Integrated post-genomic cell wall analysis reveals floating biofilm formation associated with high expression of flocculins in the pathogenCandida krusei
Authors: Alvarado María , Gómez-Navajas Jesús Alberto , Blázquez-Muñoz María Teresa , Gómez-Molero Emilia , Berbegal Carmen , Eraso Elena , Kramer Gertjan , De Groot Piet W.J. 
Published: January 2023

ABSTRACTThe pathogenic yeastCandida kruseiis more distantly related toCandida albicansthan clinically relevant CTG-cladeCandidaspecies. 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 inC. krusei. Our comparative genomic studies and experimental data indicate that the general structure of the cell wall inC. kruseiis similar toSaccharomyces cerevisiaeandC. albicansand is comprised of β-1,3-glucan, β-1,6-glucan, chitin, and mannoproteins. However, some pronounced differences withC. albicanswalls were observed, for instance, higher mannan and protein levels and altered protein mannosylation patterns. Further, despite absence of proteins with high sequence similarity toCandidaadhesins, protein structure modeling identified eleven proteins related to flocculins/adhesins inS. cerevisiaeorC. albicans. To obtain a proteomic comparison of biofilm and planktonic cells,C. kruseicells were grown to exponential phase and in static 24-h cultures. Interestingly, the 24-h static cultures ofC. kruseiyielded 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 inC. kruseiincluding its cell wall proteome, and paves the way for further investigations on the importance of flor formation and flocculins in the pathogenesis ofC. krusei.AUTHOR SUMMARYThe yeastCandida kruseiis 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 inC. kruseiand identify important architectural differences compared toC. albicanscell walls. Cell walls ofC. kruseicontain 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 otherCandidaspecies, 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 pathogenC. krusei.