Overview
Candida albicans is a normal inhabitant in our bodies, yet it can become pathogenic and cause infections that range from the superficial in healthy individuals to deadly in the immunocompromised. Comprehensive genetic analysis of C. albicans to identify mechanisms of virulence and new treatment strategies has been hampered by limited, publically accessible genomic resources. By combining the principles of Saccharomyces cerevisiae strain tagging with transposon mutagenesis to generate individually tagged mutants, we created the first entirely public resource that allows simultaneous measurement of strain fitness of ~60% of the genome in a wide range of experimental treatments. By identifying genes that confer a fitness or growth defect when reduced in copy number, we uncovered genes whose protein products represent potential antifungal targets. Moreover, screening this strain collection with chemical compounds allowed us to identify anticandidal chemicals while concurrently gaining insight into their cellular mechanism of action. This resource, combined with straightforward screening methodology, provides powerful tools to generate hypotheses for functional annotation of the genome, and our results highlight the value of direct versus model-based pathogen studies.
Abstract
Candida albicans is the most common human fungal pathogen, causing infections that can be lethal in immunocompromised patients. Although Saccharomyces cerevisiae has been used as a model for C. albicans, itlacks C. albicans’ diverse morphogenic forms and is primarily non-pathogenic. Comprehensive genetic analyses that have been instrumental for determining gene function in S. cerevisiae are hampered in C. albicans,due in part tolimited resources to systematically assay phenotypes of loss-of-function alleles. Here, we constructed and screened a library of 3633 tagged heterozygous transposon disruption mutants, using them in a competitive growth assay to examine nutrient- and drug-dependent haploinsufficiency. We identified 269 genes that were haploinsufficient in four growth conditions, the majority of which were condition-specific. These screens identified two new genes necessary for filamentous growth as well as ten genes that function in essential processes. We also screened 57 chemically diverse compounds that more potently inhibited growth of C. albicans versus S. cerevisiae. For four of these compounds, we examined the genetic basis of this differential inhibition. Notably, Sec7p was identified as the target of brefeldin A in C. albicans screens, while S. cerevisiae screens with this compound failed to identify this target. We also uncovered a new C. albicans-specific target, Tfp1p, for the synthetic compound 0136-0228. These results highlight the value of haploinsufficiency screens directly in this pathogen for gene annotation and drug target identification.