein 3 (NS3). To identify possible changes in the structure of NS3 associated with virological sustained response or non-response of patients, a model was constructed for each helicase NS3 protein coding sequence. From this, the goal was to verify the interaction between helicases variants and their ligands. Findings. Evidence was found that the NS3 helicase portion of non-responder patients contained substitutions in its ATP and RNA binding sites. K210E substitution can cause an imbalance in the distribution of loads, leading to a decrease in the number of ligations between the essential amino acids required for the hydrolysis of ATP. W501R substitution causes an imbalance in the distribution of loads, leading and forcing the RNA to interact with the amino acid Thr269, but not preventing binding of ribavirin inhibitor. Conclusions. Useful information is provided on the genetic profiling of the HCV genotype 3, specifically the coding region of the NS3 protein, improving our understanding of the viral genome and the regions of its protein catalytic site. © 2010 Rahal et al; licensee BioMed Central Ltd.

Induction pegylated interferon Alfa-2a and high dose ribavirin do not increase SVR in heavy patients with HCV genotype 1 and high viral loads

Background & Aims Patients infected with hepatitis C virus (HCV) genotype 1, body weight <85 kg, and high baseline viral load respond poorly to standard doses of pegylated interferon (peginterferon) and ribavirin. We evaluated intensified therapy with peginterferon alfa-2a plus ribavirin. Methods This double-blind randomized trial included HCV genotype 1-infected outpatients from hepatology clinics with body weight <85 kg and HCV RNA titer <400,000 IU/mL. Patients were randomized to 180 μg/wk peginterferon alfa-2a for 48 weeks plus 1200 mg/day ribavirin (standard of care) (group A, n = 191) or 1400/1600 mg/day ribavirin (group B, n = 189). Additional groups included 360 μg/wk peginterferon alfa-2a for 12 weeks then 180 μg/wk peginterferon alfa-2a for 36 weeks plus 1200 mg/day ribavirin (group C, n = 382) or 1400/1600 mg/day ribavirin (group D, n = 383). Follow-up lasted 24 weeks after treatment. Results Sustained virologic response rates (HCV RNA level <15 IU/mL at end of follow-up) in groups A, B, C, and D were 38%, 43%, 44%, and 41%, respectively. There were no significant differences among the 4 groups or between pooled peginterferon alfa-2a regimens (A + B vs C + D: odds ratio [OR], 1.08; 95% confidence interval [CI], 0.831.39; P = .584) or pooled ribavirin regimens (A + C vs B + D: OR, 1.00; 95% CI, 0.791.28; P = .974). Conclusions In patients infected with HCV genotype 1 who are difficult to treat (high viral load, body weight <85 kg), a 12-week induction regimen of peginterferon alfa-2a and/or higher-dose ribavirin is not more effective than the standard regimen. © 2010 AGA Institute.

UMP kinase from Mycobacterium tuberculosis: Mode of action and allosteric interactions, and their likely role in pyrimidine metabolism regulation

The pyrH-encoded uridine 5′-monophosphate kinase (UMPK) is involved in both de novo and salvage synthesis of DNA and RNA precursors. Here we describe Mycobacterium tuberculosis UMPK (MtUMPK) cloning and expression in Escherichia coli. N-terminal amino acid sequencing and electrospray ionization mass spectrometry analyses confirmed the identity of homogeneous MtUMPK. MtUMPK catalyzed the phosphorylation of UMP to UDP, using ATP-Mg 2+ as phosphate donor. Size exclusion chromatography showed that the protein is a homotetramer. Kinetic studies revealed that MtUMPK exhibits cooperative kinetics towards ATP and undergoes allosteric regulation. GTP and UTP are, respectively, positive and negative effectors, maintaining the balance of purine versus pyrimidine synthesis. Initial velocity studies and substrate(s) binding measured by isothermal titration calorimetry suggested that catalysis proceeds by a sequential ordered mechanism, in which ATP binds first followed by UMP binding, and release of products is random. As MtUMPK does not resemble its eukaryotic counterparts, specific inhibitors could be designed to be tested as antitubercular agents. © 2010 Elsevier Inc. All rights reserved.

