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Paracoccidioides brasiliensis PbP27 gene: knockdown procedures and functional characterization
Isaura Torres1, Orville Hernandez, Diana Tamayo
1Unidad de Biología Celular y Molecular, Corporación para Investigaciones Biológicas (CIB), Medellín, Colombia; Instituto de Biología, Universidad de Antioquia, Medellín, Colombia.
FEMS Yeast Research
|October 15, 2013
Summary
The Paracoccidioides brasiliensis PbP27 protein is crucial for fungal cell architecture and interaction with macrophages. Reducing its expression impairs yeast cell vitality and virulence during host infection.
Area of Science:
- Mycology
- Molecular Biology
- Infectious Diseases
Background:
- The Paracoccidioides brasiliensis PbP27 gene encodes a protein found in fungal cytoplasm and cell wall.
- PbP27 expression is preferential in the parasitic yeast form and varies across fungal species.
- The precise biological role of p27 during fungal growth and host infection remains undetermined.
Purpose of the Study:
- To investigate the function of the PbP27 protein in the biology and virulence of Paracoccidioides brasiliensis.
- To generate and analyze mitotically stable PbP27 mutants using antisense RNA technology.
Main Methods:
- Antisense RNA technology and Agrobacterium tumefaciens-mediated transformation were employed to create PbP27 mutants (PbP27 aRNA).
- Real-time PCR was used to confirm reduced PbP27 gene expression in the generated mutants (60-75% reduction).
- Phenotypic analyses included growth curves, morphological transition assays, cell vitality assessments, and interaction studies with IFN-γ-stimulated macrophages.
Main Results:
- PbP27 aRNA mutants exhibited no significant alterations in growth curves or mycelia-yeast transition.
- A notable reduction in cell vitality was observed in PbP27 aRNA strains.
- PbP27 aRNA yeast cells showed decreased viability when interacting with IFN-γ-stimulated macrophages.
Conclusions:
- The p27 protein plays a significant role in maintaining yeast cell architecture in Paracoccidioides brasiliensis.
- Reduced p27 expression impacts fungal cell vitality and its interaction with the host's monocyte/macrophage system.
- PbP27 is implicated as a virulence factor contributing to fungal survival during host immune responses.
