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Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens
Published on: February 14, 2011
RNA interference in Histoplasma capsulatum demonstrates a role for alpha-(1,3)-glucan in virulence
Chad A Rappleye1, Jacquelyn T Engle, William E Goldman
1Department of Molecular Microbiology, Campus Box 8230, Washington University, St Louis, MO 63110, USA.
Abstract:
Histoplasma capsulatum is a fungal pathogen that causes respiratory and systemic disease by proliferating within macrophages. While much is known about histoplasmosis, only a single virulence factor has been defined, in part because of the inefficiency of Histoplasma reverse genetics. As an alternative to allelic replacement, we have developed a telomeric plasmid-based system for silencing gene expression in Histoplasma by RNA interference (RNAi). Episomal expression of long RNAs that form stem-loop structures triggered gene silencing. To test the effectiveness of RNAi in Histoplasma, we depleted expression of a gfp transgene as well as two endogenous genes, ADE2 and URA5, and showed significant reductions in corresponding gene function. Silencing was target gene specific, stable during macrophage infection and reversible. We used RNAi targeting AGS1 (encoding alpha-(1,3)-glucan synthase) to deplete levels of alpha-(1,3)-glucan, a cell wall polysaccharide. Loss of alpha-(1,3)-glucan by RNAi yielded phenotypes indistinguishable from an AGS1 deletion: attenuation of the ability to kill macrophages and colonize murine lungs. This demonstrates for the first time that alpha-(1,3)-glucan is an important contributor to Histoplasma virulence.
Insights
A new RNA interference (RNAi) system effectively silences gene expression in Histoplasma capsulatum. This breakthrough identifies alpha-(1,3)-glucan as a key virulence factor, crucial for fungal survival and infection.
Area of Science:
- Mycology
- Molecular Biology
- Infectious Diseases
Background:
- Histoplasma capsulatum causes significant respiratory and systemic infections.
- Understanding Histoplasma virulence is limited by inefficient genetic manipulation tools.
- Defining virulence factors is crucial for developing effective treatments.
Purpose of the Study:
- To develop a novel RNA interference (RNAi) system for gene silencing in Histoplasma.
- To identify and characterize new virulence factors of Histoplasma capsulatum.
- To investigate the role of alpha-(1,3)-glucan in Histoplasma pathogenesis.
Main Methods:
- Developed a telomeric plasmid-based RNA interference (RNAi) system for gene silencing.
- Validated RNAi by targeting GFP, ADE2, and URA5 genes, confirming gene function reduction.
- Utilized RNAi to deplete alpha-(1,3)-glucan synthase (AGS1) and its product, alpha-(1,3)-glucan.
Main Results:
- The RNAi system demonstrated efficient, specific, stable, and reversible gene silencing in Histoplasma.
- Depletion of alpha-(1,3)-glucan resulted in phenotypes mirroring AGS1 deletion mutants.
- Loss of alpha-(1,3)-glucan significantly attenuated macrophage killing and lung colonization.
Conclusions:
- The developed RNAi system is a powerful tool for Histoplasma functional genomics.
- Alpha-(1,3)-glucan is a critical virulence factor for Histoplasma capsulatum.
- Targeting alpha-(1,3)-glucan synthesis may offer a novel therapeutic strategy against histoplasmosis.