Buffalo milk production in brazil and colombia: Genotype by environment interaction

The objective of this study was to determine whether there is a genotype by environment interaction (GxE) for dairy buffaloes in Brazil and Colombia. The (co)variance components were estimated by using a bi-trait repeatability animal model with the REML method. Each trait consisted in the milk yield obtained in both countries. Contemporary group (herd, year and season of parity) and age at parity (linear and quadratic covariate) fixed effects, along with the additive genetic, permanent environment, and the residual random effects were included in the model. Genetic, permanent environmental and residual variance and heritabilities were different for both countries. The genetic correlations for milk yield between Brazil and Colombia were low (between 0.10 and 0.13), indicating a GxE interaction between both countries. Knowing that this interaction influences the genetic progress of buffalo populations in Brazil and Colombia, we recommend choosing sires tested in the country they will be used, along with conducting joint genetic evaluations that consider GxE interaction effects.

Genotype by environment interaction in different birth seasons for weight at 240, 365 and 450 days of age in Tabapuã cattle

The objective of this study was to evaluate the effect of genotype by environment interaction (GEI) on the weight of Tabapuã cattle at 240 (W240), 365 (W365) and 450 (W450) days of age. In total, 35,732 records of 8,458 Tabapuã animalswhich were born in the state of Bahia, Brazil, from 1975 to 2001, from 167 sires and 3,707 dams, were used. Two birth seasons were tested as for the environment effect: the dry (D) and rainy (R) ones. The covariance components were obtainedby a multiple-trait analysis using Bayesian inference, in which each trait was considered as being different in each season. Covariance components were estimated by software gibbs2f90. As for W240, the model was comprised of contemporary groups and cow age (in classes) as fixed effects; animal and maternal genetic additive, maternal permanent environmental and residual were considered as random effects. Concerning W365 and W450, the model included only the contemporary aged cow groups as fixed effects and the genetic additive and residual effects of the animal as the random ones. The GEI was assessed considering the genetic correlation, in which values below 0.80 indicated the presence of GEI. Regarding W365 and W450, the GEI was found in both seasons. As for post-weaning weight (W240), the effect of such interaction was not observed. ©2012 Sociedade Brasileira de Zootecnia.

Intensified peginterferon α-2a dosing increases sustained virologic response rates in heavy, high viral load hepatitis c genotype 1 patients with high low-density lipoprotein

BACKGROUND AND GOAL: Patients infected with hepatitis C virus (HCV) with elevated low-density lipoprotein (LDL) levels achieve higher sustained virologic response (SVR) rates after peginterferon (PegIFN)/ribavirin treatment versus patients with lower LDL. Our aim was to determine whether SVR rates in patients with low/elevated LDL can be improved by dose intensification. STUDY: In PROGRESS, genotype 1 patients with baseline HCV RNA≥400,000 IU/mL and body weight ≥85 kg were randomized to 48 weeks of 180 μg/wk PegIFN α-2a (40 kDa) plus ribavirin (A: 1200 mg/d; B: 1400/1600 mg/d) or 12 weeks of 360 μg/wk PegIFN α-2a followed by 36 weeks of 180 μg/wk, plus ribavirin (C: 1200 mg/d; D: 1400/1600 mg/d). This retrospective analysis assessed SVR rates among patients with low (<100 mg/dL) or elevated (≥100 mg/dL) LDL. Patients with high LDL (n=256) had higher baseline HCV RNA (5.86×10 IU/mL) versus patients with low LDL (n=262; 4.02×10 IU/mL; P=0.0003). RESULTS: Multiple logistic regression analysis identified a significant interaction between PegIFN α-2a dose and LDL levels on SVR (P=0.0193). The only treatment-related SVR predictor in the nested multiple logistic regression was PegIFN α-2a dose among patients with elevated LDL (P=0.0074); therefore, data from the standard (A+B) and induction (C+D) dose arms were pooled. Among patients with low LDL, SVR rates were 40% and 35% in the standard and induction-dose groups, respectively; SVR rates in patients with high LDL were 44% and 60% (P=0.014), respectively. CONCLUSIONS: Intensified dosing of PegIFN α-2a increases SVR rates in patients with elevated LDL even with the difficult-to-cure characteristics of genotype 1, high baseline viral load, and high body weight. Copyright © 2013 by Lippincott Williams & Wilkins.