Targeted gene deletion in Candida parapsilosis demonstrates a role of CpALS3 in adhesion to human buccal epithelial cells

Among Candida non-albicans species isolated from fungemia, Candida parapsilosis has emerged as an important nosocomial pathogen, representing the second/third most common yeast isolated from blood. However, the molecular mechanisms underlying the virulence properties of this species are only beginning to be characterized. The adhesion ability to host surfaces is generally believed to play a key role in the initial stages of the infection process. In silico analysis of C. parapsilosis genomic sequence, indicates the existence of 5 potential homologues of C. albicans agglutinin-like sequence (Als) proteins, cell wall glycoproteins involved in the adhesion process. The contribution provided by each of these five genes in C. parapsilosis adhesion to biotic surfaces remains to be fully understood. During the three years of the present PhD research project, the identification of CpALS1-5 genes was performed in a collection of C. parapsilosis clinical isolates. Furthermore, RNA isolation from C. parapsilosis reference strain ATCC 22019 and RT-PCR analysis confirmed the presence of mRNA transcripts for all the 5 ALS gene homologues under basal growth conditions. CPAR2_404800 (further indicated as CpALS3) was chosen for site specific mutation on the basis of homology studies with C. albicans ALS genes. Indeed, C. albicans Als3p exhibits the highest level of adherence to the broadest array of substrates, and its role as adhesin has been extensively documented in literature. The deletion system chosen was based on the repeated use of the dominant nourseothricin resistance marker (CaSAT1) and its subsequent deletion by FLP-mediated site-specific recombination. The deletion cassette was designed according to the CpALS3 sequence and electroporated in C. parapsilosis reference strain ATCC 22019 to obtain two independent lineages of heterozygous and null mutants. Phenotypic traits of wild type strain (parental strain ATCC 22019), heterozygous strains CpALS3/Δals3 a, CpALS3/Δals3 b and null mutants CpΔals3/Δals3 a,CpΔals3/Δals3 b were analyzed and the adhesion ability of the strains was evaluated by an adhesion assay on human buccal epithelial cells (HBECs). Both null mutants exhibited a highly significant reduction in the adhesion ability to HBECs (P<0.01), which was fully restored by the re-integration of a functional copy of CpALS3 in the knock-out strain. We also investigated the impact of CpALS3 mutation on C. parapsilosis pathogenicity in an intra-hemocelic infection model of Galleria mellonella larvae. No significant difference was observed between mortality rate of G. mellonella larvae infected with WT, H, KO, or complemented strains. Collectively, these results indicate that CpALS3 deletion does not impair physiological growth of C. parapsilosis on common media, nor is it primarily involved in G. mellonella host invasion and damage, but Als3p plays an important role in the in vitro adhesion to epithelial surfaces.