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.

Hydrolytic enzymes in Paracoccidioides brasiliensis--ecological aspects

Paracoccidioides brasiliensis is a thermally dimorphic fungus that causes paracoccidioidomycosis. The yeast form of this pathogen is found in the animal host whereas the mycelial form is recovered from living and non-living organic material. The sole carbon source available in these habitats is represented by polysaccharides from the plant cell wall. Hydrolytic enzymes are necessary to convert these polymers into simple sugars for fungal metabolism. We report on the presence of ortholog genes of hydrolytic enzymes identified in the P. brasiliensis transcriptome and on hydrolytic activities in supernatants of induced P. brasiliensis cultures of mycelium and yeast cells. Enzymatic assays have shown cellulase and xylanase activities, both being higher in mycelium than in the yeast form. Amylase and chitinase activities were detected only in mycelium. Data so far reinforce the idea that mycelial P. brasiliensis is a saprobe.

Transcriptome overview of Paracoccidioides brasiliensis proteases

Proteases perform a wide variety of functions inside and outside cells, regulating many biological processes. Infectious microorganisms use proteases, either secreted or attached to their cell surface to weaken and invade their hosts. Therefore, proteases are targets for drugs against a diverse set of diseases. Paracoccidioides brasiliensis is the most prevalent fungal pathogen causing systemic mycosis in Latin America. The development of paracoccidioidomycosis depends on interactions between fungal and host components and proteases have been described as important factors implicated in the mechanism of host colonization by fungi. The primary goal for this study is to present an overview of the transcriptome sequences--identified cDNAs that encode proteases. We obtained a total of 53 cDNAs encoding proteases; 15 were classified as ATP-independent, 12 as ATP-dependent, 22 as proteasome subunits, and 4 as deubiquitinating proteases. The mechanisms and biological activity of these proteases differ in substrate specificity and in catalytic mechanisms.

Virulence insights from the Paracoccidioides brasiliensis transcriptome

Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis, is a dimorphic fungus, which is found as mycelia at 22-26 degrees C and as yeasts at 37 degrees C. A remarkable feature common to several pathogenic fungi is their ability to differentiate from mycelium to yeast morphologies, or vice-versa. Although P. brasiliensis is a recognized pathogen for humans, little is known about its virulence genes. In this sense, we performed a search for putative virulence genes in the P. brasiliensis transcriptome. BLAST comparative analyses were done among P. brasilienses assembled expressed sequence tags (PbAESTs) and the sequences deposited in GenBank. As a result, the putative virulence PbAESTs were grouped into five classes, metabolism-, cell wall-, detoxification-related, secreted factors, and other determinants. Among these, we have identified orthologs of the glyoxylate cycle enzymes, a metabolic pathway involved in the virulence of bacteria and fungi. Besides the previously described alpha- and beta-glucan synthases, orthologs to chitin synthase and mannosyl transferases, also important in cell wall synthesis and stabilization, were identified. With respect to the enzymes involved in the intracellular survival of P. brasiliensis, orthologs to superoxide dismutase, thiol peroxidase and an alternative oxidase were also found. Among the secreted factors, we were able to find phospholipase and urease orthologs in P. brasiliensis transcriptome. Collectively, our results suggest that this organism may possess a vast arsenal of putative virulence genes, allowing the survival in the different host environments.

The cell wall of Paracoccidioides brasiliensis: insights from its transcriptome

The cell wall of a human pathogenic fungus is in contact with the host, serves as a barrier against host defense mechanisms and harbors most fungal antigens. In addition, cell wall biosynthesis pathways have been recognized as essential to viability and as specific drug targets. Paracoccidioides brasiliensis is a dimorphic fungus that presents mycelium morphology in the free environment and causes infection in a yeast form. The morphogenetic conversion is correlated with changes in the cell wall composition, organization and structure. Based on transcriptome analysis, the enzymes involved in the biosynthesis and remodeling of cell wall polysaccharides, as well as several cell wall-associated molecules of P. brasiliensis, were identified and addressed in further detail.

