Functional characterization by genetic complementation of aroB-Encoded dehydroquinate synthase from Mycobactetium tuberculosis H37Rv and its heterologous expression and purification

The recent recrudescence of Mycobacterium tuberculosis infection and the emergence of multidrug-resistant strains have created an urgent need for new therapeutics against tuberculosis. The enzymes of the shikimate pathway are attractive drug targets because this route is absent in mammals and, in M. tuberculosis, it is essential for pathogen viability. This pathway leads to the biosynthesis of aromatic compounds, including aromatic amino acids, and it is found in plants, fungi, bacteria, and apicomplexan parasites. The aroB-encoded enzyme dehydroquinate synthase is the second enzyme of this pathway, and it catalyzes the cyclization of 3-deoxy-D-arabino-heptulosonate-7-phosphate in 3-dehydroquinate. Here we describe the PCR amplification and cloning of the aroB gene and the overexpression and purification of its product, dehydroquinate synthase, to homogeneity. In order to probe where the recombinant dehydroquinate synthase was active, genetic complementation studies were performed. The Escherichia coli AB2847 mutant was used to demonstrate that the plasmid construction was able to repair the mutants, allowing them to grow in minimal medium devoid of aromatic compound supplementation. In addition, homogeneous recombinant M. tuberculosis dehydroquinate synthase was active in the absence of other enzymes, showing that it is homomeric. These results will support the structural studies with M. tuberculosis dehydroquinate synthase that are essential for the rational design of antimycobacterial agents.

Parasitology of juvenile mullet (Mugil liza) and effect of formaldehyde on parasites and host

Few studies have been performed with parasites of marine and estuarine fish in southern Brazil. In the present study, unpublished results show the ways of parasitism of juvenile mullet by parasites. The toxicity of formaldehyde and the effectiveness of this chemotherapy in controlling parasites in reared juvenile mullet Mugil liza were also studied. Juvenile mullets (1 +/- 0.26 g; 4.1 +/- 0.4 cm) were exposed to different concentrations of 37% formaldehyde: control group and five formaldehyde concentrations which were tested: T1 (13.5), T2 (21.6), T3 (40.5), T4 (81) and T5 (135) mg L-1 with 8 fish per repetition in triplicate. To verify the drug effectiveness in parasitic control, juvenile mullets were exposed to 1 h prophylactic bath of 37% formaldehyde with a control group and five formaldehyde concentrations: T1 (67.5), T2 (135), T3 (270), T4 (405) and T5 (540) mg L-1, 8 fish per repetition in triplicate. Ligophorus cf. uruguayensis (Monogenoidea: Ancyrocephalidae) and Solostamenides cf. platyorchis (Monogenoidea: Microcotylidae) were identified in the gills. Digenea and Nematoda were observed in the intestines. This is the first occurrence of S. cf. platyorchis in Brazil. During the toxicity test, the LC50-96 h was estimated at 20.77 mg L-1 of formaldehyde. During the 1 h formaldehyde prophylactic bath, all parasites were eliminated in formaldehyde concentrations between 135 and 540 mg L-1. High survival rate was observed in all treatments. Values of prevalence and intensity of infestation observed in this study showed the potential damage caused by Monogenoidea to mullet. Formaldehyde baths with 135 mg L-1 are recommended to control Monogenoidea in mullet and the safe limits for formaldehyde use were presented. Besides, the endoparasites were tolerant to formaldehyde exposure. (C) 2012 Elsevier B.V. All rights reserved.

Protozoan parasites of Xiphophorus spp. (Poeciliidae) and their relation with water characteristics

Investigou-se a relação entre as características da água e a infestação de protozoários parasitos, Icthyophthirius multifiliis e Trichodina sp., em peixe espada, Xiphophorus helleri e em plati, Xiphophorus maculatus, coletados em uma piscicultura de peixes ornamentais no Estado de São Paulo, Brasil. Os peixes foram coletados mensalmente, durante um ano, dos viveiros e das caixas de estocagem. A prevalência da infestação nos peixes das caixas e dos viveiros foram, respectivamente, 34,2% e 22,5% para I. multifiliis e 13% e 54% para Trichodina sp. A elevada condutividade elétrica e o pH da água reduziram a infestação por I. multifiliis. A baixa concentração de oxigênio resultou em aumento na infestação por Trichodina sp. O uso do sal, para aumentar a condutividade elétrica da água, consistiu em um método de controle de I. multifiliis. A redução do oxigênio dissolvido e a adição de fertilizante orgânico favoreceram a reprodução de Trichodina sp.