One-step purification of 5-enolpyruvylshikimate-3-phosphate synthase enzyme from Mycobacterium tuberculosis

Currently, there are 8 million new cases and 2 million deaths annually from tuberculosis, and it is expected that a total of 225 million new cases and 79 million deaths will occur between 1998 and 2030. The reemergence of tuberculosis as a public health threat, the high susceptibility of HIV-infected persons, and the proliferation of multi-drug-resistant strains have created a need to develop new antimycobacterial agents. The existence of homologues to the shikimate pathway enzymes has been predicted by the determination of the genome sequence of Mycobacterium tuberculosis. We have previously reported the cloning and overexpression of M. tuberculosis aro A-encoded EPSP synthase in both soluble and active forms, without IPTG induction. Here, we describe the purification of M. tuberculosis EPSP synthase (mtEPSPS) expressed in Escherichia coli BL21(DE3) host cells. Purification of mtEPSPS was achieved by a one-step purification protocol using an anion exchange column. The activity of the homogeneous enzyme was measured by a coupled assay using purified shikimate kinase and purine nucleoside phosphorylase proteins. A total of 53 mg of homogeneous enzyme could be obtained from 1 L of LB cell culture, with a specific activity value of approximately 18 U mg-1. The results presented here provide protein in quantities necessary for structural and kinetic studies, which are currently underway in our laboratory. © 2002 Elsevier Science (USA). All rights reserved.

PCR-based detection of Babesia bovis and Babesia bigemina in their natural host Boophilus microplus and cattle

PCR and nested-PCR methods were used to assess the frequency of Babesia bovis and Babesia bigemina infection in Boophilus microplus engorged females and eggs and in cattle reared in an area with endemic babesiosis. Blood and the engorged female ticks were from 27 naturally infested calves and 25 crossbred cows. The frequency of both Babesia species was similar in calves and cows (P > 0.05). Babesia bovis was detected in 23 (85.2%) calves and in 25 (100%) cows and B. bigemina was detected in 25 (92.6%) calves and in 21 (84%) cows. Mixed infections with the both Babesia species were identified in 42 animals, 21 in each age category. Of female ticks engorged on calves, 34.9% were negative and single species infection with B. bigemina (56.2%) was significantly more frequent (P < 0.01) than with B. bovis (4.7%). Most of the females (60.8%) engorged on cows did not show Babesia spp. infection and the frequency of single B. bovis infection (17.6%) was similar (P > 0.05) to the frequency of single B. bigemina infection (15.9%). Mixed Babesia infection was lower (P < 0.01) than single species infection in female ticks engorged either in cows (5.7%) or in calves (4.3%). An egg sample from each female was analysed for the presence of Babesia species. Of the egg samples from female ticks infected with B. bovis, 26 (47.3%) were infected while from those from female ticks infected with B. bigemina 141 (76.6%) were infected (P < 0.01). The results showed that although the frequency of both species of Babesia was similar in calves and cows, the infectivity of B. bigemina was higher to ticks fed on calves while to those ticks fed on cows the infectivity of both Babesia species was similar. © 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

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.

Isolation of Mycobacterium gordonae from poultry slaughterhouse water in São Paulo State, Brazil

Water samples (24 untreated water, 12 treated water and 24 served water) used in different stages of the slaughter process were examined to identify a possible source of pathogenic mycobacteria. The isolates were identified based on microscopy, morphological and biochemical features, mycolic acid analysis and molecular method - PCR-restriction-enzyme analysis. Eighteen mycobacterial strains were isolated from 60 water samples: 11 from untreated water, 5 from treated water and 2 from served water. All mycobacteria isolated were identified as Mycobacterium gordonae and showed the following PRA genotypes: III (27.8%), IV (38.9%) and V (33.3%).

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.

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.

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.

