The ability of yeast to adhere to biotic and abiotic surfaces represents an essential trait during the early stages of infection. Agglutinin-like sequence (Als) cell-wall proteins play a key role in adhesion of Candida species. Candida parapsilosis genome encompasses 5 ALS members, of which only the role of CPAR2_404800 has been elucidated. The present project was aimed at investigating the contribution of C. parapsilosis Als proteins by generating edited strains lacking functional Als proteins. CPAR2_404770 and CPAR2_404780, further indicated as CpALS4770 and CpALS4780, were selected for the generation of single and double edited strains using an episomal CRISPR/Cas9 technology. Phenotypic characterization of mutant strains revealed that editing of both genes had no impact on the in vitro growth of C. parapsilosis or on morphogenesis. Notably, CpALS4770-edited strain showed a reduction of biofilm formation and adhesive properties to human buccal cells (HBECs). Conversely, single CpALS4780-edited strain did not show any difference compared to the wild-type strain in all the assays performed, while the double CpALS4770-CpALS4780 mutant revealed an increased ability to produce biofilm, a hyper-adhesive phenotype to HBECs, and a marked tendency to form cellular aggregates. Murine vaginal infection experiments indicated a significant reduction in CFUs recovered from BALC/c mice infected with single and double edited strains, compared to those infected with the wild-type strain. These finding clearly indicate that CpAls4770 plays a role in adhesion to biotic and abiotic surfaces, while both CpALS4770 and CpALS4780 genes are required for C. parapsilosis ability to colonize and persist in the vaginal mucosa.
Keywords: Agglutinin-like sequence (ALS) gene family; Candida parapsilosis; Episomal CRISPR/Cas9; Human buccal epithelial cells (HBECs); Murine vaginal candidiasis.
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