P330 Role of a D-amino acid oxidase (DAO) in an efficient human commensal Candida albicans
Authors: Das Kuladeep , Sanyal Kaustuv 
Published: September 2022

Abstract

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM

Objectives
Candida albicans is an opportunistic fungal pathogen residing as a harmless commensal component of the human microbiota. In the gut, C. albicans efficiently colonizes and successfully competes to limiting nutrient resources and overcomes the detrimental effect of an array of secondary metabolites released by the host and the dominant bacterial microflora. Among the metabolites, several free D-amino acids are released into the gut lumen, especially as a result of the degradation of bacterial cell walls. We sought to identify the genes that might confer advantages to C. albicans in metabolizing D-amino acids in diverse morphotypes, which might confer a competitive advantage.

Methods
All C. albicans genetically modified strains were constructed using a standard lithium acetate transformation method. The D-amino acid utilization and growth of C. albicans strains were assessed by spot dilution assays on minimal media agar plates at 30°C. Morphological switching between the yeast and hyphal forms was assessed in liquid media using hyphal-inducing conditions in the presence or absence of D-amino acids. Biofilm assays were performed using YNB-galactose media (with or without D-amino acids) on serum-treated 6-well plates at 30°C.

Results
C. albicans genome harbors three ORFs putatively encoding D-amino acid oxidase genes- DAO1orf19.3065, DAO2orf3365, and IFG3orf19.944. D-amino oxidase genes are lost from the genomes of several fungal species belonging to the Saccharomycetaceae family, including the free-living Saccharomyces cerevisiae. We found that C. albicans utilizes and grows on D-leucine, an amino acid known to be released by the gut bacteria into the intestinal lumen. This utilization is dependent on the growth morphotypes since the cells growing in biofilm fail to metabolize D-leucine. However, deletion of IFG3 but neither DAO1 nor DAO2 completely abrogates D-leucine utilization capabilities of C. albicans. The null ifg3 strain is moderately growth-sensitive in presence of D-alanine, an amino acid abundantly present and synthesized in the gut by bacteria for incorporation into peptidoglycan in their cell walls. By comparing Ifg3 protein levels in a leucine auxotrophic strain in different growth modes, we found that the expression of Ifg3 protein increases several folds in the biofilm growth mode in presence of D-leucine, but cells fail to grow optimally and make three-dimensional biofilm. Furthermore, ectopic expression of CaIFG3 in S. cerevisiae, which lost all D-amino oxidase genes, helps the organism to utilize D-leucine as a nutrient resource as well as overcome its inhibitory effect on growth.

Conclusion
Our results indicate that Ifg3 provides C. albicans the ability to metabolize D-leucine and helps it to overcome the growth inhibitory effect of D-alanine. Based on these primary observations we speculate a crucial role of Ifg3 in providing a competitive advantage against the resident microflora in the gut where continuous turnover of D-amino acids is crucial in maintaining host-microbe symbiotic interactions. Further in vivo commensalism experiments in mice will provide important clues on the role of this enzyme in C. albicans’s ability in gut colonization.