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.

Bacillus Thuringiensis mutant increase activity against Apodoptera Frugiperda larvae

Bacillus thuringiensis is a Gram-positive bacterium which main characteristic is the production of Cry proteins, that is toxic to some insects. These proteins, when ingested by susceptible insects, become active causing their death. In nature, it is possible to found B. thuringiensis strains which produce these proteins, but they differ in productivity (some of these isolates are more productive then others), and as to the toxicity levels of the produced proteins. Two B. thuringiensis strains that were highly effective against Spodoptera frugiperda larvae were chosen to verifying genetic mutation implication on Cry proteins productivity. One strain with a prolific spores production, while the other one only produced small amounts of spores. A genomic mutant library of these two isolates was, separately, constructed by genome Tn-5 transposon random insertion. Data analysis showed that mutation had a direct effect on the spores production, inducing an increase as well as a decrease in the production, according to the different strain observed. These results indicate, for the first time, that it is possible to use the described technique with B. thuringiensis, as well as the possibility to genetically breeding this bacteria. Another possibility introduced here is the possibility to do functional genetic studies mediated by mutagenesis in this bacterium.

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.

Expression of a new Bacillus thuringiensis Cry1Ia gene in Escherichia coli with strong activity against cotton pests

The control of cotton pests may be accomplished using Bacillus thuringiensis Cry proteins. For this purpose, the objective of this work was to evaluate the insecticidal activity of a new Cry1Ia protein against neonatal larvae of Spodoptera frugiperda and Anthonomus grandis. The complete cry1Ia gene, previously obtained by PCR with oligonucleotide primers based on the sequenced gene, was cloned into the vector pET28a(+), introduced into Escherichia coli BL21(DE3) and expressed by induction with IPTG. The expression of the Cry1Ia protein was confirmed with molecular weight of approximately 81 kDa. The results demonstrated the efficiency of the bacterial system for the expression of B. thuringiensis Cry1Ia protein, which was subsequently used in quantitative bioassays against S. frugiperda and A. grandis larvae, resulting in an extremely toxic protein for both species. This characteristic is exceptionally important for obtaining transgenic cotton plants resistant to these pests.

Keratinase production by new strain of Bacillus Amyloliquefaciens: Application of statistical experimental design for optimization of keratinase production

Feathers are rich in amino acids and can be employed as a dietary protein supplement for animal feed. Microbial degradation is an alternative technology for improving the nutritional value of feathers. Other potential applications of keratinase include use in the leather industry, detergents and medicine as well as the pharmaceutical for the treatment of acne, psoriasis and calluses. A new keratinolytic enzyme production bacterium was isolated from a poultry processing plant. To improve keratinase yield, statistically based experimental designs were applied to optimize three significant variables: temperature, substrate concentration (feathers) and agitation speed. Response surface methodology demonstrated an increase in keratinolytic activity at temperature, agitation speed and substrate concentration of 26.6°C, 150 rpm and 2%, respectively. Liquid chromatography revealed the release of amino acids in the Bacillus amyloliquefaciens culture broth, thereby demonstrating the potential of feather meal in the animal feed industry. © Global Science Publications.

Bacillus thuringiensis Cry1Ia10 and Vip3Aa protein interactions and their toxicity in Spodoptera spp. (Lepidoptera)

The polyphagous pests belonging to the genus Spodoptera are considered to be among the most important causes of damage and are widely distributed throughout the Americas'. Due to the extensive use of genetically modified plants containing Bacillus thuringiensis genes that code for insecticidal proteins, resistant insects may arise. To prevent the development of resistance, pyramided plants, which express multiple insecticidal proteins that act through distinct mode of actions, can be used. This study analyzed the mechanisms of action for the proteins Cry1Ia10 and Vip3Aa on neonatal Spodoptera frugiperda, Spodoptera albula, Spodoptera eridania and Spodoptera cosmioides larvae. The interactions of these toxins with receptors on the intestinal epithelial membrane were also analyzed by binding biotinylated toxins to brush border membrane vesicles (BBMVs) from the intestines of these insects. A putative receptor of approximately 65. kDa was found by ligand blotting in all of these species. In vitro competition assays using biotinylated proteins have indicated that Vip3Aa and Cry1Ia10 do not compete for the same receptor for S. frugiperda, S. albula and S. cosmioides and that Vip3Aa was more efficient than Cry1Ia10 when tested individually, by bioassays. A synergistic effect of the toxins in S. frugiperda, S. albula and S. cosmioides was observed when they were combined. However, in S. eridania, Cry1Ia10 and Vip3Aa might compete for the same receptor and through bioassays Cry1Ia10 was more efficient than Vip3Aa and showed an antagonistic effect when the proteins were combined. These results suggest that using these genes to develop pyramided plants may not prove effective in preventing the development of resistance in S. eridiana. © 2012 Elsevier Inc.

