Hemolytic capability and expression of a putative haem oxygenase-encoding gene by blood isolates of Candida tropicalis are influenced by iron deprivation and the presence of hemoglobin and erythrocytes.

Although hemolytic activity is known to be a putative virulence factor contributing to candidal pathogenesis, its production by Candida tropicalis, a species closely related to Candida albicans, is poor understood. The present study was undertaken to evaluate the hemolytic activity and the expression level of a putative haem oxygenase encoding gene by blood isolates of C. tropicalis following growth in iron deprivation, and in the presence of hemoglobin and erythrocytes. The lowest values of hemolytic activity were observed in cell-free culture supernatants of isolates growing in iron-restricted medium (RPMI medium and RPMI medium supplemented with iron chelator bathophenanthrolindisulphonic acid). Hemolysis was increased in the presence of either hemoglobin or erythrocytes. Reverse transcriptase PCR analysis showed that the putative haem oxygenase encoding gene (CtHMX1), potentially related with iron uptake, was up-regulated (p < 0.001) following growth in iron deprivation and in the presence of hemoglobin; CtHMX1 was repressed in the presence of human erythrocytes (p < 0.001). Our data suggest that hemoglobin had positive effect in the production of hemolytic factor and gene expression related to iron uptake in C. tropicalis.

Production of cuticle-degrading proteases by Nomuraea rileyi and its virulence against Anticarsia gemmatalis / Produção de proteases degradadoras de cutícula por Nomuraea rileyi e sua virulência contra Anticarsia gemmatalis

Nomuraea rileyi represents an important natural control agent of Anticarsia gemmatalis preventing populations from reaching economic threshold levels in soybean. During the processes of host infection, entomopathogenic fungi produce extracellular proteases, which degrade the host cuticle and are suggested to be virulence determinants. It was examined the production of subtilisin-like (Pr1) and trypsin-like (Pr2) proteases in two strains (NR458 and CG434) of N. rileyi and its possible role in the process of pathogenicity to this caterpillar. Fungal growth was performed in a mineral medium containing nitrate, and supplemented with the cuticle or exuviae from A. gemmatalis, or with the non-cuticular substrate casein. In medium containing nitrate as sole nitrogen source, no detectable Pr1-like activity occurred in the culture supernatants of the two fungal strains. However, both strains of N. rileyi produced Pr1-like protease in all medium amended with exogenous nitrogen source, and it was highly expressed in the presence of insect cuticle. Pr2-like activity was significantly inferior to Pr1-like activity and it was detected only in some of the media culture and incubation periods tested. In the NR458 culture supernatant the highest activity was observed in medium containing nitrate as nitrogen source. Correlation analysis between the percentage of A. gemmatalis mortality in bioassays and Pr1-like protease activity of strain NR458 suggests a positive correlation for these variables.

O objetivo deste estudo foi avaliar a produção de proteases dos tipos subtilisina [Pr1] e tripsina [Pr2] por duas linhagens do fungo entomopatogênico Nomuraea rileyi e a correlação entre a atividade de Pr1 e a patogenicidade contra Anticarsia gemmatalis. O crescimento do fungo foi realizado em meio mínimo contendo nitrato e suplementado com a cutícula ou exúvia de A. gemmatalis, ou com substrato não cuticular caseína. Em meio contendo nitrato, nenhuma atividade de Pr1 foi detectada nos sobrenadantes das culturas. Entretanto, as duas linhagens de N. rileyi produziram Pr1 em meio suplementado com fonte exógena de nitrogênio, e alta atividade foi verificada na presença da cutícula do inseto. A atividade de Pr2 foi inferior à atividade de Pr1. A análise de correlação entre a atividade de Pr1 da linhagem NR458 e mortalidade de A. gemmatalis sugere uma correlação positiva para essas variáveis. A avaliação da atividade de enzimas em diferentes condições pode ajudar na compreensão do processo infeccioso de N. rileyi em A. gemmatalis.

Genetic relationships among Brazilian strains of Aspergillus ochraceus based on RAPD and ITS sequences.

Ochratoxin A (OA) is a mycotoxin that has been found in coffee beans and coffee beverages. Its toxicological profile includes carcinogenicity, nephrotoxicity, and immunotoxicity. Aspergillus ochraceus is the major species responsible for OA production in Brazilian coffee beans. The genetic relationships among 25 A. ochraceus strains collected from Brazilian coffee-bean samples were determined based on RAPD and internal transcribed spacer (ITS) sequence data. The isolates were resolved into 2 distinct groups, one with 4 strains (group A) and the other with 21 strains (group B). Specific nucleotide variations characterizing group A and B were found for both ITS1 and ITS2 regions. Group B is a new group proposed here to accommodate the majority of the Brazilian isolates. Each group was found to contain both toxigenic and nontoxigenic strains, indicating that there is no association between molecular genotypes and the ability to produce OA.

Genetic variation in the cuticle-degrading protease activity of the entomopathogen Metarhizium flavoviride

Extracellular proteases have been shown to be factors of virulence in fungal entomopathogenicity. We examined the production of the cuticle-degrading extracellular proteases chymoelastase (Pr1) and trypsin (Pr2) in isolates of the fungus Metarhizium flavoviride. Fungal growth was in a mineral medium (MM) containing nitrate, and in MM supplemented with either cuticle from Rhammatocerus schistocercoides or with the non-cuticular substrate casein. The substrates used for growth influenced the expression of both analyzed proteases, the highest protease activities of nearly all isolates having been observed in the medium containing insect cuticle, with more Pr1 than Pr2 being produced. There was a natural variability in the production of cuticle-degrading proteases among isolates, although this was less evident for Pr2. Our data support the hypothesis that the production of Pr1 on insect cuticle is a useful characteristic for the analysis of intraspecific variability of M. flavoviride isolates