Adhesion of Candida tropicalis to polystyrene and epithelial cell lines: Insights of correlation of the extent of adherent yeast cells among distinct surfaces.

Candida tropicalis is an emerging fungal pathogen associated with high mortality. We aimed to compare adherence capability of C. tropicalis to polystyrene and epithelial cell lines (HeLa and Vero), and determine whether adherent blastoconidia is cell-type specific. Blastoconidia adhesion to epithelial cells and polystyrene were determined by crystal violet assay. The percentage of epithelial cells with adhered blastoconidia and the number of adhered blastoconidia per cell line were determined by light microscopy. The correlation between adhesion surfaces was assessed by Pearson's correlation coefficient. The adhesiveness of C. tropicalis to polystyrene was greater than that observed for ephitelial cells. High correlation values (r2 0.9999222, p 0.007941) were found for the adhesion capability between biotic and polystyrene surface for isolates 100.10 (obtained from blood) and 335.07 (obtained from tracheal secretion). The number of adherent blastoconidia per HeLa cell was greater in comparison to that observed for Vero cells (P<0.05). Further, high correlation (r2 1, p 0.0001) was found for the adhesion ability between HeLa cells and Vero cells. The results suggest a correlation of C. tropicalis adhesion capability among different surfaces, and that the adhesion to epithelial cells is specific to the cell type.

Production of haemolytic factor by clinical isolates of Candida tropicalis.

Although haemolytic factor is known to be a putative virulence factor contributing to pathogenicity in Candida species, its production by Candida tropicalis is poorly understood. In this study, we analysed the culture conditions under which C. tropicalis can display haemolytic factor on plate assay and the secretion of haemolytic factor in liquid medium by clinical isolates obtained from different specimens. All the tested isolates exhibited an internal translucent ring, resembling beta-haemolysis, surrounding by a peripheral greenish-grey halo on sheep blood agar medium. Similar haemolytic pattern was observed on human blood enriched medium. Furthermore, incubation either under normal atmosphere or under increased CO(2) had no effect on haemolysis. Overall, no differences were observed on beta-haemolytic activities (P > 0.05) among tested isolates of C. tropicalis. In glucose-limited medium (RPMI 1640 with 0.2% glucose), none of the isolates induced haemolysis on red blood cells. Similarly to found on plate assays, there were no significant differences (P > 0.05) in the activity of secreted haemolytic factor in liquid medium among C. tropicalis isolates. However, after growth, the number of yeast cells varied among isolates revealing different efficiencies of haemolytic factor production. Haemolytic activity was neither inhibited by heat treatment (100 °C) nor by the addition of pepstatin A. The obtained results extend our knowledge about haemolytic factor production by Candida species.

Haemolytic and proteinase activities in clinical isolates of Candida parapsilosis and Candida tropicalis with reference to the isolation anatomic site.

The aim of this study was to determine in vitro haemolytic and protease activities of Candida parapsilosis and Candida tropicalis isolates, obtained from anatomically distinct sites. Analysis of haemolytic activity of C. parapsilosis and C. tropicalis isolates obtained from the same anatomic site revealed that C. tropicalis isolates from blood had statistically higher activity (P < 0.05) than C. parapsilosis. On comparison of haemolytic activities of Candida isolates obtained from different anatomic sites, C. parapsilosis isolates from tracheal secretion were found to have higher activity than blood isolates. Protease activity was detected in the majority of the isolates analysed. Analysis of proteinase activity of C. parapsilosis and C. tropicalis isolates obtained from the same anatomic site revealed that C. parapsilosis isolates from tracheal secretion had statistically higher activity than C. tropicalis isolates. On comparison of proteinase activities of Candida isolates obtained from different anatomic sites, C. parapsilosis isolates from tracheal secretion were found to have higher activity than blood and superficial lesions isolates. Furthermore, C. tropicalis isolates from superficial lesions had higher activity than tracheal secretion isolates. Our results show the potential of C. parapsilosis and C. tropicalis isolates, obtained from distinct anatomic sites, to produce haemolytic factor and proteinases. Anatomic sites of isolation seem to be correlated with these activities, particularly for C. parapsilosis isolates.