Detection of the basement membrane-degrading proteolytic activity of Paracoccidioides brasiliensis after SDS-PAGE using agarose overlays containing Abz-MKALTLQ-EDDnp

We have characterized, in the Paracoccidioides brasiliensis yeast phase, an exocellular SH-dependent serine proteinase activity against Abz-MKRLTL-EDDnp and analogous fluorescent-quenched peptides, and showed that it is also active against constituents of the basement membrane in vitro. In the present study, we separated the components of P. brasiliensis culture filtrates by electrophoresis and demonstrated that the serine-thiol exocellular proteinase has a diffuse and heterogeneous migration by SDS-PAGE, localizing in a region between 69 and 43 kDa. The hydrolytic activity was demonstrable after SDS-PAGE using buffered agarose overlays of Abz-MKALTLQ-EDDnp, following incubation at 37oC, and detection of fluorescent bands with a UV transilluminator. Hydrolysis was more intense when incubation was carried out at basic pH, and was completely inhibited with 2.5 mM PMSF and partially with sodium 7-hydroxymercuribenzoate (2.5 mM p-HMB), suggesting its serine-thiol nature. A proteolytic band with similar characteristics was observed in conventional gelatin zymograms, but could not be correlated with a silver-stained component. Detection of the serine-thiol proteinase in substrate gels after SDS-PAGE provides a useful way of monitoring purification of the basement membrane degrading enzyme

Variabilidade microbiólogica de isolados de Paracoccidioides brasiliensis obtidos de pacientes com diferentes formas clínicas / Study of microbiological variability of Paracoccidioides brasiliensis strains from patients with diferents clinical forms

Diversos trabalhos têm demonstrado variabilidade biológica em Paracoccidioides brasiliensís. As múltiplas manifestações clínicas da PCM e a depressão da resposta imune celular, especialmente do tipo 1, que é recuperada após terapêutica efetiva, são indicativos de que os processos de infecção e evolução da doença dependem também de fatores relacionados à virulência do agente. Com o objetivo de investigar possíveis mercadores que pudessem estar associados ao papel do agente na determinação da forma clínica da PCM foi proposto um estudo de algumas características microbiológicas comparando-se 30 isolados de P. brasilíensís. O primeiro grupo "A', foi constituído por 15 cultivos obtidos de pacientes que desenvolveram a forma aguda da doença. O grupo "C" representou os isolados de pacientes com PCM forma crônica. Os fungos, recebidos de várias procedências, eram mantidos em laboratório por períodos variáveis. A fim de recuperar o potencial de virulência, supostamente atenuado durante os subcultivos ín vitro, todos os fungos foram inicialmente reisolados após desenvolvimento de orquite em hamsters. O estudo foi basicamente desenvolvido com a forma leveduriforme do fungo, obtida por cultivos em PYGA, a 35º C. Os parâmetros incluíram: morfologia microscópica (morfometria da célula-mãe e número de brotamentos)- velocidade de transformação in vitro da forma miceliana para a forma leveduriforme; determinação de curvas de crescimento; expressão da glicoproteína de 43KDa (gp43); polimorfismo isoenzimático e determinação do perfil de susceptibilidade, ín vitro, dos isolados frente aos antifúngicos empregados na terapêutica da PCM. Em todos os aspectos analisados ficou evidente a expressiva variabilidade biológica entre os isolados. Foi possível padronizar o teste de susceptibilidade aos antifúngicos através de adaptações à técnica de referência proposta pelo NCCLS, (l997). Os resultados mostraram-se seguros e reprodutíveis desde que se utilizem cultivos com alto grau de viabilidade celular. Foram testadas anfotericina B, 5-fluorocitosina e os azólicos ítraconazol, cetoconazol e fluconazol. Frente à 5-fiuorocitosina, todos os isolados foram resistentes a concentraçäo iguais ou superiores a 64mg/mL da droga. Em relação aos demais antifúngicos, houve variabilidade nos títulos das ClMs não se caracterizando, porém, nenhum caso de resistência microbiana in vitro. As curvas de crescimento, determinadas através de ...(au)

Regulaciones bioquímicas en el dimorfismo y la virulencia de hongos patogenos para humanos / Biochemical regulations in the dimorphism and virulence of pathogenic fungi for humans

Se revisan diversos mecanismos bioquímicos involucrados en el control de la virulencia y el dimorfismo de hongos patógenos para humanos. Entre ellos, la participación de grupos sulfidricos y disulfuros, los receptores hormonales y las proteinas intra- y extracelulares en histoplasma capsulatum, paracoccidioides brasiliensis y Coccidioides immitis