Avaliação de dois diferentes meios de cultura para o desenvolvimento de biopesticidas à base de Bacillus thuringiensis e sua aplicação em larvas de Aedes aegypti

The bacteria Bacillus thuringiensis var. israelensis (Bti) generates certain toxins with pesticide action, which can be used on the control of transmissible diseases by culicides, specially Aedes aegypti, the dengue vector. This biopesticide has been produced by submerged fermentation and, in Brazil, this production has been made by very little research centers and, more recently, by a unique small enterprise. For the implementation of a viable vectors control program through biopesticides, some studies about culture media are essential in order to join efficiency and low costs. In this way, agroindustrial wastes or by-products have been used as a nutrient source for the culture media production. In this study, corn steep liquor, a corn industrial processing by-product and tryptose, both with/without sugar addition, were compared as culture media. Cellular growth was evaluated by optical density at 620 nm, spore production by total viable cell count and LC50 by bioassays against 4th instar larvae. Among the four examined substrates, the medium composed by glucose plus corn steep liquor presented the best spore production and bioassay results.

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.

Estimaciones indirectas de la prevalencia y mortalidad por enfermedad de Chagas, malaria y tuberculosis en Bolivia: dos aplicaciones a la evaluación de programas de salud

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Study of a series of cobalt(II) sulfonamide complexes: Synthesis, spectroscopic characterization, and microbiological evaluation against M. tuberculosis. Crystal structure of [Co(sulfamethoxazole)2(H 2O)2]·H2O

Nowadays, the research for new and better antimicrobial compounds is an important field due to the increase of immunocompromised patients, the use of invasive medical procedures and extensive surgeries, among others, that can affect the incidence of infections. Another big problem associated is the occurrence of drug-resistant microbial strains that impels a ceaseless search for new antimicrobial agents. In this context, a series of heterocyclic- sulfonamide complexes with Co(II) was synthesized and characterized with the aim of obtaining new antimicrobial compounds. The structural characterization was performed using different spectroscopic methods (UV-Vis, IR, and EPR). In spite of the fact that the general stoichiometry for all the complexes was Co(sulfonamide)2·nH2O, the coordination atoms were different depending on the coordinated sulfonamide. The crystal structure of [Co(sulfamethoxazole)2(H2O)2]·H 2O was obtained by X-ray diffraction showing that Co(II) is in a slightly tetragonal distorted octahedron where sulfamethoxazole molecules act as a head-to-tail bridges between two cobalt atoms, forming polymeric chains. Besides, the activity against Mycobacterium tuberculosis, one of the responsible for tuberculosis, and the cytotoxicity on J774A.1 macrophage cells were evaluated. © 2012 Elsevier B.V. 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.

Avaliação de microrganismos antagônicos, Saccharomyces cerevisiae e Bacillus subtilis para o controle de Penicillium Digitatum

Citrus fruits are affected by diverse diseases, mainly the fungal infections, which affect productivity and quality, especially when it targets the market of fresh fruit. Among the fungal diseases that occur in postharvest, there is the green mold caused by Penicillium digitatum. The control measures are based mainly in the treatment of fruits with different combinations of fungicides in packing-house. Due to restrictions on the presence of residues of fungicides in citrus fruits and the increasing development of resistant strains of pathogens to the fungicide used, it is necessary to search for control alternatives such as biological control. Therefore, this study aimed to: (i) verify the antagonistic effect of biological control agents (BCA), being 13 isolates of Bacillus subtilis and 06 isolates of Saccharomyces cerevisiae against P. digitatum, (ii) study in vitro interactions between pathogen and BCA (iii) determine the effect of integration of antagonists with sodium bicarbonate and carnauba wax in the control of green mold. The results showed that the majority of the isolates, and all yeast isolates inhibited the mycelial growth of the phytopathogen. Only one isolate of B. subtilis (ACB-84) was able to inhibit the germination of P. digitatum (72% of inhibition), whereas ACB-K1 and ACB-CR1 (S. cerevisiae) were the most effective with inhibition from 78 and 85.7% respectively. The addition of sucrose (0.5%) favored the inhibition of conidia germination by the yeast isolates. The results from the in vivo control showed the viability of S. cerevisiae ACB-K1 and ACB-CR1 to control P. digitatum in 'Tahiti' lime fruits and orange 'Hamlin' fruits, respectively. The combination of sodium bicarbonate and biocontrol agents did not result in improvements in the curative control of the green mold. Carnauba wax (18% of TSS) favored the antagonistic activity of S. cerevisiae, and this effect depended on the variety of fruits in the study and of the yeast isolate used for the biocontrol.