RESUMO
Paracoccidioidomycosis (PCM), caused by Paracoccidioides species, is the main cause of death due to systemic mycoses in Brazil and other Latin American countries. Therapeutic options for PCM and other systemic mycoses are limited and time-consuming, and there are high rates of noncompliance, relapses, toxic side effects, and sequelae. Previous work has shown that the cyclopalladated 7a compound is effective in treating several kinds of cancer and parasitic Chagas disease without significant toxicity in animals. Here we show that cyclopalladated 7a inhibited the in vitro growth of Paracoccidioides lutzii Pb01 and P. brasiliensis isolates Pb18 (highly virulent), Pb2, Pb3, and Pb4 (less virulent) in a dose-response manner. Pb18 was the most resistant. Opportunistic Candida albicans and Cryptococcus neoformans were also sensitive. BALB/c mice showed significantly lighter lung fungal burdens when treated twice a day for 20 days with a low cyclopalladated 7a dose of 30 µg/ml/day for 30 days after intratracheal infection with Pb18. Electron microscopy images suggested that apoptosis- and autophagy-like mechanisms are involved in the fungal killing mechanism of cyclopalladated 7a. Pb18 yeast cells incubated with the 7a compound showed remarkable chromatin condensation, DNA degradation, superoxide anion production, and increased metacaspase activity suggestive of apoptosis. Autophagy-related killing mechanisms were suggested by increased autophagic vacuole numbers and acidification, as indicated by an increase in LysoTracker and monodansylcadaverine (MDC) staining in cyclopalladated 7a-treated Pb18 yeast cells. Considering that cyclopalladated 7a is highly tolerated in vivo and affects yeast fungal growth through general apoptosis- and autophagy-like mechanisms, it is a novel promising drug for the treatment of PCM and other mycoses.
Assuntos
Antifúngicos/farmacologia , Compostos Organometálicos/farmacologia , Paládio/farmacologia , Paracoccidioides/efeitos dos fármacos , Paracoccidioidomicose/tratamento farmacológico , Animais , Antifúngicos/síntese química , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Cadaverina/análogos & derivados , Cadaverina/biossíntese , Candida albicans/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Caspases/genética , Caspases/metabolismo , Cromatina/efeitos dos fármacos , Cromatina/patologia , Cromatina/ultraestrutura , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/crescimento & desenvolvimento , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Pulmão/efeitos dos fármacos , Pulmão/microbiologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Compostos Organometálicos/síntese química , Paládio/química , Paracoccidioides/genética , Paracoccidioides/crescimento & desenvolvimento , Paracoccidioides/ultraestrutura , Paracoccidioidomicose/microbiologia , Paracoccidioidomicose/patologia , Superóxidos/metabolismo , Vacúolos/efeitos dos fármacos , Vacúolos/patologia , Vacúolos/ultraestruturaRESUMO
BACKGROUND: Paracoccidioides brasiliensis and P. lutzii cause paracoccidioidomycosis (PCM). P. brasiliensis main diagnostic antigen is glycoprotein gp43, and its peptide sequence is 81% identical with a P. lutzii ortholog here called Plp43. P. lutzii ("Pb01-like") apparently predominates in Midwestern/Northern Brazil, where high percentages of false-negative reactions using P. brasiliensis antigens have recently been reported. The aim of this work was to produce recombinant Plp43 to study its antigenic identity with gp43. METHODOLOGY: We expressed rPlp43 as a secreted major component in Pichia pastoris and studied its reactivity in immunoblot with PCM patients' sera from Southwestern and Midwestern Brazil. PRINCIPAL FINDINGS: We showed that rPlp43 is not glycosylated and bears glucanase activity. The protein did not react with anti-gp43 monoclonal antibodies in immunoblot, suggesting absence of the corresponding gp43 epitopes. Nevertheless, common epitope(s) might exist, considering that gp43-positive PCM sera recognized rPlp43 in immunoblot, while gp43-negative sera (33 out of 51) from patients resident in Midwestern Brazil were also rPlp43-negative. Two genotyped P. lutzii were from patients with gp43-negative sera, suggesting that non-reactive sera are from patients infected with this species. CONCLUSION: Our data suggest that gp43 and Plp43 bear one or only a few common epitopes and that gp43 cannot be used in diagnosis of PCM patients infected with P. lutzii probably because Plp43 is poorly expressed during infection.
