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.

The Mode of Action of Recombinant Mycobacterium tuberculosis Shikimate Kinase: Kinetics and Thermodynamics Analyses

Tuberculosis remains as one of the main cause of mortality worldwide due to a single infectious agent, Mycobacterium tuberculosis. The aroK-encoded M. tuberculosis Shikimate Kinase (MtSK), shown to be essential for survival of bacilli, catalyzes the phosphoryl transfer from ATP to the carbon-3 hydroxyl group of shikimate (SKH), yielding shikimate-3-phosphate and ADP. Here we present purification to homogeneity, and oligomeric state determination of recombinant MtSK. Biochemical and biophysical data suggest that the chemical reaction catalyzed by monomeric MtSK follows a rapid-equilibrium random order of substrate binding, and ordered product release. Isothermal titration calorimetry (ITC) for binding of ligands to MtSK provided thermodynamic signatures of non-covalent interactions to each process. A comparison of steady-state kinetics parameters and equilibrium dissociation constant value determined by ITC showed that ATP binding does not increase the affinity of MtSK for SKH. We suggest that MtSK would more appropriately be described as an aroL-encoded type II shikimate kinase. Our manuscript also gives thermodynamic description of SKH binding to MtSK and data for the number of protons exchanged during this bimolecular interaction. The negative value for the change in constant pressure heat capacity (ΔCp) and molecular homology model building suggest a pronounced contribution of desolvation of non-polar groups upon binary complex formation. Thermodynamic parameters were deconvoluted into hydrophobic and vibrational contributions upon MtSK:SKH binary complex formation. Data for the number of protons exchanged during this bimolecular interaction are interpreted in light of a structural model to try to propose the likely amino acid side chains that are the proton donors to bulk solvent following MtSK:SKH complex formation. © 2013 Rosado et al.

In Vitro and In Vivo Activities of Ruthenium(II) Phosphine/Diimine/Picolinate Complexes (SCAR) against Mycobacterium tuberculosis

Rifampicin, discovered more than 50 years ago, represents the last novel class of antibiotics introduced for the first-line treatment of tuberculosis. Drugs in this class form part of a 6-month regimen that is ineffective against MDR and XDR TB, and incompatible with many antiretroviral drugs. Investments in R&D strategies have increased substantially in the last decades. However, the number of new drugs approved by drug regulatory agencies worldwide does not increase correspondingly. Ruthenium complexes (SCAR) have been tested in our laboratory and showed promising activity against Mycobacterium tuberculosis. These complexes showed up to 150 times higher activity against MTB than its organic molecule without the metal (free ligand), with low cytotoxicity and high selectivity. In this study, promising results inspired us to seek a better understanding of the biological activity of these complexes. The in vitro biological results obtained with the SCAR compounds were extremely promising, comparable to or better than those for first-line drugs and drugs in development. Moreover, SCAR 1 and 4, which presented low acute toxicity, were assessed by Ames test, and results demonstrated absence of mutagenicity. © 2013 Pavan et al.

New isoniazid complexes, promising agents against mycobacterium tuberculosis

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

A photoelectrocatalytic process that disinfects water contaminated with Mycobacterium kansasii and Mycobacterium avium

Nontuberculous mycobacteria are resistant to conventional water treatment; indeed, they have been recovered from a wide variety of environmental sources. Here, we applied the photoelectrocatalytic technique using a Ti/TiO2-Ag photoanode to inactivate mycobacteria. For a mycobacteria population of 5 × 108 CFU mL-1, we achieved 99.9 and 99.8% inactivation of Mycobacterium kansasii and Mycobacterium avium with rate constant of 6.2 × 10-3 and 4.2 × 10-3 min-1, respectively, after 240 min. We compared the proposed method with the photolytic and photocatalytic methods. Using a mycobacteria population of 7.5 × 104 CFU mL-1, the proposed Ti/TiO2-Ag photoanode elicited total mycobacteria inactivation within 3 min of treatment; the presence of Ag nanoparticles in the electrode provided 1.5 larger degradation rate constant as compared with the Ti/TiO2 anode (1.75 × 10-2 for M. kansassi and 1.98 × 10-2 for M. avium). We monitored the degradation of the metabolites released during cellular lysis by TOC removal, sugar release, chromatography, and mass spectrometry measurements; photoelectrocatalysis and Ti/TiO2-Ag photoanodes furnished the best results. © 2013 Elsevier Ltd. All rights reserved.

Prevalência sorológica e molecular de Babesia bovis e Babesia bigemina em búfalos (Bubalus bubalis) na Ilha de Marajó, Pará

The aim of the study was to estimate the prevalence of Babesia bovis and Babesia bigemina in water buffaloes of the Marajó Island, State of Pará, Brazil. We used an indirectenzyme- linked immunosorbent assay (iELISA), with total antigen containing proteins outer surface, and polymerase chain reaction (qPCR), involving the use of SYBR Green based on amplification of a small fragment of the cytochrome b gene. The prevalence of positive animals in iELISA to B. bovis B. bigemina and mixed infection was 24.87% (199/800), 20.75% (166/800) and 18.75% (150/800), respectively. Using the PCR, the presence of B. bovis wasdetected in 15% (18/199) and B. bigemina in 16% (19/199) of animals, and of these, 58% (11/19) presented co-infected by the two agents. The results show a low prevalence of antibodies anti-B. bovis and anti-B. bigemina in water buffaloes from Marajó Island. However, it was observed that the agents of bovine babesiosis circulate in buffaloes, and these may act as reservoirs.

Molecular and serological prevalence of Babesia bovis and Babesia bigemina in water buffaloes in the north region of Brazil

Bovine babesiosis is a tick-borne disease caused mainly by Babesia bovis and Babesia bigemina, which are associated to considerable economic losses in cattle herds worldwide. Approximately 60% of buffalo herds in South America are located in Northern Brazil. Little is known about the impact of babesiosis on buffalo herds in Brazil. The present work aimed to verify the occurrence of B. bovis and B. bigemina in 542 water buffaloes in the state of Pará, Northern Brazil, using molecular and serological techniques. The percentage of seropositive animals for B. bovis and B. bigemina was 41.2% and 19.0%, respectively, by ELISA. B. bovis and B. bigemina DNA were detected in 15 and 16% of sampled buffaloes, respectively. A high correlation (Kappa index of 0.9) between serological and molecular tests suggests that the combination of the utilized techniques in the present study is suitable for babesiosis diagnosis in an endemic unstable area. Significantly difference of positivity for serological and molecular assays was verified to localities and reproductive status of sampled animals, but not between buffalo breeds. The immune status of sampled buffaloes associated to the circulation of babesiosis agents in sampled population suggests that the studied area is at risk to clinical babesiosis outbreaks. Furthermore, this study demonstrated that this region can be classified as endemically unstable. © 2013 Elsevier B.V.

Modelagem molecular da Chiquimato quinase de Mycobacterium leprae

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)