Cuticle-degrading proteases produced by the entomopathogenic fungus Beauveria bassiana in the presence ofcoffee berry borer cuticle / Proteases degradadoras de cutícula produzidas pelo fungo entomopatogênico Beauveria bassiana em presença de cutícula de broca do café

A Brazilian isolate of Beauveria bassiana (CG425) that shows high virulence against the coffee berry borer (CBB) was examined for the production of subtilisin-like (Pr1) and trypsin-like (Pr2) cuticle-degrading proteases. Fungal growth was either in nitrate-medium or in CBB cuticle-containing medium under both buffered and unbuffered conditions. In unbuffered medium supplemented with cuticle, the pH of cultures dropped and Pr1 and Pr2 activities were detected in high amounts only at a pH of 5.5 or higher. In buffered cultures, Pr1 and Pr2 activities were higher in medium supplemented with cuticle compared to activities with nitrate-medium. The Pr1 and Pr2 activities detected were mostly in the culture supernatant. These data suggest that Pr1 and Pr2 proteases produced by strain CG425 are induced by components of CBB cuticle, and that the culture pH influences the expression of these proteases, indicating the occurrence of an efficient mechanism of protein secretion in this fungus. The results obtained in this study extend the knowledge about protease production in B. bassiana CG425, opening new avenues for studying the role of secreted proteases in virulence against the coffee berry borer during the infection process.

O isolado brasileiro de Beauveria bassiana (CG425) que apresenta alta virulência contra a broca do café (CBB) foi analisado quanto à produção de proteases degradadoras de cutícula, tipo-subtilisina (Pr1) e tipo-tripsina (Pr2). O crescimento fúngico foi realizado em meio contendo nitrato e em meio contendo cutícula da broca em condições de pH tamponado e não tamponado. Em meio não tamponado, suplementado com cutícula, o pH da cultura caiu e as atividades de Pr1 e Pr2 foram detectadas somente em valores de pH igual ou superior a 5,5. Em culturas tamponadas, as atividades Pr1 e Pr2 foram superiores em meio suplementado com cutícula, comparativamente as atividades em meio contendo nitrato. As atividades Pr1 e Pr2 ocorreram predominantemente no sobrenadante de cultivo. Os dados obtidos sugerem que Pr1 e Pr2 produzidas pelo isolado CG425 são induzidas por componentes da cutícula da broca do café (CBB), e que o pH da cultura influencia a expressão destas proteases, indicando a ocorrência de um mecanismo eficiente de secreção por este fungo. Os resultados obtidos neste estudo aumentam o conhecimento a respeito da produção de proteases por B. bassiana CG425, abrindo novos caminhos para o estudo do papel de proteases na virulência contra a broca do café durante o processo de infecção.

Cuticle-degrading proteases produced by the entomopathogenic fungus Beauveria bassiana in the presence of coffee berry borer cuticle.

A Brazilian isolate of Beauveria bassiana (CG425) that shows high virulence against the coffee berry borer (CBB) was examined for the production of subtilisin-like (Pr1) and trypsin-like (Pr2) cuticle-degrading proteases. Fungal growth was either in nitrate-medium or in CBB cuticle-containing medium under both buffered and unbuffered conditions. In unbuffered medium supplemented with cuticle, the pH of cultures dropped and Pr1 and Pr2 activities were detected in high amounts only at a pH of 5.5 or higher. In buffered cultures, Pr1 and Pr2 activities were higher in medium supplemented with cuticle compared to activities with nitrate-medium. The Pr1 and Pr2 activities detected were mostly in the culture supernatant. These data suggest that Pr1 and Pr2 proteases produced by strain CG425 are induced by components of CBB cuticle, and that the culture pH influences the expression of these proteases, indicating the occurrence of an efficient mechanism of protein secretion in this fungus. The results obtained in this study extend the knowledge about protease production in B. bassiana CG425, opening new avenues for studying the role of secreted proteases in virulence against the coffee berry borer during the infection process.

