• CRIPR-Cas9
• gene editing
• host-pathogen interactions
• Candida parapsilosis
• virulence factors
• adhesion
• ALS genes

Candida parapsilosis is an increasingly important opportunistic human pathogen, responsible for both mucosal and systemic infections. Epidemiologic studies indicate that this species ranks as the second/third most frequently isolated Candida species form blood cultures in Europe, Latin America and North America, following Candida albicans and, in some areas, Candida glabrata. While C. albicans has been extensively characterized, little is still known about virulence determinants of C. parapsilosis. Among these factors, adhesion to host surfaces is a key event during the first phase of infection. The Agglutinin-Like Sequence (ALS) gene family in C. albicans encodes for 8 cell wall glycoproteins known to play a major role in adhesion to biotic and abiotic surfaces. In C. parapsilosis genome (http://cgob3.ucd.ie/), five putative homologues (CpALS) have been identified (CPAR2_404800, CPAR2_404770, CPAR2_404780, CPAR2_404790, CPAR2_500660), three of which have been previously demonstrated to have a role in adhesion and pathogenicity. This thesis project is part of a wider study aimed at dissecting the role of CpALS genes in the virulence of C. parapsilosis, through the characterization of mutant strains obtained by gene disruption and gene editing strategies. In particular, during this thesis internship, a plasmid-based CRISPR-Cas9 system was used to mutate the two uncharacterized CpALS genes, CPAR2_404770 and CPAR2_404780, henceforth named CpALS11 and CpALS12, respectively. Together with the plasmid bearing the CRISPR/Cas9 elements, C. parapsilosis was transformed with a repair template aimed at introducing two stop codons at the double strand break site, induced by Cas9, through homologous recombination.

For CpALS12 editing, a highly adhesive clinical isolate of C. parapsilosis (Cp50) was used as parental strain. Due to an unexpected genetic variability in the region targeted by the designed gRNA, C. parapsilosis reference strain ATCC 22019 could not be used as genetic background.
Editing of CpALS11 was performed on both the wild-type reference strain ATCC 22019 and the Cp50 clinical isolate. In addition, the same strategy was performed on the CpALS12 mutant strain, leading to the creation of a double CpALS12-CpALS11 mutant strain. Gene editing of both single and double CpALS12-CpALS11 mutants was confirmed using allele specific-PCR, enzymatic digestions and sequencing, demonstrating that correct gene editing occurred at the desired loci according to the experimental design.
 
Phenotypic characterization of the mutant strains revealed that the editing of CpALS11 did not have effect on pseudohyphae formation, but it induced an alteration in fungal cell morphology in some of the conditions tested. Furthermore, a reduction of both biofilm formation and ad-hesive properties was observed in the single CpALS11 mutant strain, suggesting a direct role of CpAls11 in both adhesion to biotic or abiotic surfaces and cell wall organization.

While single CpALS12 mutant strain did not show any difference, when compared to the wild-type strain, in any of the assays performed, the double CpALS12-CpALS11 mutant strain had a marked increase in the ability to produce biofilm and to adhere to HBECs, as well as an altera-tion in fungal cell morphology in all of the conditions tested. These results suggest an indirect role of CpAls12 in cell wall organization or in the regulation of other genes.

Further studies will be performed to shed more light on the role of CpALS11 and CpALS12 in C. parapsilosis virulence and pathogenicity, in order to better understand the pathogenic mechanisms used by C. parapsilosis in the onset of mucosal candidiasis.