Regulation of thigmotropism in human pathogenic fungi
Microscopical examination of Candida albicans grown on contoured artificial surfaces provided evidence that hyphae responded thigmotropically to features of the growth substrate. Hyphae of C. albicans followed grooves and ridges on various artificial membranes and penetrated pores of Nucleopore filters. The thigmotropic response in C. albicans was attenuated by gadolinium ions and by verapamil suggesting that calcium uptake may be involved in thigmotropic regulation. Thigmotropism was also observed for the first time in three genera of dermatophytic fungi (Epidermophyton, Trichophyton and Microsporum) and two saprophytic fungi (Mucor mucedo and Neurospora crassa). Therefore thigmotropism may be a general feature of fungal hyphae that must forage for nutrients on surfaces and within solid materials. Since Ca2+ appears to be involved in the regulation of thigmotropism attempts were made to construct strains expressing the Ca2+ sensitive photoprotein aequorin. The apoaequorin d gene was cloned in to C. albicans and S. cerevisiae using the YPB-ADHpt expression vector. Southern analysis indicated low copy number of the plasmid in C. albicans as compared with S. cerevisiae. Aequorin was reconstituted in protein extracts of C. albicans and S. cerevisiae by supplementing them with coelenterazine. The level for C. albicans was ten times higher than for Neurospora crassa, the only filamentous fungus to be transformed with this gene so far. Aequorin was successfully reconstituted in transformed living cells, and the luminescence levels were sufficiently high to be detected when external Ca2+ was added to the growth medium. Transformed C. albicans cells undergoing the dimorphic transition from yeast-to-hyphal form exhibited higher resting levels of luminescence indicating that cells induced to form hyphae have higher [Ca2+] than yeast cells. The work presented in this thesis presents first evidence of construction of strains expressing the luminescence photoprotein aequorin in a pathogenic fungus. This method provides a non-toxic, non-invasive method for monitoring [Ca2+] in C. albicans.