Abstract
A large proportion of Candida albicans cell surface proteins are decorated post-translationally by glycosylation. Indeed N-glycosylation is critical for cell wall biogenesis in this major fungal pathogen and for its interactions with host cells. A detailed understanding of N-glycosylation will yield deeper insights into host-pathogen interactions. However, the analysis of N-glycosylation is extremely challenging because of the complexity and heterogeneity of these structures. Therefore, in an attempt to reduce this complexity and facilitate the analysis of N-glycosylation, we have developed new synthetic C. albicans reporters that carry a single N-linked glycosylation site derived from Saccharomyces cerevisiae Suc2. These glycosylation reporters, which carry C.albicans Hex1 or Sap2 signal sequences plus carboxy-terminal FLAG3 and His6 tags, were expressed in C.albicans from the ACT1 promoter. The reporter proteins were successfully secreted and hyperglycosylated by C.albicans cells, and their outer chain glycosylation was dependent on Och1 and Pmr1, which are required for N-mannan synthesis, but not on Mnt1 and Mnt2 which are only required for O-mannosylation. These reporters are useful tools for the experimental dissection of N-glycosylation and other related processes in C.albicans, such as secretion.
Highlights
Candida albicans is an opportunistic pathogen that inhabits the oral cavity, gastrointestinal and urogenital tracts of many healthy individuals
Our goal was to develop a new reporter to facilitate the analysis of N-glycosylation in C. albicans
Cell surface N-glycosylation is essential for pathogenicity (Bates et al, 2006, 2005; Mora-Montes et al, 2007, 2009; Munro et al, 2005) and contributes significantly to the recognition of C. albicans cells by host immunological defences (Mora-Montes et al, 2010, 2007; Netea et al, 2008; Torosantucci et al, 1990; Wang et al, 1998)
Summary
Candida albicans is an opportunistic pathogen that inhabits the oral cavity, gastrointestinal and urogenital tracts of many healthy individuals This fungus is a frequent cause of mucosal infections (e.g. oral and vaginal thrush), and in severely immunocompromised patients it can cause life-threatening systemic infections of the bloodstream and internal organs (Calderon et al, 2003; Kabir and Hussain, 2009; Odds et al, 1988). The C. albicans cell commits significant resources to the synthesis of its cell wall, which comprises approximately 30% of its dry weight.
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