The regulation of hyphal orientation in Candida albicans
Candida albicans hyphae grow cathodally in an applied electric field. The chelation of exogenous Ca2+ ions attenuated this response. Similarly, the blockade of L-type calcium channels resulted in a diminished galvanotropic response suggesting the importance of calcium ion uptake in the C. albicans galvanotropic response. Following this rationale experiments were initiated to use molecular genetics to perturb calcium homeostasis in C. albicans. CaCch1p, the C. albicans calcium channel that is homologous to the mammalian L-type a1 calcium channel subunit was identified in silico and subsequently analysed. The primary amino acid sequence of the CaCch1p was analysed to ascertain those involved in blockade by calcium channel blocking drugs. Attempts were made to create a CaCCH1 deletion mutant for analysis in hyphal orientation response. Heterozygous mutants responded normally to applied electric fields. CaMID1, the C. albicans homologue of the S. cerevisiae stretch-activated cation channel, was cloned into the expression vector YPB-ADH1pt. The overexpression of CaMID1 was confirmed by northern analysis. The overexpression of CaMID1 did not have an effect on the galvanotropic response of C. albicans, however thigmotropic responses were enhanced (S. Shanks, personal communication). A series of mutants with disruptions in signal transduction pathways were screened for attenuations in galvanotropic alignment. The serine threonine protein kinase CaCst20p was demonstrated to be involved in the C. albicans hyphal orientation response but proteins of the Ras-cAMP were apparently not involved in the C. albicans hyphal orientation response. This thesis provides evidence that galvanotropic alignments of hyphae in C. albicans are dependent on a functional voltage-sensitive calcium channel and may be regulated via a pathway that includes CaCst20p and possibly other components of the polarisome complex.