Distinct chitinases are expressed during various growth phases of the human pathogen Paracoccidioides brasiliensis

The aim of this work was the partial purification and subsequent evaluation of chitinase expression during the various growth phases of Paracoccidioides brasiliensis. Initially, PbCTS1r was expressed as a recombinant protein and displayed enzymatic activity against 4-MU-[N-acetylglucosamine (GlcNAc)]3 and 4-MU-(GlcNAc)2. Two proteins, 45 kDa and 39 kDa in size, were partially purified from P. brasiliensis yeast crude extract using cation-exchange chromatography coupled with HPLC and were characterised as PbCTS1 and PbCTS2, respectively. Anti-PbCTS1r antibody recognised two proteins in the crude extracts of yeast and the transitional stage between mycelial and yeast phases. In crude extracts of mycelium, only the 45 kDa protein was detected. However, quantitative real-time polymerase chain reaction led to the detection of small quantities of Pbcts2 transcript in the mycelial phase. In the yeast cell wall extract, only the 39 kDa protein was detected. Moreover, both proteins were secreted by the yeast parasitic phase, suggesting that these proteins participate in the modulation of the fungal environment. Phylogenetic analysis of the predicted PbCTS1 and PbCTS2 proteins indicated that they code for distinct chitinases in P. brasiliensis. During evolution, P. brasiliensis could have acquired the paralogues Pbcts1 and Pbcts2 for growth and survival in diverse environments in both saprophytic and parasitic phases.

Comparative genome analysis of proteases, oligopeptide uptake and secretion systems in Mycoplasma spp

Mycoplasmas are very fastidious in their nutritional requirements for in vitro growth and have limited biosynthetic capacity, a reflection of their reduced genomes. As a result, these bacteria depend upon external metabolites for nutrition and growth and have developed dependence on their hosts for survival and maintenance. Protein degradation and peptide importation play an important role in Mycoplasma spp. nutrition, and proteases can play a role in host adaptation and pathogenicity. Here, we present a general survey on the genes involved in protein degradation, secretion and importation, comparing all available Mollicute genomes.

Aspectos fenotípicos, genotípicos e de diagnóstico da bactéria A. pleuropneumoniae / Phenotypic, genotypic and diagnostic aspects of the bacterium A. pleuropneumoniae

A pleuropneumonia suína (PPS) provoca prejuízos significativos na suinocultura no mundo. O agente etiológico é a bactéria Actinobacillus pleuropneumoniae (App), que apresenta 15 sorotipos descritos, os quais variam consideravelmente em relação a sua patogenicidade. Nesse sentido, a precisa caracterização patotípica desta bactéria é de grande importância para a adoção de medidas de controle e profilaxia. O diagnóstico e a sorotipificação deste patógeno são realizados pelas técnicas microbiológicas convencionais. Entretanto, problemas nestes esquemas podem ser observados, especialmente em isolados de rebanhos sem histórico de PPS. No Brasil, diversos esforços vêm sendo aplicados no sentido de desenvolver técnicas moleculares que auxiliem no diagnóstico da infecção crônica ocasionada por este agente, principalmente em rebanhos presumidamente sadios e com infecção subclínica. Nesta revisão, são discutidos os resultados obtidos na caracterização de isolados de A. pleuropneumoniae e espécies relacionadas provenientes tanto de suínos com PPS, como de animais presumidamente isentos da infecção. Apresentamos, ainda, perspectivas para o desenvolvimento de metodologias que possibilitem o diagnóstico precoce e a melhor compreensão dos mecanismos de virulência deste patógeno.

Isolation of streptomycetes strain inhibitors of toxigenic fungi: partial characterization of their chitinolytic system

En la búsqueda de biocontroladores potenciales de hongos toxicogénicos, se aislaron desde el suelo 27 cepas de streptomycetes spp. y se desarrollaron pruebas de confrontación contra cepas toxicogénicas de: aspergillus parasiticus (productor de aflatoxinas t-2 y ht-2) y fusarium graminearum (productor de deoxinivalenol y nivalenol). El desarrollo de al menos uno de los hongos toxicogénicos fue inhibido por el 63 porciento de los streptomyces aislados, 41 porciento de las cepas de streptomyces inhibieron el crecimiento de al menos dos de los hongos probados, y el 36 porciento de los streptomyces fue efectivo contra los tres hongos. Además, fue analizada la capacidad de los aislamiento de degradar quitina coloidal y posteriormente fueron caracterizados los complejos quitinolíticos de dos de las cepas de streptomyces. El 66 porciento de los streptomyces spp. degradaron quitina coloidal en las pruebas en placa. La secreción de quitinasas de dos aislamientos fue ensayada empleando métodos colorimétricos y geles de actividad, y los productos de degradación a partir de diferentes sustratos fueron analizados por cromatografía en capa delgada. Los resultados indicaron que uno de los streptomyces (c112), posee actividad endoquitinasa pero no n-acetilglucosaminidasa y que en el complejo de enzimas quitinolíticas de la cepa (c103) una actividad de n-acetil-glucosaminidasa

Superoxide dismutases in the entomopathogenic fungus Metarhizium anisopliae

Control of gene expression is a key subject in Molecular Biology. Superoxide dismutases are essential enzymes to protect living organisms against toxicity of radicals generated by the metabolism and represent an ideal system to study gene regulation. Filamentous fungi are extensively used as model eukaryotic systems and some representatives are important microorganisms in the biological control of insects in agriculture. Metarhizium anisopliae is employed at a commercial scale to control insects in sugar-cane plantations and pastures in Brazil and is currently the best studied entomopathogenic fungus. It possesses three SOD activities, CuZnSOD, MnSOD and Fe SOD. The iron enzyme is found in fungi for the first time. A gene coding for SOD was cloned by PCR amplification, partially sequenced and is under characterization. Transformation systems are developed but rendering poor efficiencies. Homologous genes have been isolated and should increase transformation yields.