Histone H3 lysine 56 acetylation and deacetylation pathways as targets for novel antifungal therapies in Candida albicans

Candida albicans is a major opportunistic fungal pathogen that is harmless to healthy individuals but causes life-threatening systemic infections in individuals whose immune system is compromised. Invasive infection caused by C. albicans in immunocompromised patients leads to a significant mortality. Current therapeutic options are limited by the emergence of drug resistant strains or adverse side effects. Hence, there is an urgent need to find novel fungal-specific drug targets to overcome the limitations of existing drugs. This thesis consists of two research articles.  The first article studies histone H3 lysine 56 (H3K56) acetylation, an abundant post-translational modification that plays an important role in the survival of DNA damage and promotes virulence in C. albicans. H3K56 is acetylated by the histone acetyltransferase Rtt109 (HAT), and deacetylated by Hst3 (HDAC). Both of these enzymes have fungal-specific properties and are promising antifungal targets. Previous studies in Saccharomyces cerevisiae have shown that the histone chaperones ScAsf1 and ScVps75 control ScRtt109 activity and substrate specificity. ScRtt109 and ScAsf1 act in a concerted fashion to acetylate H3K56, whereas the ScRtt109-Vps75 complex acetylates several lysine residues in the N- terminal tail of histone H3. In C. albicans, the roles of Rtt109 and H3K56ac in DNA damage survival are conserved. However, the functional and substrate specificities of CaRtt109 when it co-operates with the histone chaperones CaAsf1 or CaVps75 have not been studied. In this study, we used genetic, biochemical and mass spectrometry methods to identify and characterize the roles of Asf1 or Vps75 when they function with Rtt109 in C. albicans. Unexpectedly, C. albicans cells lacking Vps75 exhibit lower levels of H3K56ac than wild-type cells and, consistent with this, are more resistant than wild-type cells to nicotinamide, an inhibitor of the sirtuin Hst3 that kills C. albicans cells by causing hyperacetylation of H3K56 and irreparable DNA damage. We provide evidence that the CaRtt109-Vps75 holoenzyme can directly acetylate H3K56 in vitro. We also show that the unexpectedly low levels of H3K56ac found in cells lacking Vps75 can be attributed, at least in part, to instability of Rtt109 in the absence of its partner Vps75. Our studies also show that, apart from assisting Rtt109 to promote H3K56 acetylation, Asf1 is essential for viability in C. albicans where it plays a role in the maintenance of chromosome integrity.  Hst3 belongs to the class of NAD+-dependant deacetylases called sirtuins, which can be inhibited by the reaction product nicotinamide (NAM). Hst3 inhibition by NAM leads to hyper-acetylation of H3K56, catastrophic DNA   damage and lethality. In an effort to enhance the clinical potential of NAM, we studied its metabolic fate in C. albicans. NAM is a precursor of a canonical salvage pathway that generates NAD+. We hypothesized that cells lacking the Pnc1 nicotinamidase, which converts NAM into nicotinic acid (NA), would be more sensitive to NAM than wild-type cells. Despite the fact that Pnc1 was the sole nicotinamidase in C. albicans, NAM concentrations and cytotoxicity were, at best, modestly higher in pnc1 mutants than in wild-type cells. These results suggest the existence of a Pnc1-independent mechanism that limits the accumulation of NAM and, therefore, restricts its ability to inhibit Hst3. To identify the underlying mechanism, we generated several mutants lacking enzymes involved in the biosynthesis of NAD+ and performed experiments with heavy isotope-labeled NAM and mass spectrometry. Our results demonstrate the existence of a non- canonical, Pnc1-independent conversion of NAM into NAD+ that has never been reported in fungi. We show that this conversion occurs, at least in part, through reversal of sirtuin activity and provide the evidence of sirtuins reversal in vivo. However, we were able to demonstrate that this pathway cannot serve as the only source of NAD+ and sustain viability at physiological NAM concentrations.

Keyphrases: Candida albicans, Acétylation de l'histone H3 lysine 56 (H3K56ac), Rtt109, Nicotinamide, Histone H3 lysine 56 acetylation (H3K56ac)