Development of a simple and rapid Agrobacterium tumefaciens-mediated transformation system for the entomopathogenic fungus Metarhizium anisopliae var. acridum.

AIMS: To examine the ability of Agrobacterium to attach to Metarhizium anisopliae var. acridum strain CG423 under co-cultivation and to develop an Agrobacterium-mediated method of gene delivery into strain CG423, a promising agent for biological control of grasshoppers. METHODS AND RESULTS: The co-cultivation of Agrobacterium tumefaciens and M. anisopliae var. acridum was analysed under scanning electron microscopy. We observed that Agrobacterium attached to and formed aggregates around Metarhizium conidia and germ tubes. We also observed the occurrence of fibril-like structures connecting neighbouring bacterial-fungal cells. The Agrobacterium-mediated transformation was applied using two binary vectors carrying a benomyl resistance gene as a selection marker. The efficiency of transformation was up to 53 transformants per 10(5) target conidia. High mitotic stability of the transformants (89-97%) was demonstrated after five successive transfers on non-selective media. Molecular analysis revealed the occurrence of high frequency of gene conversion. CONCLUSIONS: In our study, we report that A. tumefaciens strain AGL-1 attaches to and genetically transforms the entomopathogenic fungus Metarhizium anisopliae var. acridum. SIGNIFICANCE AND IMPACT OF THE STUDY: We report for the first time, the attachment of Agrobacterium to fungal cells opening new avenues for the study of this essential step of the T-DNA transfer process. Considering the efficiency of the transformation protocol herein described, this is a useful tool for gene disruption in M. anisopliae var. acridum.

Transformation of the entomopathogenic fungus Paecilomyces fumosoroseus with Agrobacterium tumefaciens.

AIMS: To test the suitability of the Agrobacterium tumefaciens-mediated transformation (AMT) method with Paecilomyces fumosoroseus, a fungal pathogen that causes diseases in a wide range of insects including whiteflies. METHODS AND RESULTS: Conidia of P. fumosoroseus were successfully transformed to hygromycin B resistance using the hph gene of Escherichia coli as the selectable marker. Transformation frequencies were 58.3 +/- 18.5, 98.3 +/- 24.8 and 169.7 +/- 35.5 (+/-SEM) transformants per 10(5), 10(6) and 10(7) target conidia respectively. After confirmation by PCR, transformants were subjected to Southern analysis, and the results revealed that 45% (four of nine) of the transformants contained single-copy integration of the T-DNA. CONCLUSIONS: In our AMT system, we efficiently transformed conidia of P. fumosoroseus. The employment of this method circumvents time-consuming protoplast preparation and allows the isolation of transformants containing single-copy integration of the T-DNA. SIGNIFICANCE AND IMPACT OF THE STUDY: Considering the efficiency of Ag. tumefaciens-mediated transformation, this method represents a useful tool for insertional mutagenesis to characterize genes that are important for the pathogenicity of P. fumosoroseus.

Cuticle-degrading proteases from the entomopathogen Metarhizium flavoviride and their distribution in secreted and intracellular fractions.

AIMS: The aim of this study was to analyse a native isolate of Metarhizium flavoviride (CG423) which is being developed as a myco-insecticide against grasshoppers in Brazil for the production of the cuticle-degrading subtilisin-like (Pr1), and trypsin-like (Pr2) proteases. METHODS AND RESULTS: The results show that Pr1 activity occurred only in medium supplemented with grasshopper cuticle (Schistocerca pallens). In contrast, Pr2 was detected in higher amounts on defined growth substrate than on cuticle-supplemented medium. Both activities were detected after 48 h of growth, suggesting that in S. pallens cuticle-containing medium these protease types are not co-ordinately expressed. Low levels of enzyme activity were detected when pre-grown mycelium was used to investigate the induction of Pr1 proteases. CONCLUSIONS: The analysis of Pr1 and Pr2 distribution in both secreted and intracellular fractions revealed high percentage of extracellular activity, which may suggest the occurrence of an efficient mechanism of protein secretion by this fungus, probably related to substrate degradation which provides nutrients for fungal growth.

