Candida tropicalis oligopeptide transporters assist in the transmembrane transport of the antimicrobial peptide CGA-N9.

Candida tropicalis is often reported as the second or third most common pathogen causing fungal infections. Antimicrobial peptides (AMPs) have attracted increasing attention for their broad-spectrum antimicrobial properties and low cytotoxicity. Our previous studies have shown that CGA-N9, a non-membrane-rupturing AMP, crosses the cell membrane to exert anticandidal activity. We speculate that there are some related transporters that assist in the transmembrane transport of CGA-N9. In this study, the relationship between CGA-N9 lethality kinetics and its real-time transmembrane amount in C. tropicalis cells was investigated. The results demonstrated that there was a positive correlation between its candicidal activity and transmembrane amount. A total of 12 oligopeptide transporter (OPT) coding sequences (CDSs) were cloned from C. tropicalis by using the conservative OPT gene sequences of Candida spp. to design primers and were named C. tropicalis OPTs (CtOPTs). The results of RT‒qPCR demonstrated that the expression levels of CtOPT1, CtOPT9 and CtOPT12 were correlated with the CGA-N9 transmembrane amount in a time-dependent manner. The results of molecular docking demonstrated that CtOPT1, CtOPT9 and CtOPT12 interact strongly with CGA-N9. Therefore, CtOPT1, CtOPT9 and CtOPT12 were predicted to assist in the transmembrane transport of the AMP CGA-N9.

Rational design, synthesis, antifungal evaluation and docking studies of antifungal peptide CGA-N12 analogues based on the target CtKRE9.

Candida tropicalis is a major non-albicans species that causes invasive candidiasis. CGA-N12, an anti-Candida peptide found by our group, disrupted cell wall architecture by inhibiting the activity of the protein killer-resistant 9 (KRE9), a ß-1,6-glucan synthase specific to Candida spp. and plants. Herein, a set of CGA-N12 analogues were rationally designed based on the interaction networks between CGA-N12 and C. tropicalis KRE9 (CtKRE9). Seven CGA-N12 analogues with significantly improved antifungal activity against C. tropicalis were screened by reducing the docking energy of CGA-N12 and CtKRE9 and increasing the number of positive charges on CGA-N12 based on a stable three-dimensional model of CtKRE9. CGA-N12 and its analogues exhibited antifungal activity against C. tropicalis and its persist cells; they also inhibited biofilm formation and eradicated preformed biofilms. Compared with fluconazole, they displayed higher activities against the growth of persister cells and more effective preformed biofilm eradication. Among them, CGA-N12-0801, CGA-N12-0902 and CGA-N12-1002 displayed much higher activity and anti-proteinase digestion stability than CGA-N12. Specifically, CGA-N12-0801 was the optimal analogue, with a minimum inhibitory concentration of 3.46 µg/mL and a therapeutic index of 158.07. The results of electronic microscopy observations and KRE9 activity inhibition assays showed that CGA-N12 and its analogues killed C. tropicalis by disrupting the architecture of the cell wall and the integrity of the cell membrane. In conclusion, for the first time, we provide a simple and reliable method for the rational design of antimicrobial peptides and ideal candidates for treating Candida infections that not effectively eliminated by azole drugs.