Candida parapsilosis is an important non-albicans species in hospital settings and is one of the leading causes of invasive candidal disease, with neonates, immuno-suppresed, and patients in intensive care units at highest risk. C.parapsilosis is less susceptible to mainline antifungal treatments such as the echinocandins and azoles. In previous studies, Phr1, a β-(1,3)-glucan transglycosylase (Cazy GH72) from C. parapsilosis, was identified as an important virulence factor. Changes in expression levels of Phr1 result in distinct changes to the cell surface proteome that impact the ability of C. parapsilosis to adhere to host tissues. This finding makes Phr1 a potentially attractive anti-virulence target and is conserved in Candida sp. To evaluate potential inhibitors of Phr1, a viable, sensitive, and rapid assay for measuring transglycosylase activity is required. We prepared and evaluated sulforhodamine (SR) labeled laminaroligosaccharides (Lam) as potential substrates for Phr1. Thin-layer chromatography coupled with compact mass spectrometry was used to assess Phr1 ability to hydrolyze Lam-SR oligosacchrides or utilize them as acceptors in transglycosylation reactions. Assays with recombinant C.parapsilosis Phr1 demonstrated it is capable of hydrolyzing sulforhodamine-laminarihexaose (Lam6-SR) to give Lam3-SR. In the presence of β-(1,3)-glucan and Lam6-SR, Phr1 catalyzed transglycosylation products were observed. Mass spectrometry analysis of TLC spots of soluble transglycosylation products identified oligosaccharides up to Lam11-SR. Preliminary results demonstrate that Lam-SR oligosaccharides are accepted by Phr1 and can be used to monitor hydrolysis or transglycosylation reactions.

Development of a fluorescent based assay for characterization of Candida sp. β-(1,3)-glucan transglycosylase Phr1

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ACS Fall 2022

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