Patrick Reijnst:
Functional analysis of Candida albicans genes encoding SH3-domain containing proteins

Date: 15-07-2010    Supervisor: Steen Holmberg

Actin dependent endocytosis in fungi is an essential and well studied process where a set of 20-30 highly conserved proteins coordinate rapid remodeling of the plasma membrane to internalize extra-cellular material. Studies in Saccharomyces cerevisiae have shown that many of the proteins involved in endocytosis bear SH3 domains. The human genome codes roughly 300 SH3 domains while fungal genomes generally code between 25-30 domains. The role of SH3 domains is not fully understood but they are thought to function as protein-protein interaction domains. The dimorphic fungus Candida albicans is a model organism and one of the major fungal human pathogens with increasing occurrence in immune-compromised patients.

An important virulence factor of C. albicans is the ability to switch between different growth forms, which in turn is affected by endocytosis, membrane traffic and transport of vesicles. Thus, the involvement of SH3 domains in endocytosis plays a potentially important role in the virulence of C. albicans. Endocytosis in S. cerevisiae may occur at distinct locations marked by a protein complex termed eisosomes, rather than appearing at random locations. Eisosomes have so far only been described in S. cerevisiae. Due to its dimorphic nature, the involvement of eisosomes in endocytosis makes them an attractive target to study in C. albicans. The aim of this project was to elucidate the role of 12 previously uncharacterized genes coding SH3-domain proteins in C. albicans and to expand the knowledge of eisosomes from S. cerevisiae to investigate their role in C. albicans, especially during filamentous growth.

Deletion of both alleles of BBC1, BUD14, FUS1, HSE1, PIN3, RVS167-2, and SHO1 in the diploid C. albicans reference strain did not affect the morphogenesis and the strains behaved wild type-like during all growth conditions. Overexpression of the SH3 domains from the corresponding genes did also not result in altered cell morphologies.

Deletion of CYK3 was not possible, suggesting it is an essential gene. Promoter shut-down experiments using a strain with CYK3 regulated by the inducible MET3 promoter showed severe cytokinesis defects and abnormal chitin localization when grown under repressive conditions. This is supported by the localization of Cyk3 at the mother-bud neck during cell division.

Deletion of SLA1 and RVS167 resulted in an altered actin cytoskeleton comparable with deletion mutants of the corresponding orthologs in S. cerevisiae. The sla1 and rvs167 null mutants exhibit slower filamentous growth which is thought to be a result of endocytosis defects. The three SH3 domains in Sla1 were found to be essential for the function of the protein, especially SH3 domain #1 and #2. This is different from S. cerevisiae where it is instead SH3 domain #3 that is important for Sla1 function.

Deletion of BOI2 and NBP2 resulted in failure to fuse vesicles and forming a large vacuole during filamentous growth. This is a novel function that has not been described in other fungi. The filamentous growth of nbp2 mutants was affected but boi2 mutants have a wild type phenotype despite lacking a large vacuole. This indicates that fragmented vacuoles per se are not sufficient to abolish or even affect hyphal growth.

Two major components of eisosomes are Pil1 and Lsp1. The C. albicans homologs of these genes were tagged with different fluorescent proteins and localization studies showed complete colocalization of these two proteins but surprisingly showed no co-localization with several endocytosis markers. Thus, contradictory to what has previously been described in S. cerevisiae, eisosomes may not represent sites of actin-mediated endocytosis. Deletion of PIL1 could not be achieved which suggests that PIL1 is an essential gene. This opens a new view on eisosomes whose cellular function therefore needs to be investigated in more detail. The results of the work presented in this Ph. D. thesis contribute to the general understanding of how endocytosis is regulated in C. albicans, specifically with regard to the effect of SH3 domains and eisosomes. The yeast-hyphal switch in C. albicans is a major factor in its pathogenicity and this work describes several new factors involved in this process.