The fungal pathogen, Candida albicans is able to form strongly adherent biofilms on inert surfaces such as those used for medical devices and on biological surfaces such as the skin, nails and mucous membranes. This attribute represents a key trait, which facilitates the persistence and dissemination of this opportunist, accounting in part for its increasing emergence as an important pathogen in immunocompromised individuals, neonates and the elderly. While it is widely known that microbial communities grow as biofilms and display distinct phenotypes [e.g. acquired drug resistance] as compared with their planktonic counterparts, the molecular basis through which these phenotypes are manifest remains unknown. Recent work shows that upon contact with an abiotic surface, C. albicans specifically and rapidly up regulates pathways for sulfur assimilation and glutathione biosynthesis and a number of other genes, many of which are of unknown function (103). These observations led us to ask how, at the molecular level, does a eukaryotic organism sense and respond to contact with a foreign (abiotic or biotic) surface and then transduce this information into altered patterns of gene expression and cell behavior. The immediate objective of this application is to identify key components, which regulate contact-dependent gene expression in C. albicans. To accomplish this we will use the MET3 gene, which encodes an ATP sulfurylase catalyzing the first step of the sulfur assimilation pathway. The MET3 gene is up-regulated over 20-fold within 50 minutes of cell contact to the abiotic surface and thus can be used to develop an experimental system to elucidate contact-dependent gene expression.
In Specific Aim 1, we will define the c/s-acting regions, which regulate the differential expression of MET3 upon surface contact. Completion of this Aim will result in the identification of specific sequence elements in the DNA, which are required for contact- dependent expression of MET3 and will further produce a detailed map of the MET3 promoter in C. albicans, for which few have been characterized to this level.
In Specific Aim 2, trans-acting elements required for contact-dependent gene expression will be identified. Completion of this Aim will result in the elucidation of the regulatory elements required for both planktonic and adherence-associated expression of MET3 and define transcription factors, which are uniquely responsive to the contact stimulus. Finally, in Specific Aim 3 we will assess the role of MET3 and the sulfur assimilation pathway in pathogenesis. The Candida sulfur assimilation pathway is qualitatively different from that of its mammalian host, providing promising targets for the development of new therapeutics. Understanding the underlying mechanism of contact sensing should provide novel approaches for interfering with cell adherence and thus controlling biofilm formation at the point of initial contact.
