Characterization of the ESCRT pathway in Candida albicans.

Abstract

iv Abstract The human opportunistic pathogen Candida albicans has a signal transduction pathway unique to fungi, called the Rim101 pathway. The Rim101 pathway regulates the proteolytic activation of the transcription factor, Rim101, the activation of which is required for growth at neutral-alkaline pH. Many genes regulated by Rim101 play a role in C. albicans virulence, including genes involved in filamentation, cell wall structure, adhesion, and nutrient acquisition. The Rim101 pathway consists of two complexes: a signaling complex at the plasma membrane and a processing complex inside the cell, and both of these complexes are required for Rim101 activation. Rim101 activation also requires members of a second pathway, the endosomal sorting complex required for transport (ESCRT) pathway. The ESCRT pathway is required to generate multivesicular bodies prior to vesicle fusion with the vacuole. The ESCRT pathway consists of several polyprotein complexes recruited sequentially to the endosomal membrane to generate an intraluminal vesicle. The role of the ESCRT pathway has not been well characterized in C. albicans, and study of the ESCRT pathway is complicated by the secondary effect many ESCRT mutations have on Rim101 processing. These studies sought to separate iv Abstract The human opportunistic pathogen Candida albicans has a signal transduction pathway unique to fungi, called the Rim101 pathway. The Rim101 pathway regulates the proteolytic activation of the transcription factor, Rim101, the activation of which is required for growth at neutral-alkaline pH. Many genes regulated by Rim101 play a role in C. albicans virulence, including genes involved in filamentation, cell wall structure, adhesion, and nutrient acquisition. The Rim101 pathway consists of two complexes: a signaling complex at the plasma membrane and a processing complex inside the cell, and both of these complexes are required for Rim101 activation. Rim101 activation also requires members of a second pathway, the endosomal sorting complex required for transport (ESCRT) pathway. The ESCRT pathway is required to generate multivesicular bodies prior to vesicle fusion with the vacuole. The ESCRT pathway consists of several polyprotein complexes recruited sequentially to the endosomal membrane to generate an intraluminal vesicle. The role of the ESCRT pathway has not been well characterized in C. albicans, and study of the ESCRT pathway is complicated by the secondary effect many ESCRT mutations have on Rim101 processing. These studies sought to separate iv Abstract The human opportunistic pathogen Candida albicans has a signal transduction pathway unique to fungi, called the Rim101 pathway. The Rim101 pathway regulates the proteolytic activation of the transcription factor, Rim101, the activation of which is required for growth at neutral-alkaline pH. Many genes regulated by Rim101 play a role in C. albicans virulence, including genes involved in filamentation, cell wall structure, adhesion, and nutrient acquisition. The Rim101 pathway consists of two complexes: a signaling complex at the plasma membrane and a processing complex inside the cell, and both of these complexes are required for Rim101 activation. Rim101 activation also requires members of a second pathway, the endosomal sorting complex required for transport (ESCRT) pathway. The ESCRT pathway is required to generate multivesicular bodies prior to vesicle fusion with the vacuole. The ESCRT pathway consists of several polyprotein complexes recruited sequentially to the endosomal membrane to generate an intraluminal vesicle. The role of the ESCRT pathway has not been well characterized in C. albicans, and study of the ESCRT pathway is complicated by the secondary effect many ESCRT mutations have on Rim101 processing. These studies sought to separate ESCRT function from Rim101 function, and to investigate ESCRT pathway function in C. albicans virulence. In these studies, ESCRT and Rim101 pathway separation is demonstrated (1) at distinct domains on a single protein known to be part of both pathways by using alanine scanning mutagenesis and (2) at ESCRT pathway complexes by using deletion mutagenesis. The ESCRT pathway is demonstrated here to play a wholly Rim101-independent role in C. albicans virulence.