Characterization of the genetic diversity of Mycobacterium tuberculosis in São Paulo city, Brazil

Background: Tuberculosis is a major health problem in São Paulo, Brazil, which is the most populous and one of the most cosmopolitan cities in South America. To characterize the genetic diversity of Mycobacterium tuberculosis in the population of this city, the genotyping techniques of spoligotyping and MIRU were applied to 93 isolates collected in two consecutive years from 93 different tuberculosis patients residing in São Paulo city and attending the Clemente Ferreira Institute (the reference clinic for the treatment of tuberculosis). Findings. Spoligotyping generated 53 different spoligotype patterns. Fifty-one isolates (54.8%) were grouped into 13 spoligotyping clusters. Seventy- two strains (77.4%) showed spoligotypes described in the international databases (SpolDB4, SITVIT), and 21 (22.6%) showed unidentified patterns. The most frequent spoligotype families were Latin American Mediterranean (LAM) (26 isolates), followed by the T family (24 isolates) and Haarlem (H) (11 isolates), which together accounted for 65.4% of all the isolates. These three families represent the major genotypes found in Africa, Central America, South America and Europe. Six Spoligo-International- types (designated SITs by the database) comprised 51.8% (37/72) of all the identified spoligotypes (SIT53, SIT50, SIT42, SIT60, SIT17 and SIT1). Other SITs found in this study indicated the great genetic diversity of M. tuberculosis, reflecting the remarkable ethnic diversity of São Paulo city inhabitants. The MIRU technique was more discriminatory and did not identify any genetic clusters with 100% similarity among the 93 isolates. The allelic analysis showed that MIRU loci 26, 40, 23 and 10 were the most discriminatory. When MIRU and spoligotyping techniques were combined, all isolates grouped in the 13 spoligotyping clusters were separated. Conclusions: Our data indicated the genomic stability of over 50% of spoligotypes identified in São Paulo and the great genetic diversity of M. tuberculosis isolates in the remaining SITs, reflecting the large ethnic mix of the São Paulo city inhabitants. The results also indicated that in this city, M. tuberculosis isolates acquired drug resistance independently of genotype and that resistance was more dependent on the selective pressure of treatment failure and the environmental circumstances of patients. © 2011 Leite et al; licensee BioMed Central Ltd.

Perfil Fermentativo e Valor Nutritivo de Silagem de Capim-Elefante Inoculada com Streptococcus Bovis

The experiment was developed to evaluate the effect of strains of Streptococcus bovis (HC5 and JB1) on pH, ammonia production and nutritional value in elephant-grass silages (Pennisetum purpureum Schum). The experimental design was entirely randomized, with three treatments: T1 - elephant- grass, T2 - grass-elephant inoculated with Streptococcus bovis HC5, T3 - elephant grass inoculated with Streptococcus bovis JB1, totaling five replicates per treatment. The smallest value of ammonia was observed in the silage treated with inoculante the base Streptococcus bovis JB1 (5.90% N-total). Inoculation increased the levels of lactic acid and decreased levels of acetic, propionic and butyric acids, and the silages inoculated with Streptococcus bovis JB1 and HC5 were those with the highest values of lactic acid. There were not statistical differences for the tenors of dry matter (MS) and crude protein (PB). For the values of the fibers, the grass-elephant silage inoculated with Streptococcus bovis JB1, it presented the smallest value of neutral detergent fiber (59.77 %) and Hemicellulose (26.71 %). The largest value of in vitro dry matter digestibility (DIVMS) was observed in the silage with Streptococcus bovis JB1, showing superiority of four percentile points (64.23 %) in relation to the treatment without bacterial inoculante (60.35 %). The use Streptococcus bovis JB1 was efficient in the process fermentativo besides promoting would get better in the quality of the fiber and DIVMS in the studied silage.

