Inhibition of Yeast to Hyphal form Transition in Candida Albicans Identification of Potential Targets

Abstract

Dicyclomine is a human muscarinic acetylcholine receptor antagonist used for the treatment of abdominal cramps. Dicyclomine inhibited adhesion, early biofilm, mature biofilm, and planktonic growth of Candida albicans. Yeast to hyphal form transition of C. albicans in various inducer media such as serum, proline, glucose, and N-acetylglucosamine was inhibited. Dicyclomine killed C. albicans cells within 15 min of exposure. Dicyclomine appears to inhibit the yeast to hyphal form conversion by affecting signal transduction pathway. RtPCR analysis showed that dicyclomine targets both cAMP pathway as well as MAPK cascade. Eight genes were upregulated. Out of these, three major upregulated genes were Bcy1, Tup1, and Mig1. Dicyclomine downregulated Ume6, Ece1, and Pde2 genes which are involved in cAMP signaling pathway and also downregulated the DNA binding protein gene, Rfg1. Dicyclomine significantly upregulated the master negative regulator of hyphal formation, Tup1. A BLAST protein analysis revealed that a C. albicans protein Rrp9 shares 54 % identity and 71 % similarity with human muscarinic M1 receptor at 24 amino acids overlap. Global alignment between human muscarinic M1 receptor and C. albicans Rrp9 showed 19 % identity and 33 % similarity at 570 amino acid residues overlap. Our molecular docking study of dicyclomine with C. albicans, Rrp9, Gpr1, Ste2 and Ste3 showed that dicyclomine could bind only with Rrp9 by forming hydrogen bond interactions with VAL386 and ILE313 amino acid residues in the active site region. Dicyclomine bound with human muscarinic receptor M1 by forming hydrogen bond interaction with ARG123 at the active site region. We have studied the effect of human muscarinic receptor antagonists, dicyclomine, solifenacin and flavoxate on C. albicans morphogenesis induced by muscarinic receptors agonists, acetylcholine and bethanechol. Muscarinic receptor antagonists inhibited this transition in a concentration dependent manner. Dicyclomine inhibited yeast to hyphal form transition induced by bethane