The Putative Cytochrome b 5 Domain-Containing Protein CaDap1 Homologue Is Involved in Antifungal Drug Tolerance, Cell Wall Chitin Maintenance, and Virulence in Candida albicans

Abstract

Candida albicans (Ca), a prominent opportunistic fungal pathogen in humans, has garnered considerable attention due to its infectious properties. Herein, we have identified and characterized CaCDAP1 (Ca orf19.1034), a homolog of ScDAP1 found in Saccharomyces cerevisiae. CaCDAP1 encodes a 183-amino acid protein with a conserved cytochrome b5-like heme-binding domain. The deletion of CaDAP1 renders Ca cells susceptible to caspofungin and terbinafine. CaDAP1 deletion confers resistance to Congo Red and Calcofluor White, and sensitivity to sodium dodecyl sulfate. The deletion of CaDAP1 results in a 50% reduction in chitin content within the cell wall, the downregulation of phosphorylation levels in CaMkc1, and the upregulation of phosphorylation levels in CaCek1. Notably, CaDAP1 deletion results in the abnormal hyphal development of Ca cells and diminishes virulence in a mouse systemic infection model. Thus, CaDAP1 emerges as a critical regulator governing cellular responses to antifungal drugs, the synthesis of cell wall chitin, and virulence in Ca.

Keywords: CaDAP1; Candida albicans; chitin content; drug susceptibility; virulence.

Figure 1

Deletion of two CaDAP1 alleles. (A) Sequentially targeted disruption of two CaDAP1 alleles in WT RM1000 to generate Cadap1Δ/Δ (Cadap1::HIS1/Cadap1::FRT). Strain designations are depicted on the right, while primer sites are highlighted with arrows. (B) PCR verification of the homozygous mutant Cadap1Δ/Δ genotypes. Lane 1: A 0.25 kb fragment from RM1000 was amplified using the primers DAP1 − ORF − UP/DOWN. Lane 2: No fragment was amplified from Cadap1Δ/Δ (Cadap1::HIS1/Cadap1::FRT) using the primers DAP1 − ORF − UP/DOWN. Lane 3: A 1.4 kb fragment was amplified from RM1000 using primers DAP1 − UP − CHECK/DOWN-CHECK. Lane 4: The 0.98 kb and 2.6 kb fragments containing FRT and HIS1, respectively, were amplified from Cadap1Δ/Δ using the primers DAP1 − UP − CHECK/DOWN − CHECK.

Figure 2

CaDAP1 is involved in conferring resistance to terbinafine and caspofungin. Growth phenotypes of the WT, Cadap1/Cadap1 mutant, and CaDAP1 RS were evaluated following exposure to caspofungin (0.25 μg/mL) and terbinafine (5 μg/mL). Overnight cell cultures were then grown in YPD to the exponential phase at 30 °C. Tenfold serial dilutions were spotted onto YPD plates supplemented with caspofungin or terbinafine, and incubated at 30 °C for 48–72 h.

Figure 3

The deletion of CaDAP1 leads to reduced chitin content in cell walls, accompanied by decreased phosphorylation levels of CaMkc1 and slightly increased phosphorylation levels of CaCek1. (A) The deletion of CaDAP1 renders Ca cells resistant to CR and CFW, but sensitive to SDS. Growth phenotypes of WT, Cadap1/Cadap1 mutant, and CaDAP1 RS following exposure to 150 μg/mL CR and 150 μg/mL CFW, along with 0.03% SDS. Knockout of CaDAP1 results in a 50% decrease in chitin content (B) and a 9% increase in phosphomannan content (C) of the cell wall. (D) The intensity of CFW fluorescence of WT, Cadap1/Cadap1 mutant, and CaDAP1 RS stained with 25 μg/mL CFW. (E) Loss of CaDAP1 downregulates the expression level of CaCHS3. The WT strain (white bar), Cadap1/Cadap1 mutant (gray bar), and RS (black bar) were cultured in YPD medium to the exponential phase at 30 °C. CaCHS3 expression is depicted as a fold increase compared to the WT strain. (F) The deletion of CaDAP1 downregulates the phosphorylation levels of CaMkc1 and slightly upregulates the phosphorylation levels of CaCek1. Exponential phase cultures were harvested, and protein extracts separated using SDS-PAGE, followed by immunoblotting with antibodies against phosphorylated CaMkc1 or CaCek1. Immunoblotting with antibodies against the α-tubulin served as a loading control. An asterisk (*) denotes a significant difference (p < 0.05) between the Cadap1/Cadap1 mutant and WT or RS.

Figure 4

The deletion of CaDAP1 affects hyphal development and colony morphology. Induction of filamentation was visualized in YPD medium supplemented with 10% FBS for both WT RM1000 and the Cadap1/Cadap1 mutant, which contained either the integrated CIp10 vector or CIp10-CaDAP1 plasmid (A). Filament length was quantified for each strain (B), and their colony-formation ability assessed (C). The difference in filament length between WT RM1000 and Cadap1/Cadap1 mutant (B) was significant. An asterisk (*) denotes a significant difference (p < 0.05) between the Cadap1/Cadap1 mutant and WT or RS.

Figure 5

Assessment of virulence. (A) The survival rates of mice (n = 10) were evaluated after infection with WT RM1000, homozygous Cadap1/Cadap1 mutant, and RS. Daily monitoring for morbidity and survival tracking was conducted over a 30-day period. (B) Kidney tissues from mice infected with WT strain, Cadap1/Cadap1 homozygous mutant, and RS were subjected to histopathological examination. Saline buffer injection (CK) served as a negative control. Sections from infected kidney tissues were stained with periodic acid–Schiff’s reagent. Representative images of 5 kidney cross-sections (n = 2 mice/strain) were captured at 40× magnification.