Babesia bovis infection in cattle in the southwestern Brazilian Amazon

The present study provides the first epidemiological data on infection with Babesia bovis in cattle raised in the southwestern Brazilian Amazon. Blood clot samples were filtered through nylon cloth before being submitted to DNA extraction. PCR and nested-PCR were applied to assess the frequency of infection with B. bovis in calves with ages from 4 to 12 months bred in 4 microregions each in the states of Rondônia and Acre. After the DNA was extracted from the samples, the infection in cattle was investigated by amplification of the rap1 gene from B. bovis. The DNA amplification results revealed a frequency of infection with B. bovis of 95.1% (272/286) in the samples from Rondônia and 96.1% (195/203) in those from Acre. The high frequency of B. bovis infection in the animals with ages from 4 to 12 months indicates a situation of enzootic stability in the regions studied. The infection rates are comparable to those detected by immunodiagnostic techniques in other endemic regions of Brazil. © 2012 Elsevier GmbH.

Case report: Disseminated Mycobacterium tuberculosis infection in a dog

An uncommon disseminated Mycobacterium tuberculosis infection is described in a 12-year-old female dog presenting with fever, dyspnea, cough, weight loss, lymphadenopathy, melena, epistaxis, and emesis. The dog had a history of close contact with its owner, who died of pulmonary tuberculosis. Radiographic examination revealed diffuse radio-opaque images in both lung lobes, diffuse visible masses in abdominal organs, and hilar and mesenteric lymphadenopathy. Bronchial washing samples and feces were negative for acid-fast organisms. Polymerase chain reaction (PCR)-based species identification of bronchial washing samples, feces, and urine revealed M. tuberculosis using PCR-restriction enzyme pattern analysis-PRA. Because of public health concerns, which were worsened by the physical condition of the dog, euthanasia of the animal was recommended. Rough and tough colonies suggestive of M. tuberculosis were observed after microbiological culture of lung, liver, spleen, heart, and lymph node fragments in Löwenstein-Jensen and Stonebrink media. The PRA analysis enabled diagnosis of M. tuberculosis strains isolated from organs. Copyright © 2013 by The American Society of Tropical Medicine and Hygiene.

New ruthenium(II)/phosphines/diimines complexes: Promising antitumor (human breast cancer) and Mycobacterium tuberculosis fighting agents

The synthesis and characterization of ruthenium compounds of the type [RuCl2(P)2(N-N)] [(P)2 = (PPh3) 2, dppb = 1,4-bis(diphenylphosphino)butano; dppp = 1,3-bis(diphenylphosphino)propane; N-N = 5,5′-dimethyl-2,2′dipyridyl (5,5′-mebipy) or 4,4′-dimethyl-2,2′dipyridyl (4,4′-mebipy)] are described. The complexes were characterized using elemental analysis, UV-Vis and infrared spectroscopies, cyclic voltammetry, and X-ray crystallography. In vitro evaluation of the complexes, using the MTT methodology, revealed their cytotoxic activities in a range of 5.4-15.7 μM against the MDA-MB-231 breast tumor cells and showed that, in this case, they are more active than the reference metallodrug cisplatin. The in vitro antimycobacterial activities of the complexes had their Minimum Inhibitory Concentration (MIC) for MTB cell growth measured, by the REMA method. The MICs for these complexes were found to be between 12.5 and 25.0 μg/mL. The results are comparable with the second line drug cycloserine (MIC = 12.5-50.0 μg/mL), commonly used in the treatment of TB. © 2013 Elsevier Ltd. All rights reserved.

Comparison of three molecular typing methods to assess genetic diversity for Mycobacterium tuberculosis

This study describes the comparison of three methods for genotyping of Mycobacterium tuberculosis, namely MIRU-VNTR (mycobacterial interspersed repetitive units-variable number of tandem repeats), spoligotyping and, for the first time, MLST (Multilocus Sequence Typing). In order to evaluate the discriminatory power of these methods, a total of 44 M. tuberculosis isolates obtained from sputum specimens of patients from Brazil were genotyped. Among the three methods, MLST showed the lowest discriminatory power compared to the other two techniques. MIRU-VNTR showed better discriminatory power when compared to spoligotyping, however, the combination of both methods provides the greatest level of discrimination and therefore this combination is the most useful genotyping tool to be applied to M. tuberculosis isolates. © 2013 Elsevier B.V.