Double-stranded RNA in the entomopathogenic fungus metarhizium flavoviride.

Bands of dsRNA were detected in five out of seven isolates of the entomopathogenic fungus Metarhizium flavoviride. The identity of these bands was proven by RNase and S1-nuclease treatments. The transference of dsRNA between isolates (from CG291 to CG442) was successfully carried out through forced heterokaryons. Isogenic strains, with or without dsRNA, were submitted to virulence tests against the grasshopper Rhammatocerus schistocercoides. In contrast to what has been found in some phytopathogenic fungi, these dsRNA fragments did not cause hypovirulence to M. flavoviride.

Genetic analysis of Aspergillus nidulans unstable transformants obtained by the biolistic process.

Mitotically unstable Aspergillus nidulans argB+ transformants obtained by the biolistic process were studied in the present work. Hybridization signals from undigested DNA and pulsed-field chromosomal bands of the transformants suggested the introduced plasmid occurred as free concatenated molecules. Fifteen vigorous growth sectors released from the transformants were analysed in order to understand the mechanisms involved in their formation. All sectors showed the integration of exogenous genes into the fungal genome by homologous or heterologous recombinant events.

Compressäo venosa ilíaco-femoral por cisto sinovial simulando trombose venosa profunda / Iliofemoral vein compression by sinovial cyst simulating deep vein thrombosis

Os autores relatam um caso raro de compressäo das veias femoral e ilíaca externa esquerda por um cisto sinovial do quadril, simulando trombose venosa profunda. Houve regressäo completa do quadro clínico após ressecçäo cirúrgica do cisto

Intraspecific hybridisation of Trichoderma pseudokoningii by anastomosis and by protoplast fusion.

Double auxotrophic and morphological mutants of Trichoderma pseudokoningii Rifai were fused by anastomosis and by protoplast fusion. The recovery of recombinants from heterokaryons on different selective media and from heterokaryotic colonies indicated the occurrence of parasexual events. Prototrophic colonies growing on minimal medium produced binucleate spores, green in colour, revealing a non-autonomous system for conidial pigmentation. Recombinants were obtained from these dikaryotic colonies suggesting the occurrence of a highly unstable diploid phase.

Virulence factors of Escherichia coli in urinary isolates.

1. Escherichia coli strains isolated from 100 urine samples taken from patients with urinary tract infections (UTI) and from 20 normal fecal (NF) samples were examined for serum resistance, mannose-resistant hemagglutination of human erythrocytes (MRHA) and for production of aerobactin, hemolysin and colicin. 2. Among the UTI E. coli strains, 79% produced aerobactin, 69% showed serum resistance, 44% produced MRHA, 32% were beta-hemolytic and 22% were colicinogenic. A greater proportion of UTI E. coli strains produced aerobactin, colicin V, beta-hemolysis and MRHA when compared to NF strains. Production of MR hemagglutinins was significantly correlated with that of aerobactin and hemolysin. 3. These results suggest that the presence of aerobactin may be a significant etiological factor in UTI, and that the production of MR adhesins and of hemolysin also might contribute to the virulence of these strains.

Virulence factors of Eschericha coli in urinary isolates

Escherichia coli strains isolated from 100 urine samples taken from patients with urinary tract infections (UTI) and from 20 normal fecal (NF) samples were examined for serum resistance, mannose-resistant hemagglutination of human erythrocytes (MRHA) and for production of aerobactin, hemolysis and colicin. Among the UTI E. coli strains, 79% produced aerobactin, 69% showed serum resistance, 44% produced MRHA, 32% were beta-hemolytic and 22% were colicinogenic. A greater proportion of UTI E. coli strains produced aerobactin, colicin V, beta-hemolysis and MRHA when compared to NF strains. Production of MR hemagglutins was significant correlated with that of aerobactin and hemolysin. These results suggest that the presence of aerobactin may be a significant etiological factor in UTI, and that the production of MR adhesins and of hemolysin also might contribute to the virulence of these strains