A key structural domain of the Candida albicans Mdr1 protein

Area of Science:

• Molecular Biology
• Mycology
• Biochemistry

Background:

• The MDR1 transporter in Candida albicans, a member of the Major Facilitator Superfamily (MFS), is implicated in azole drug resistance.
• MDR1 facilitates the efflux of antifungal drugs, contributing to multidrug resistance (MDR) in fungal infections.

Purpose of the Study:

• To investigate the role of the largest interdomain intracellular loop 3 (ICL3) of the MDR1 protein in Candida albicans.
• To determine the functional significance of ICL3 in the MDR1 transporter's activity and drug resistance.

Main Methods:

• Site-directed mutagenesis was employed to introduce deletions and insertions into the ICL3 region of the MDR1 gene.
• Overexpression of mutant MDR1 variants and assessment of drug resistance phenotypes.
• Trypsinization assays to evaluate protein stability of mutant variants.
• Homology modeling to analyze the structural impact of ICL3 mutations.

Main Results:

• Progressive deletions in ICL3 led to a loss of drug resistance when MDR1 variants were overexpressed.
• Restoring ICL3 length through insertional mutagenesis did not restore transporter functionality.
• Most ICL3 insertion and deletion variants showed increased susceptibility to trypsinization, indicating altered protein structure.
• Homology modeling revealed perturbations in the molecular surface-charge distribution of ICL3 mutant variants.

Conclusions:

• The central cytoplasmic loop (CCL), specifically ICL3, of the fungal MFS transporter (DHA1 family) is critical for MDR function.
• ICL3's role is essential for MDR1 transporter function, independent of substrate specificity or protein recruitment.
• These findings highlight ICL3 as a key determinant of MDR1 activity in Candida albicans.