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RNA interference in Histoplasma capsulatum demonstrates a role for α‐(1,3)‐glucan in virulence
Author(s) -
Rappleye Chad A.,
Engle Jacquelyn T.,
Goldman William E.
Publication year - 2004
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2004.04131.x
Subject(s) - biology , rna interference , gene silencing , virulence , histoplasma , rna silencing , gene , gene expression , microbiology and biotechnology , rna , histoplasmosis , genetics , immunology , histoplasma capsulatum
Summary 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 α‐(1,3)‐glucan synthase) to deplete levels of α‐(1,3)‐glucan, a cell wall polysaccharide. Loss of α‐(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 α‐(1,3)‐glucan is an important contributor to Histoplasma virulence.