Assuntos
Antígenos de Fungos , Proteínas Fúngicas , Glicoproteínas , Glicosídeo Hidrolases , Paracoccidioides , Sequência de Aminoácidos , Antígenos de Fungos/química , Antígenos de Fungos/imunologia , Antígenos de Fungos/metabolismo , Epitopos , Proteínas Fúngicas/química , Proteínas Fúngicas/imunologia , Proteínas Fúngicas/metabolismo , Glicoproteínas/química , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/imunologia , Glicosídeo Hidrolases/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Paracoccidioides/química , Paracoccidioides/classificação , Paracoccidioides/enzimologia , Paracoccidioides/imunologia , Paracoccidioidomicose/imunologia , Paracoccidioidomicose/microbiologiaRESUMO
Glycoprotein gp43 is an immunodominant diagnostic antigen for paracoccidioidomycosis caused by Paracoccidioides brasiliensis. It is abundantly secreted in isolates such as Pb339. It is structurally related to beta-1,3-exoglucanases, however inactive. Its function in fungal biology is unknown, but it elicits humoral, innate and protective cellular immune responses; it binds to extracellular matrix-associated proteins. In this study we applied an antisense RNA (aRNA) technology and Agrobacterium tumefaciens-mediated transformation to generate mitotically stable PbGP43 mutants (PbGP43 aRNA) derived from wild type Pb339 to study its role in P. brasiliensis biology and during infection. Control PbEV was transformed with empty vector. Growth curve, cell vitality and morphology of PbGP43 aRNA mutants were indistinguishable from those of controls. PbGP43 expression was reduced 80-85% in mutants 1 and 2, as determined by real time PCR, correlating with a massive decrease in gp43 expression. This was shown by immunoblotting of culture supernatants revealed with anti-gp43 mouse monoclonal and rabbit polyclonal antibodies, and also by affinity-ligand assays of extracellular molecules with laminin and fibronectin. In vitro, there was significantly increased TNF-α production and reduced yeast recovery when PbGP43 aRNA1 was exposed to IFN-γ-stimulated macrophages, suggesting reduced binding/uptake and/or increased killing. In vivo, fungal burden in lungs of BALB/c mice infected with silenced mutant was negligible and associated with decreased lung ΙΛ-10 and IL-6. Therefore, our results correlated low gp43 expression with lower pathogenicity in mice, but that will be definitely proven when PbGP43 knockouts become available. This is the first study of gp43 using genetically modified P. brasiliensis.
Assuntos
Antígenos de Fungos/genética , Antígenos de Fungos/imunologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/imunologia , Glicoproteínas/genética , Glicoproteínas/imunologia , Paracoccidioides/genética , Paracoccidioides/imunologia , Fatores de Virulência/genética , Fatores de Virulência/imunologia , Animais , Antígenos de Fungos/metabolismo , Proteínas Fúngicas/metabolismo , Expressão Gênica/genética , Expressão Gênica/imunologia , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Vetores Genéticos/metabolismo , Glicoproteínas/metabolismo , Interleucina-6/genética , Interleucina-6/imunologia , Interleucina-6/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação/genética , Mutação/imunologia , Paracoccidioides/metabolismo , Paracoccidioidomicose/genética , Paracoccidioidomicose/imunologia , Paracoccidioidomicose/metabolismo , Paracoccidioidomicose/microbiologia , RNA Antissenso/genética , RNA Antissenso/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Fatores de Virulência/metabolismoRESUMO
Melioidosis has been considered an emerging disease in Brazil since the first cases were reported to occur in the northeast region. This study investigated two municipalities in Ceará state where melioidosis cases have been confirmed to occur. Burkholderia pseudomallei was isolated in 26 (4.3%) of 600 samples in the dry and rainy seasons.