Haploinsufficiency-based large-scale forward genetic analysis of filamentous growth in the diploid human fungal pathogen C.albicans

Abstract

Candida albicans is the most prevalent human fungal pathogen. Here, we take advantage of haploinsufficiency and transposon mutagenesis to perform large-scale loss-of-function genetic screen in this organism. We identified mutations in 146 genes that affect the switch between its single-cell (yeast) form and filamentous forms of growth; this switch appears central to the virulence of C.albicans. The encoded proteins include those involved in nutrient sensing, signal transduction, transcriptional control, cytoskeletal organization and cell wall construction. Approximately one-third of the genes identified in the screen lack homologs in Saccharomyces cerevisiae and other model organisms and thus constitute candidate antifungal drug targets. These results illustrate the value of performing forward genetic studies in bona fide pathogens.

Fig. 1. Method for transposon mutagenesis of C.albicans. Linearized C.albicans genomic DNA fragments generated by restriction enzyme digestion were added to the donor plasmid containing a modified Tn7 transposon and Tn7 transposase. The modified transposon contains a promoterless Streptococcus thermophilus lacZ (Uhl and Johnson, 2001), C.albicans URA3 (Gillum et al., 1984) and the ampicillin resistance gene (bla) and origin of replication from pBluescriptKS+ (Stratagene). The sacB gene located on the donor plasmid external to the Tn7 repeats allows for selection against the donor plasmid (see Materials and methods). Following the transposition reaction (Biery et al., 2000), mutagenized genomic DNA was ligated and transformed into E.coli. The library was amplified, linearized by digestion with BsrGI and transformed in batch into C.albicans strain CAI4 (ura3/ura3). The transformed DNA was allowed to integrate into the C.albicans genome by homologous recombination, and successful integrants were selected as URA+ transformants.

Fig. 2. Examples of mutants identified in the screen. (A) A portion of a replica plate containing nine independent members of the insertion library grown on Spider medium for 4 days at 30°C. The center patch has a significantly less wrinkled appearance relative to its neighbors and was scored as a mutant reduced for filamentous growth. At this time point, the patches have not yet produced peripheral hyphae. (B) Examples of three other types of mutants obtained from the screen. From left to right, wild-type strain CAF2-1 (URA3/ura3:: imm434) (Fonzi and Irwin, 1993), a mutant reduced for filamentous growth, a mutant reduced for filamentous growth in the center of the patch but increased for production of peripheral hyphae, and a strongly hyperfilamentous mutant. Strains were grown for 4 days on Spider medium.

Fig. 3. Comparison of phenotypes of original transposon insertion mutants with those of reconstructed hemizygous deletion mutants. In each case, the deletion mutant was constructed in a fresh strain (RM1000) by deleting one copy of the indicated gene. These genes were identified by the site of transposon insertion in the original mutants. Colonies were grown on the indicated media for the indicated length of time and photographed at 2.5× magnification. Note the complex and individualistic colony morphologies, particularly those of the ZAP1/zap1 and FGR22/fgr22 mutants.

Fig. 4. Similarity of C.albicans genes that affect filamentous growth to genes in S.cerevisiae. (A) Roughly two-thirds of the C.albicans genes identified in the screen for filamentous growth mutants have closely related genes in S.cerevisiae. Nearly 30% lack significant similarity (as indicated by a BLAST score less than –15) to genes in any available databases. (B) The overall distribution of C.albicans genes relative to the S. cerevisiae genome. (C) Putative functions for the C.albicans genes that affect filamentous growth and have a close relative in S.cerevisiae. Each of these C.albicans genes was assigned a function based on the S.cerevisiae counterpart (see Supplementary table 1 for individual assignments). Categories are derived from the Saccharomyces Genome Database (http://genome-www.stanford.edu/Saccharomyces/).