Systematic identification of Candida albicans secreted proteins required for virulence

Abstract

The fungal secretome is uniquely important to pathogenic lifestyles, providing a range of factors, including nutrient scavenging proteins, effectors, and toxins, which directly interact with the host. The human fungal pathogen Candida albicans releases a number of these factors into the extracellular environment, where they contribute to virulence. However, despite their importance, relatively few of the proteins C. albicans are predicted to secrete have been characterised. This study aimed to identify and characterise secreted proteins required for virulence. Through in silico analysis, we identified a total of 135 putative secreted proteins, including 82 grouped across 25 families and 53 singletons. The gene families mainly included proteins with known or predicted functions in nutrient acquisition and cell wall remodelling. Proteins with characterised roles included hydrolytic enzymes such as the secreted aspartyl proteinases, lipases, phospholipases, and arginases, as well as enzymes involved in cell wall remodelling. Uncharacterised proteins included esterases, sphingolipid phosphodiesterases, ribonucleases and nucleoside scavengers. Relatively few of the singletons had been characterised, but those studied include Ece1 (candidalysin), Hex1, Pho100, Kre9, Pra1, Sel1 and MFalpha. Moreover, 31 predicted singletons had no recognised functional domains. We generated a library of barcoded null mutants for the predicted singletons, using the CRISPR/Cas9 transient system, and examined their relative fitness in a mouse model of systemic infection using a Barcode analysis by sequencing (Bar-seq) approach. From this, we identified 5 mutants with reduced fitness in the brain and/or the kidneys and confirmed attenuated virulence for 2 of these mutants in a 3-day mouse model of systemic infection. We also screened the library in vitro for phenotypic differences under various growth conditions, for defects in the interaction with epithelial cells and in biofilm formation. We identified 2 mutants with phenotypic differences compared to the wild-type (WT) strain, 1 mutant with reduced ability to adhere to epithelial cells and consequently modestly reduced in damage and 2 mutants with reduced ability to form biofilms in specific-media conditions. Together these findings contribute to a better understanding of fungal virulence and could ultimately provide novel targets for antifungal drugs and vaccines.