Enrichment and Isolation of Fungal mRNA from Candida albicans–Induced Murine Keratitis for Differential Gene Expression Analysis

Abstract

Abstract: : Purpose: To determine the feasibility of isolating fungal mRNA from Candida albicans–infected mouse corneas for fungal gene expression microarray analysis in experimental keratitis. Methods: Corneas of immunocompetent BALB/c mice were scarified and either inoculated with 1x10⁶ CFU of Candida albicans (strain SC5314) or mock inoculated with phosphate–buffered saline. The animals were sacrificed 24 hours post infection and the eyes were enucleated and homogenized. Total RNA was extracted directly from the homogenates or the Candida RNA was enriched by hypotonic lysis of the murine host cells prior to extraction. RNA extraction was performed using Trizol reagent. The yeast RNA recovery was quantitated by RT–PCR using specific primers targeting the Candida EFB1 gene product and normalized to the host RNA levels using murine APRT mRNA as the internal sample control. The RT–PCR products were confirmed by Southern blot analysis. Results: Candida albicans EFB1 mRNA expression was detected in the Candida–infected mouse eyes by RT–PCR analysis and quantitated to be approximately 10⁷ copies per infected eye. While normal levels of host APRT mRNA expression were detected in both infected and mock–infected eyes, no fungal mRNA expression was detected in the mock–infected eyes. Enrichment for Candida RNA by hypotonic lysis of host cells significantly increased the relative ratio of Candida RNA compared to host RNA without a significant reduction in the overall yield of yeast RNA. Conclusions:Fungal RNA can be isolated from experimentally infected mouse eyes, can be enriched by lysis of host cells prior to RNA extraction, and should be amenable to amplification and fluorescent labeling for subsequent microarray analysis. Gene expression microarray analysis combined with experimental manipulation of the mouse model for Candida keratitis will allow the identification of genes differentially expressed under various conditions of disease severity. This approach will allow the identification of possible virulence factors contributing to fungal keratitis and may help identify potential therapeutic targets.

Keywords: keratitis • fungal disease • gene/expression