Rad6 and Bre1 ubiquitin ligase negatively regulate biofilm formation and virulence in Candida glabrata

Background

Candida glabrata is an opportunistic human fungal pathogen causing infections due to its innate antifungal drug resistance and ability to adhere to mucocutaneous surfaces. Epigenetic pathways may be important factors in the development of drug resistance. Our previous studies showed that deubiquitination of H2B, regulated by a module comprised of Ubp8, Sgf11, Sgf73, and Sus1, plays important roles in oxidative stress tolerance and biofilm formation of C. glabrata. However, the roles of the Rad6 and Bre1 ligase in regulating the ubiquitination of H2B in C. glabrata remain unclear.

Methods

We characterized the functions of Rad6 and Bre1 in C. glabrata by generating deletion mutants (rad6, bre1, and rad6 bre1). We analyzed biofilm formation, gene expression of key adhesins (EPA1, EPA6, EPA20) and protease (YPS4), antifungal drug susceptibility, stress responses, and virulence in a murine model of systemic candidiasis.

Results

Deletion of RAD6 and BRE1 resulted in enhanced biofilm formation, correlating with upregulation of key adhesin genes and the protease gene YPS4. The mutants showed distinct patterns of antifungal drug susceptibility: rad6 and rad6 bre1 mutants exhibited increased sensitivity to azoles, while bre1 mutant showed enhanced resistance to azoles in solid YPD agar plates but no significant difference in liquid RPMI medium. All mutants demonstrated decreased resistance to echinocandins and amphotericin B, associated with altered expression of ergosterol biosynthesis genes (ERG11) and glucan synthase genes (FKS1, FKS2). The mutants also displayed decreased resistance to oxidative and cell wall stresses despite elevated basal expression of antioxidant genes (SOD1, GPX2, CTA1). In a murine model of systemic candidiasis, both rad6 and bre1 mutants exhibited enhanced virulence compared to the wild type.

Conclusion

Rad6 and Bre1 in C. glabrata function as negative regulators of biofilm formation and adhesion, and their related-genes expression, while RAD6 deletion also suppresses macrophage ROS production and enhances fungal survival. The enhanced virulence observed in the rad6 and bre1 mutants is primarily attributed to these combined effects of increased biofilm formation, enhanced adhesion capability, and macrophage immune evasion.