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Molecular detection and identification of Paracoccidioides brasiliensis.

ABSTRACT: Nearly 800 nucleotides from the 5' terminus of the 28S ribosomal gene of Paracoccidioides brasiliensis were sequenced, and a 14-base DNA probe specific for this species was identified. Hybridization results showed that the probe identified P. brasiliensis ribosomal DNA in a panel of ribosomal DNAs representing a total of 48 species of fungi.

SUBMITTER: Sandhu GS

PROVIDER: S-EPMC229867 | biostudies-other | 1997 Jul

REPOSITORIES: biostudies-other

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Molecular detection and identification of Paracoccidioides brasiliensis.

Sandhu G S GS Aleff R A RA Kline B C BC da Silva Lacaz C C

Journal of clinical microbiology 19970701 7

Nearly 800 nucleotides from the 5' terminus of the 28S ribosomal gene of Paracoccidioides brasiliensis were sequenced, and a 14-base DNA probe specific for this species was identified. Hybridization results showed that the probe identified P. brasiliensis ribosomal DNA in a panel of ribosomal DNAs representing a total of 48 species of fungi. ...[more]

PMID: 9196219

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Molecular identification of Paracoccidioides brasiliensis by PCR amplification of ribosomal DNA.

Project description:We have amplified and sequenced the 5.8S and 28S ribosomal DNA genes and intergenic regions of Paracoccidioides brasiliensis, strain Pb01. Using primers specifically designed for both ribosomal DNA regions, we were able to discriminate between P. brasiliensis and other human pathogenic fungi by PCR. The use of this molecular marker could be important for paracoccidiodomycosis diagnosis and ecological and molecular epidemiological studies of P. brasiliensis in Latin America.

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Ecological study of Paracoccidioides brasiliensis in soil: growth ability, conidia production and molecular detection.

Project description:BACKGROUND: Paracoccidioides brasiliensis ecology is not completely understood, although several pieces of evidence point to the soil as its most probable habitat. The present study aimed to investigate the fungal growth, conidia production and molecular pathogen detection in different soil conditions. METHODS: Soils samples of clayey, sandy and medium textures were collected from ground surface and the interior of armadillo burrows in a hyperendemic area of Paracoccidioidomycosis. P. brasiliensis was inoculated in soil with controlled humidity and in culture medium containing soil extracts. The molecular detection was carried out by Nested PCR, using panfungal and species specific primers from the ITS-5.8S rDNA region. RESULTS: The soil texture does not affect fungus development and the growth is more abundant on/in soil saturated with water. Some soil samples inhibited the development of P. brasiliensis, especially those that contain high values of Exchangeable Aluminum (H+Al) in their composition. Some isolates produced a large number of conidia, mainly in soil-extract agar medium. The molecular detection was positive only in samples collected from armadillo burrows, both in sandy and clayey soil. CONCLUSION: P. brasiliensis may grow and produce the infectious conidia in sandy and clayey soil, containing high water content, mainly in wild animal burrows, but without high values of H+Al.

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Molecular characterization of siderophore biosynthesis in Paracoccidioides brasiliensis.

Project description:Iron is an essential nutrient for all organisms. For pathogenic fungi, iron is essential for the success of infection. Thus, these organisms have developed high affinity iron uptake mechanisms to deal with metal deprivation imposed by the host. Siderophore production is one of the mechanisms that fungal pathogens employ for iron acquisition. Paracoccidioides spp. present orthologous genes encoding the enzymes necessary for the biosynthesis of hydroxamates, and plasma membrane proteins related to the transport of these molecules. All these genes are induced in iron deprivation. In addition, it has been observed that Paracoccidioides spp. are able to use siderophores to scavenge iron. Here we observed that addition of the xenosiderophore ferrioxamine B FOB) to P. brasiliensis culture medium results in repression (at RNA and protein levels) of the SidA, the first enzyme of the siderophore biosynthesis pathway. Furthermore, SidA activity was reduced in the presence of FOB, suggesting that P. brasiliensis blocks siderophores biosynthesis and can explore siderophores in the environment to scavenge iron. In order to support the importance of siderophores on Paracoccidioides sp. life and infection cycle, silenced mutants for the sidA gene were obtained by antisense RNA technology. The obtained AsSidA strains displayed decreased siderophore biosynthesis in iron deprivation conditions and reduced virulence to an invertebrate model.

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Argentilactone Molecular Targets in Paracoccidioides brasiliensis Identified by Chemoproteomics.

Project description:Paracoccidioidomycosis (PCM) is the cause of many deaths from systemic mycoses. The etiological agents of PCM belong to the Paracoccidioides genus, which is restricted to Latin America. The infection is acquired through the inhalation of conidia that primarily lodge in the lungs and may disseminate to other organs and tissues. The treatment for PCM is commonly performed via the administration of antifungals such as amphotericin B, co-trimoxazole, and itraconazole. The antifungal toxicity and side effects, in addition to their long treatment times, have stimulated research for new bioactive compounds. Argentilactone is a compound that was isolated from the Brazilian savanna plant Hyptis ovalifolia, and it has been suggested to be a potent antifungal, inhibiting the dimorphism of P. brasiliensis and the enzymatic activity of isocitrate lyase, a key enzyme of the glyoxylate cycle. This work was developed due to the importance of elucidating the putative mode of action of argentilactone. The chemoproteomics approach via affinity chromatography was the methodology used to explore the interactions between P. brasiliensis proteins and argentilactone. A total of 109 proteins were identified and classified functionally. The most representative functional categories were related to amino acid metabolism, energy, and detoxification. Argentilactone inhibited the enzymatic activity of malate dehydrogenase, citrate synthase, and pyruvate dehydrogenase. Furthermore, argentilactone induces the production of reactive oxygen species and inhibits the biosynthesis of cell wall polymers.

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Serological detection of antibodies against Paracoccidioides brasiliensis in dogs with leishmaniasis.

Project description:The aim of this study was to detect antibodies against Paracoccidioides brasiliensis in dogs seropositive and seronegative for leishmaniasis. Sera from 836 dogs (449 positive and 387 negative to leishmaniasis) were analysed by ELISA and the immunodiffusion test using gp43 and exoantigen, respectively. The analysis of the 836 serum samples by ELISA and the immunodiffusion test showed a positivity of 67.8 % and 7.3%, respectively, for P. brasiliensis infection. The dogs positive to leishmaniasis showed a higher reactivity to gp43 (79.9%) and exoantigen (12.7%) than the negative ones (54.0% and 1.0%, respectively). The higher reactivity to P. brasiliensis antigens may be due to cross-reactivity or a co-infection of dogs by Leishmania and P. brasiliensis. The lower correlation (0.187) observed between reactivity to gp43 and Leishmania antigen reinforces the latter hypothesis.

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