Strains and Strategies for Large-Scale Gene Deletion Studies of the Diploid Human Fungal Pathogen <i>Candida albicans</i>

TLDR

Candida albicans is the most common human fungal pathogen, causing significant morbidity and mortality, yet its pathogenesis remains poorly understood and current gene‑deletion procedures are time‑consuming, inefficient, and can alter virulence through URA3 expression effects. The study aims to develop reference strains with wild‑type virulence and new disruption marker cassettes plus a fusion PCR protocol to enable rapid, highly efficient generation of homozygous knockout mutants for large‑scale genetic analysis. By exploiting the diploid nature of C. albicans, the authors constructed leu2Δ/leu2Δ, his1Δ/his1Δ, and arg4Δ/arg4Δ reference strains, introduced novel disruption markers, and employed a fusion PCR strategy to replace target genes without relying on the URA3 selectable marker.

Abstract

Candida albicans is the most common human fungal pathogen and causes significant morbidity and mortality worldwide. Nevertheless, the basic principles of C. albicans pathogenesis remain poorly understood. Of central importance to the study of this organism is the ability to generate homozygous knockout mutants and to analyze them in a mammalian model of pathogenesis. C. albicans is diploid, and current strategies for gene deletion typically involve repeated use of the URA3 selectable marker. These procedures are often time-consuming and inefficient. Moreover, URA3 expression levels-which are susceptible to chromosome position effects-can themselves affect virulence, thereby complicating analysis of strains constructed with URA3 as a selectable marker. Here, we describe a set of newly developed reference strains (leu2Delta/leu2Delta, his1Delta/his1Delta; arg4Delta/arg4Delta, his1Delta/his1Delta; and arg4Delta/arg4Delta, leu2Delta/leu2Delta, his1Delta/his1Delta) that exhibit wild-type or nearly wild-type virulence in a mouse model. We also describe new disruption marker cassettes and a fusion PCR protocol that permit rapid and highly efficient generation of homozygous knockout mutations in the new C. albicans strains. We demonstrate these procedures for two well-studied genes, TUP1 and EFG1, as well as a novel gene, RBD1. These tools should permit large-scale genetic analysis of this important human pathogen.