Integrated post-genomic cell wall analysis reveals floating biofilm formation associated with high expression of flocculins in the pathogen Candida krusei.

Abstract

The pathogenic yeast Candida krusei is more distantly related to Candida albicans than clinically relevant CTG-clade Candida species. Its cell wall, a dynamic organelle that is the first point of interaction between pathogen and host, is relatively understudied, and its wall proteome remains unidentified to date. Here, we present an integrated study of the cell wall in C. krusei. Our comparative genomic studies and experimental data indicate that the general structure of the cell wall in C. krusei is similar to Saccharomyces cerevisiae and C. albicans and is comprised of {beta}-1,3-glucan, {beta}-1,6-glucan, chitin, and mannoproteins. However, some pronounced differences with C. albicans walls were observed, for instance, higher mannan and protein levels and altered protein mannosylation patterns. Further, despite absence of proteins with high sequence similarity to Candida adhesins, protein structure modeling identified eleven proteins related to flocculins/adhesins in S. cerevisiae or C. albicans. To obtain a proteomic comparison of biofilm and planktonic cells, C. krusei cells were grown to exponential phase and in static 24-h cultures. Interestingly, the 24-h static cultures of C. krusei yielded formation of floating biofilm (flor) rather than adherence to polystyrene at the bottom. The proteomic analysis of both conditions identified a total of 32 cell wall proteins. In line with a possible role in flor formation, increased abundance of flocculins, in particular Flo110, was observed in the floating biofilm compared to exponential cells. This study is the first to provide a detailed description of the cell wall in C. krusei including its cell wall proteome, and paves the way for further investigations on the importance of flor formation and flocculins in the pathogenesis of C. krusei. AUTHOR SUMMARYThe yeast Candida krusei is among the five most prevalent causal agents of candidiasis but its mechanisms underlying pathogenicity have been scarcely studied. This is also true for its cell wall structure, an essential organelle that governs primary host-pathogen interactions and host immune responses. Solid knowledge about cell wall synthesis and dynamics is crucial for the development of novel antifungal strategies against this pathogenic yeast. Here, through a combination of comparative genomics, protein structure modeling, and biochemical and proteomic analysis of purified walls, we present a detailed study of the cell wall composition in C. krusei and identify important architectural differences compared to C. albicans cell walls. Cell walls of C. krusei contain higher mannan and protein levels with altered mannan branching patterns, governed by expansions and reductions in gene families encoding mannosyltransferases. We also show that, in contrast to other Candida species, static cultures produce floating biofilms. Comparative wall proteomic studies of these biofilms show increased abundance of flocculins and hydrolytic enzymes, protein classes implicated in biofilm formation and primary host-pathogen interactions leading to tissue colonization. In conclusion, our study uncovers important keys towards a better molecular understanding of the virulence mechanisms of the important pathogen C. krusei.