Research Project
8030219
DESCRIPTION (provided by applicant): The pathogenic potential of Candida albicans is intimately related with the way this organism senses and reacts to its surrounding environment, which, in the case of C. albicans, is the host. Candidiasis now represents the third most frequent nosocomial infection in hospitals both in the US and worldwide and C. albicans remains the most frequent causative agent of fungal infections. C. albicans is an increasingly common threat to human health as a consequence of AIDS, steroid therapy, organ and tissue transplantation, cancer therapy, broad spectrum antibiotics and other immune defects. Unfortunately these infections carry unacceptably high morbidity, mortality rates and important economic repercussions (estimated total direct cost of approximately 2 billion dollars in 1998 in US hospitals alone). C. albicans can grow as yeast cells, pseudohyphae or hyphae with its form being dictated by its surrounding conditions. The ability to form hyphal cells has been fundamentally linked to the disease potential of this organism. In fact, cells which cannot make the transition are avirulent. Hyphal formation is regulated by a complex network of signaling pathways. Research has centered on parts of a small number of specific signal transduction pathways that are known to mediate its environmental sensing and host-pathogen interactions. This has left substantial gaps in our knowledge including the originating signal and the nature of crosstalk within these very pathways. This is compounded by the possibility of other transduction pathways and the use of phosphorylation to modulate the activity of the proteins responsible for regulating filamentation. Although phosphorylation is important for a number of cellular and developmental processes, most studies have focused on a few specific proteins and there are limited proteome-wide data on signal transduction pathway proteins or phosphoproteins in general. This application will study the C. albicans phosphoproteome with an emphasis on signal transduction pathways. This project focuses on a central themes: i) that Candida filamentation is a virulence trait strongly associated with environmental sensing and that by studying the phosphoproteome we can identify environmental sensing molecules and pathways vital for this process. The basis of the proposed experimentation is to use Stable Isotope Labeling with Amino acids in Cell culture (SILAC) in conjunction with Immobilized Metal Affinity Chromatography (IMAC) enrichment of phosphoproteins followed by mass spectrometric analysis. The presence of the stable isotope causes a mass change allowing the direct quantitative comparison to an unlabeled sample on a mass spectrometer. PUBLIC HEALTH RELEVANCE: Candida albicans is the main causative agent of candidiasis, the most frequent fungal infection and the fourth leading cause of infections in US hospitals, with high mortality rates and significant economic burden. It frequently infects AIDS patients. Here we propose using a phosphoproteomic approach to investigate the sensing and signalling that mediate key virulence associated events, hopefully leading to preventative strategies against these frequent nosocomial infections.
