Objectives:
The adhesion of microbial pathogens to the host cells—a critical step for the host infection—depends on an arsenal of proteins, collectively called adhesins that are exposed on the pathogen surface and bind numerous host proteins. Besides typical adhesins, some pathogen proteins are loosely associated with the cell surface and identical with well known intracellular enzymes, involved in the crucial conserved metabolic routes. Secreted via yet unknown mechanism(s), these “moonlighting proteins” perform a new function of adhesins.
Candida glabrata is the second, after Candida albicans, most common fungal pathogen of humans that causes various infectious diseases ranging from superficial candidiasis such as oral thrush to life-threatening bloodstream infections. Unlike other species from the Candida genus, C. glabrata is haploid yeast unable to convert into invasive hyphal forms, but still possesses a wide repertoire of virulence attributes, including the presence of adhesive proteins at the fungal cell surface.
The present study aimed to identify C. glabrata surface-exposed proteins that can be classified as moonlighting proteins with a putative adhesin activity.

Methods:
Candida glabrata was cultured in various media, some of them simulating the conditions at the host niches, often infected by this pathogen. The fungal “surfaceome” was analyzed by a rapid method based on the “cell surface shaving” with trypsin, combined with the “shotgun” proteomic approach. Some of the identified yeast surface proteins that appeared identical with enzymes known to normally reside in the cytoplasm, were extracted from the yeast cell wall with 1,6-glucanase, followed by purification by ion-exchange chromatography and gel filtration. Interactions of these purified proteins with human extracellular matrix proteins—fibronectin and vitronectin—were quantitatively characterized using the surface plasmon resonance (SPR) measurements.

Results:
Under each of the conditions tested, a large number of various surface-exposed proteins were detected, including typical cell wall proteins such as the epithelial adhesin 6 (Epa6) and cell wall mannoprotein Cwp1, several heat shock proteins and a wide variety of moonlighting proteins. Two of the latter—enolase (Eno1) and glyceraldehyde-3-phosphate dehydrogenase (Tdh3)—belonged to the most abundant surface-exposed C. glabrata proteins after growth in all culture media used. After isolation and purification, these glycolytic enzymes were found to bind vitronectin and fibronectin, with the dissociation constants within a 10-8-10-7 M range, suggesting their adhesin function.
Several other moonlighting proteins were also identified at the cell surface of C. glabrata cultured under specific conditions. These included the enzymes that in the cytoplasm are involved in the glycolysis (triosephosphate isomerase, phosphoglycerate mutase, phosphoglucose isomerase, phosphoglycerate kinase and fructose 1,6-bisphosphate aldolase), fermentation (pyruvate decarboxylase and alcohol dehydrogenase) and the pentose phosphate pathway (transaldolase and 6-phosphogluconate dehydrogenase).

Conclusion:
The presence of numerous diverse moonlighting proteins at the surface of C. glabrata cells suggests their potential collective involvement in the pathogenesis of infections caused by these yeasts.
This work was supported in part by the National Science Centre of Poland (grant no. 2016/23/B/NZ6/00089 awarded to A.K.).

“Moonlighting” adhesins at the cell surface of Candida glabrata pathogenic yeasts