Type

Text

Type

Thesis

Advisor

Dean, Neta | Konopka, James

Date

2017-12-01

Keywords

Biochemistry | Candida albicans | Cell wall | Cytology | CRISPR/Cas9 | Molecular biology | Glycosylation | Mannosyltransferase | VAN1

Department

Department of Biochemistry and Structural Biology

Language

en_US

Source

This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree.

Identifier

http://hdl.handle.net/11401/78226

Publisher

The Graduate School, Stony Brook University: Stony Brook, NY.

Format

application/pdf

Abstract

The outer layer of the cell wall of Candida albicans contains glycoproteins made up of mannose-rich glycans that are critical for host-fungal interactions, including adhesion, virulence, and drug resistance. Asparagine (N)-linked glycans are added to a core oligosaccharide by mannosyltransferases in the Golgi to create the long outer chain α1,6-linked mannose “backbone” that branches with α1,2-, α1,3-, and β1,2- linked mannoses. In Saccharomyces cerevisiae, the mannosyltransferases responsible for these modifications have been well studied and characterized. Orthologs of these mannosyltransferases have been identified in C.albicans and in this study we examine VAN1, a mannosyltransferase that acts very early in elongation of the backbone. To determine the role that VAN1 plays in protein glycosylation, we used CRISPR/Cas9 to delete VAN1 in C. albicans . Phenotypic analyses demonstrated this mutation caused severe morphological, growth, and cell wall phenotypes. van1Δ mutants are defective in hyphal formation, produce increased levels of cell wall chitin, and have abnormal size and shape. Analyses of glycoprotein reporters specific for N-linked glycosylation showed loss of VAN1 resulted in severe truncation of N-linked glycans in vivo. Together, these results demonstrate the importance of VAN1 in N-glycan outer chain synthesis and the integrity of the cell wall. | 50 pages

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