Abstract
Purpose :
To establish the functionality of HWP1 gene which codes for major hyphal cell wall protein in biofilm formation in Candida albicans isolated from a keratitis patient.
Methods :
Expression of 27 different genes was monitored during adhesion, expansion, maturation and dispersal phase of biofilm formation in ocular C. albicans. The results indicated that HWP1 gene involved in adhesion was up regulated during biofilm formation in ocular C. albicans. To establish whether HWP1 is required for biofilm formation attempts were made to mutate the gene by CRISPR-Cas9 method. The method involved transformation of C. albicans with linearized pV1093-HWP1-gRNA vector along with the donor template.CAS9 and the small guide RNA would bind and cleave 51-73 bp of HWP1 from the genomic DNA. This cleaved region would be replaced by a fragment containing stop codon and a restriction site by homologous recombination. The unique restriction site would help for rapid screening of mutants. Mutants were validated by restriction digestion, Real time PCR and subjected to biofilm formation by XTT method and confocal laser scanning microscope for measuring thickness of biofilm.
Results :
Transformation of Candida albicans with pv1093-HWP1-gRNA yielded 45 transformants. Ten of the 45 transformants showed no amplification of HWP1 gene indicating that the gene was mutated. Restriction digestion by EcoRI resulted in two bands in the mutants and only one in the wild type indicating integration of restriction site from donor template in to the HWP1 gene. XTT assay showed no biofilm formation in the mutants whereas the wild type formed a luxuriant biofilm. The thickness of the wild type biofilm was 17.2 µm and mutants appeared as isolated cells with clear morphology and no biofilm formation.
Conclusions :
CRISPR–Cas9 protocol was used for mutating the HWP1 gene in an ocular Candida albicans. The resultant mutant was devoid of its ability to form biofilm and after 72 h isolated cells with clear morphology were observed. In contrast in the wild type cells, a luxuriant biofilm of 17.2 µm thickness was observed. Our results indicate that HWP1 gene is required for biofilm formation in ocular C. albicans.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.