Inhibition of PbGP43 Expression May Suggest that gp43 is a Virulence Factor in Paracoccidioides brasiliensis

Autor: Orville Hernández, Isaura Torres, Ana María García, Natanael P. Leitão, Angela Restrepo, Juan G. McEwen, Rosana Puccia, Diana Tamayo, José F. Muñoz
Jazyk: angličtina
Rok vydání: 2013
Předmět:
Mutant
Gene Expression
lcsh:Medicine
Paracoccidioides
Mice
Gene expression
Molecular Cell Biology
Pathology
lcsh:Science
Lung
Genetics Paracoccidioidomycosis
Fungal protein
Mice
Inbred BALB C
Multidisciplinary
biology
Fungal Diseases
Immunology Genetic Vectors
Host-Pathogen Interaction
Infectious Diseases
Medical Microbiology
Medicine
Genetic Engineering
Research Article
Biotechnology
Clinical Pathology
Antigens
Fungal
Virulence Factors
Genetic Vectors
Immunology
Mycology
Microbiology
Fungal Proteins
Diagnostic Medicine
Extracellular
Genetics
Animals
RNA
Antisense
Biology
Microbial Pathogens
Glycoproteins
Paracoccidioides brasiliensis
Immunology Tumor Necrosis Factor-alpha
Paracoccidiomycosis
Interleukin-6
Tumor Necrosis Factor-alpha
lcsh:R
Wild type
Fungi
Genetics Gene Expression
biology.organism_classification
Molecular biology
In vitro
Yeast
Clinical Microbiology
Mutation
lcsh:Q
Gene Function
Paracoccidioidomycosis
Zdroj: Repositorio UdeA
Universidad de Antioquia
instacron:Universidad de Antioquia
PLoS ONE, Vol 8, Iss 7, p e68434 (2013)
PLoS ONE
Popis: 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.
Databáze: OpenAIRE
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