Studies on the relationship between the pathogenicity of Paracoccidioides brasiliensis in mice and its growth rate under different oxygen atmospheres

We performed comparative studies of the pathogenicity of six strains of Paracoccidioides brasiliensis (Bt-9, Bt-4, Pb-9, Pb-18, Bt-7 and B-1183) for young adult male ddY mice and the growth rate of each strain under different oxygen atmospheres (aerobic, micro-aerobic and anaerobic atmospheres) at 37 °C. 10⁶ units of yeast cells were intravenously injected into each mouse. The pathogenicity of each isolate was determined by a scoring system based on organ culture and histopathological findings. The growth rates under different oxygen atmospheres were determined by a scoring system in which 300 fungal units per strain were counted. The strain Bt-9 showed the greatest pathogenicity, followed by Bt-4. Pb-9 and Pb-18 had on intermediate rank of pathogenicity. Bt-7 and B-1183 were the least pathogenic of the strains tested. Except for strain Bt-7 all strains showed an excellent growth under an aerobic atmosphere. Bt-4 and Bt-9 also showed excellent growth under a micro-aerobic atmosphere, followed by Pb-9, whereas the growth of Pb-18, Bt-7 and B-1183 was limited. There was a correlation between the growth rate under a micro-aerobic atmosphere and the pathogenicity of a strain. The growth rate of P. brasiliensis under a micro-aerobic atmosphere strongly correlated to its pathogenicity.     

Interaction between Paracoccidioides brasiliensis conidia and the coagulation system: involvement of fibrinogen

The infectious process starts with an initial contact between pathogen and host. We have previously demonstrated that Paracoccidioides brasiliensis conidia interact with plasma proteins including fibrinogen, which is considered the major component of the coagulation system. In this study, we evaluated the in vitro capacity of P. brasiliensis conidia to aggregate with plasma proteins and compounds involved in the coagulation system. We assessed the aggregation of P. brasiliensis conidia after incubation with human serum or plasma in the presence or absence of anticoagulants, extracellular matrix (ECM) proteins, metabolic and protein inhibitors, monosaccharides and other compounds. Additionally, prothrombin and partial thromboplastin times were determined after the interaction of P. brasiliensis conidia with human plasma. ECM proteins, monosaccharides and human plasma significantly induced P. brasiliensis conidial aggregation; however, anticoagulants and metabolic and protein inhibitors diminished the aggregation process. The extrinsic coagulation pathway was not affected by the interaction between P. brasiliensis conidia and plasma proteins, while the intrinsic pathway was markedly altered. These results indicate that P. brasiliensis conidia interact with proteins involved in the coagulation system. This interaction may play an important role in the initial inflammatory response, as well as fungal disease progression caused by P. brasiliensis dissemination.     

Chitinase from Paracoccidioides brasiliensis: molecular cloning, structural, phylogenetic, expression and activity analysis

A full-length cDNA encoding a chitinase (Pbcts1) was cloned by screening a cDNA library from the yeast cells of Paracoccidioides brasiliensis. The cDNA consists of 1888 bp and encodes an ORF of 1218 bp corresponding to a protein of 45 kDa with 406 amino acid residues. The deduced PbCTS1 is composed of two signature family 18 catalytic domains and seems to belong to fungal/bacterial class. Phylogenetic analysis of PbCTS1 and other chitinases suggests the existence of paralogs of several chitinases to be grouped based on specialized functions, which may reflect the multiple and diverse roles played by fungi chitinases. Glycosyl hydrolase activity assays demonstrated that P. brasiliensis is able to produce and secrete these enzymes mainly during transition from yeast to mycelium. The fungus should be able to use chitin as a carbon source. The presence of an endocytic signal in the deduced protein suggests that it could be secreted by a vesicular nonclassical export pathway. The Pbcts1 expression in mycelium, yeast, during differentiation from mycelium to yeast and in yeast cells obtained from infected mice suggests the relevance of this molecule in P. brasiliensis electing PbCTS1 as an attractive drug target.     

Mice Immunization with Radioattenuated Yeast Cells of <Emphasis Type="Italic">Paracoccidiodes brasiliensis</Emphasis>: Influence of the Number of Immunizations

Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, the most prevalent mycosis in Latin America. Up to the moment no vaccine has been reported. The aim of this study was to evaluate the influence of the number of immunizations on the protection elicited by radioattenuated yeast cells of P. brasiliensis. BALB/c mice were divided into two groups that were immunized once (Group 1) or twice (Group 2), respectively. In each group, mice were divided into sub-groups that were challenged 30, 45, or 60 days after the second immunization. Organ colony-forming units (CFUs) was determined 90 days post-challenge. A significant reduction in CFUs recovery was verified in both groups, but it was higher in Group 2. Histologic alterations were observed only in Group 1. The cytokines IL-4, IL-10, and IFN-γ were produced in mice of Group 1. In Group 2, only IFN-γ was significantly detected. IgG2a predominance relative to IgG1 was also observed in Group 2. Altogether, our results indicated that mice immunized once developed a mixed Th1/Th2 response, which was less efficient in the infection control, while a trend to a Th1 pattern was obtained with two immunizations, promoting optimal elimination of P. brasiliensis yeast cells from mice tissues.     

