ABSTRACT
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Subject(s)
Female , Humans , Young Adult , Paracoccidioidomycosis/metabolism , Paracoccidioidomycosis/physiopathology , Mycoses/complications , Mycoses/metabolism , Fungi/cytology , Paracoccidioidomycosis/transmission , Paracoccidioidomycosis/virology , Mycoses/diagnosis , Mycoses/transmission , Fungi/metabolism , Paraguay/ethnologyABSTRACT
The aim of this study was to demonstrate the DNA of Paracoccidioides brasiliensis in human serum samples of patients with paracoccidioidomycosis using the polymerase chain reaction (PCR). The diagnosis of paracoccidioidomycosis (PCM) was defined by microscopic observation of the fungus on direct exam or histopathology, culture, and serological positivity. DNA from serum of 33 patients with PCM was extracted and submitted to nested-PCR using primers from the gp 43 gene. Only one sample was positive on nested-PCR. We conclude that the prevalence of fungemia in patients with different clinical forms of PCM is low, limiting the use of serum DNA detection as an alternative diagnostic tool.
Subject(s)
Antigens, Fungal/genetics , DNA, Fungal/analysis , Fungal Proteins/genetics , Fungemia/diagnosis , Glycoproteins/genetics , Paracoccidioides/genetics , Paracoccidioidomycosis/blood , Polymerase Chain Reaction/methods , Adult , Aged , Child , DNA Primers , DNA, Fungal/blood , Female , Humans , Male , Middle Aged , Paracoccidioidomycosis/virology , Sensitivity and SpecificityABSTRACT
The aim of this study was to demonstrate the DNA of Paracoccidioides brasiliensis in human serum samples of patients with paracoccidioidomycosis using the polymerase chain reaction (PCR). The diagnosis of paracoccidioidomycosis (PCM) was defined by microscopic observation of the fungus on direct exam or histopathology, culture, and serological positivity. DNA from serum of 33 patients with PCM was extracted and submitted to nested-PCR using primers from the gp 43 gene. Only one sample was positive on nested-PCR. We conclude that the prevalence of fungemia in patients with different clinical forms of PCM is low, limiting the use of serum DNA detection as an alternative diagnostic tool.
Subject(s)
Humans , Male , Female , Child , Adult , Middle Aged , Antigens, Fungal/genetics , DNA, Fungal/analysis , Fungemia/diagnosis , Glycoproteins/genetics , Paracoccidioides/genetics , Paracoccidioidomycosis/blood , Fungal Proteins/genetics , Polymerase Chain Reaction/methods , DNA, Fungal/blood , DNA Primers , Paracoccidioidomycosis/virology , Sensitivity and SpecificityABSTRACT
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.
Subject(s)
DNA, Complementary/analysis , Gene Expression Regulation, Fungal/genetics , Paracoccidioides/enzymology , Peptide Hydrolases/genetics , Transcription, Genetic/genetics , Base Sequence , DNA, Complementary/genetics , Expressed Sequence Tags , Humans , Molecular Sequence Data , Paracoccidioides/genetics , Paracoccidioides/pathogenicity , Paracoccidioidomycosis/virology , VirulenceABSTRACT
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.
Subject(s)
Expressed Sequence Tags/metabolism , Paracoccidioides/pathogenicity , Transcription, Genetic/genetics , Animals , Base Sequence , DNA, Complementary , DNA, Fungal , Gene Expression Regulation, Fungal , Humans , Molecular Sequence Data , Paracoccidioides/enzymology , Paracoccidioides/genetics , Paracoccidioidomycosis/virology , Transcription, Genetic/physiology , Virulence/geneticsABSTRACT
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.
Subject(s)
Humans , DNA, Complementary/analysis , Paracoccidioides/enzymology , Peptide Hydrolases/genetics , Gene Expression Regulation, Fungal/genetics , Transcription, Genetic/genetics , DNA, Complementary/genetics , Molecular Sequence Data , Expressed Sequence Tags , Paracoccidioides/genetics , Paracoccidioides/pathogenicity , Paracoccidioidomycosis/virology , Base Sequence , VirulenceABSTRACT
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.
Subject(s)
Humans , Animals , Expressed Sequence Tags/metabolism , Paracoccidioides/pathogenicity , Transcription, Genetic/genetics , DNA, Complementary , DNA, Fungal , Molecular Sequence Data , Paracoccidioides/enzymology , Paracoccidioides/genetics , Paracoccidioidomycosis/virology , Gene Expression Regulation, Fungal , Base Sequence , Transcription, Genetic/physiology , Virulence/geneticsABSTRACT
O presente estudo trata dos resultados obtidos, do ponto de vista micologico e imunoquimico, de duas amostras de Paracoccidioides consideradas como pertencentes a espécie cerebriformis, criada por MOORE em 1935 e mantidas desde aquela época, através de repiques em agar-Sabouraud, na Micoteca do Instituto de Medicina Tropical de Säo Paulo. Após cerca de 60 anos, tais amostras conservavam as mesmas características descritas por MOORE (1935). Näo foram registradas lesöes experimentais em cobaias inoculadas por via intratesticular, näo se demonstrando também, pelas técnicas SDS PAGE e Western blotting...
