The DEAD-box RNA helicase Vad1 regulates multiple virulence-associated genes in Cryptococcus neoformans
John Panepinto, … , Brian Wickes, Peter R. Williamson
John Panepinto, … , Brian Wickes, Peter R. Williamson
Published March 1, 2005
Citation Information: J Clin Invest. 2005;115(3):632-641. https://doi.org/10.1172/JCI23048.
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The DEAD-box RNA helicase Vad1 regulates multiple virulence-associated genes in Cryptococcus neoformans

Abstract

The study of fungal regulatory networks is essential to the understanding of how these pathogens respond to host environmental signals with effective virulence-associated traits. In this study, a virulence-associated DEAD-box RNA helicase–encoding gene (VAD1) was isolated from a mutant defective in the virulence factor laccase. A Δvad1 mutant exhibited a profound reduction in virulence in a mouse model that was restored after reconstitution with WT VAD1. Loss of VAD1 resulted in upregulation of NOT1, a gene encoding a global repressor of transcription. NOT1 was found to act as an intermediary transcriptional repressor of laccase. Vad1 was located within macromolecular complexes that formed cytoplasmic granular bodies in mature cells and during infection of mouse brain. In addition, VAD1 was shown by in situ hybridization to be expressed in the brain of an AIDS patient coinfected with C. neoformans. To understand the role of VAD1 in virulence, a functional genomics approach was used to identify 3 additional virulence determinants dependent on VAD1: PCK1, TUF1, and MPF3, involved in gluconeogenesis, mitochondrial protein synthesis, and cell wall integrity, respectively. These data show that fungal virulence-associated genes are coordinately regulated and that an analysis of such transcriptomes allows for the identification of important new genes involved in the normal growth and virulence of fungal pathogens.

Authors

John Panepinto, Lide Liu, Jeanie Ramos, Xudong Zhu, Tibor Valyi-Nagy, Saliha Eksi, Jianmin Fu, H. Ari Jaffe, Brian Wickes, Peter R. Williamson

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Figure 6

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Expression of PCK1 is dependent on VAD1. Deletion of PCK1 results in def...
Expression of PCK1 is dependent on VAD1. Deletion of PCK1 results in defective growth on lactic acid, normal growth on glucose, and attenuated virulence. (A) Northern blot analysis of RNA from the indicated cells incubated for 3 hours in asparagine media with or without glucose. (B) Southern blot analysis of DNA of the indicated cells digested with BamHI and KpnI, hybridized with a fragment of PCK1. (C) Cells of the WT (a), Δpck1 mutant (b), or complemented Δpck1mutant (c) grown on asparagine agar (pH 6.5) containing either 2% glucose (top panel) or 2% lactic acid (bottom panel). (D) Survival plot of mice injected with 106 CFU of the indicated strains. Mice were sacrificed when moribund.