基因枪法获得GAI转基因橡胶树植株的研究

将含有Camv35S启动子、卡那霉素抗性基因和GUS报告基因,目的基因为GAI基因的转化载体质粒pBI121,通过基因枪轰击巴西橡胶树(Hevea brasiliensis Muell-Arg.)花药愈伤组织,50 mg L~(-1)卡那霉素的继代培养基进行抗性筛选.获得了抗性再生植株,经过PCR、Southern检测结果表明:GAI基因已经成功转入橡胶树基因组中.     

Immunogenicity and biophysical properties of a Nocardia brasiliensis protease involved in pathogenesis of mycetoma

We isolated and purified to homogeneity a caseinolytic protease from a Nocardia brasiliensis cell extract. Preparative polyacrylamide gel electrophoresis and electroelution were employed for purification. This purified protease was injected in BALB/c mice and induced IgM and IgG anti-protease antibodies. Active immunization of mice with this protease prevented mycetoma development in experimentally infected animals. Passive immunization with hyperimmune sera containing a high anti-protease antibody titer conferred partial but transient protection when collected 30 days after donor's immunization. The protective effect of hyperimmune sera was lost when obtained from donors after 60 days from their immunization despite its higher anti-protease antibody concentration. Cytokines are good candidates to explain these findings.     

Comparison between human and armadillo Paracoccidioides brasiliensis isolates by random amplified polymorphic DNA analysis

Sixty-three Paracoccidioides brasiliensis isolates obtained from three nine-banded armadillos ( Dasypus novemcinctus), one Amazonian armadillo's and 19 clinical isolates were compared by random amplified polymorphic DNA analysis with the primer OPG-19. The isolates were divided into three major clusters, I, II and III. Coincidences between human and armadillo isolates were observed in clusters I and II. Cluster III consisted only of armadillos' isolates. The results suggested that (I) humans may acquire P. brasiliensis infection by contact with armadillo's environment, (II) there may be P. brasiliensis genotypes peculiar to the animal, and (III) individual armadillos may be infected with P. brasiliensis cells with different genotypes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Antimicrobial susceptibilities of strains of Nocardia brasiliensis isolated from soil of Tucumán

The activity of antimicrobial agents against soil isolates of N. brasiliensis was studied by determination of the minimum inhibitory concentrations (MICs) and disk diffusion technique, according to the National Committee for Clinical Laboratory Standards (NCCLS). The objectives were: (a) to study the patterns of sensitivity among regional strains of N. brasiliensis isolated from natural sources (soil) of different zones of Tucumán province; (b) to correlate these results with those previously obtained with regional strains of N. brasiliensis isolated from human mycetomas, as a contribution in the evaluation of the importance of the natural reservoir area of the potentially pathogen strains. The results obtained by both methods identified strains of N. brasiliensis from soils with similar patterns of susceptibility to the strains N. brasiliensis isolated from human mycetomas. This showed strains sensitive and resistant to antibiotics. The majority of the isolates of N. brasiliensis from soils showed higher susceptibilities to antibiotics than the strains isolated from human mycetomas. Among the antibiotics studied, cefotaxime, ceftriaxone and gentamicin were the most effective against all the regional strains tested, and these results are correlated with those obtained with regional strains that cause human mycetomas. This revised version was published online in June 2006 with corrections to the Cover Date.     

Presence of carnitine acetyltransferase in peroxisomes and in mitochondria of oleic acid-grown Saccharomyces cerevisiae

Abstract Paracoccidiodes brasiliensis , the agent of paracoccidioidomycosis, when grown in a synthetic medium, expresses at the cell surface of both yeast and mycelial forms acidic glycoconjugates containing N -acetlyneuraminic acid units. Sialic acids were extracted using mild hydrolytic conditions, and were identified by thin-layer and gas chromatography, standard colorimetry, reaction with periodate-resorcinol and mass spectrometry. Their surface location was inferred from fluorescent-lectin ( Limulus polyphemus agglutinin) binding to whole cells abrogated by previous treatment with neuraminidase. Expression of sialic acids on virulent yeast forms of P. brasiliensis (3.7 × 10⁶ residues per cell) may inhibit fungal phagocytosis during early infection, when the immunological response is still being built up.