Microscopical examination of <I>Candida albicans</I> grown on contoured artificial surfaces provided evidence that hyphae responded thigmotropically to features of the growth substrate. Hyphae of <I>C. albicans</I> followed grooves and ridges on various artificial membranes and penetrated pores of Nucleopore filters. The thigmotropic response in <I>C. albicans</I> 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 <I>(Epidermophyton, Trichophyton </I>and <I>Microsporum)</I> and two saprophytic fungi <I>(Mucor mucedo </I>and <I>Neurospora crassa</I>). Therefore thigmotropism may be a general feature of fungal hyphae that must forage for nutrients on surfaces and within solid materials. Since Ca<sup>2+</sup> appears to be involved in the regulation of thigmotropism attempts were made to construct strains expressing the Ca<sup>2+ </sup>sensitive photoprotein aequorin. The apoaequorin d gene was cloned in to <I>C. albicans</I> and <I>S. cerevisiae</I> using the YPB-ADHpt expression vector. Southern analysis indicated low copy number of the plasmid in <I>C. albicans</I> as compared with <I>S. cerevisiae. </I>Aequorin was reconstituted in protein extracts of <I>C. albicans</I> and <I>S. cerevisiae</I> by supplementing them with coelenterazine. The level for <I>C. albicans </I>was ten times higher than for <I>Neurospora crassa</I>, 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 Ca<sup>2+</sup> was added to the growth medium. Transformed <I>C. albicans</I> 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 [Ca<sup>2+</sup>] 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 [Ca<sup>2+</sup>] in <I>C. albicans.</I>
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:327040 |
| Date | January 1998 |
| Creators | Perera, Thanuja Harshini |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU098400 |
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