Chromatin Modifications in Fungal Virulence and Pathogenesis

In all eukaryotes, the genetic information contained in DNA double strands are packaged around nucleosomes to form chromatin, which regulates numerous pathophysiological processes, ranging from normal development to cancer and infections by microbial pathogens. Chromatin is subject to dynamic post-translational modifications of its core constituents, histone proteins, which modulate chromatin condensation and determine how chromatin exerts its regulatory functions on target genes. The reversible acetylation of lysine residues by a so-called histone acetyltransferase (HAT) is often associated with transcriptional activation, whereas histone deacetylase (HDAC) function is normally associated with gene repression. In all eukaryotes, including the most prevalent dimorphic human fungal pathogen C. albicans, several HATs/HDACs exist that appear to influence morphogenesis. Candida spp are opportunistic pathogens that can cause severe chronic mucocutaneous, as well as invasive candidemia of staggering mortalities exceeding 40% when the host immune surveillance is malfunctioning. Invasive Candida spp are under constant pressure to cope and to evade the host immune surveillance. Chromatin appears to play a major role in fungal pathogenesis, since a changing histone modification status modulates morphogenesis, as well as antifungal drug susceptibility. This determines whether cells grow in unicellular or multicellular filamentous morphologies, both of which interact differently with the host immune cells or colonize distinct host niches. At present, clinically relevant antifungal drugs to cure Candida infections are mainly limited to azoles and to the fungicidal echinocandins, targeting ergosterol and cell wall biogenesis, respectively. However, the potential of specifically inhibiting HDAC/HAT activities as a novel therapeutic strategy is just beginning to be recognized, since filamentation and morphogenesis are considered key virulence traits of C. albicans. While most data on HAT/HDAC functions stem the non-pathogenic yeast S. cerevisiae, still little is known about mechanisms by which HATs/HDACs control virulence in pathogenic fungi. Hence, there is a need to better understand the mechanisms of dual layer regulation that connects transcriptional regulation with dynamic chromatin modifications in response to host immune response. In this FWF project, we will therefore decipher the mechanisms by which the paradigm fungal Hat1/Hat2 HAT, controls DNA repair, and we will answer the question how Hat1/Hat2-mediated chromatin remodelling at respective promoters or other genomic regions specifically controls antifungal drug resistance, virulence and stress response. Moreover, we will address the question how a prototype HDAC in C. albicans, the Set3/Hos2 complex Set3C, regulates target genes necessary for integrating stimuli from signalling pathways that control filamentation, morphogenesis, drug susceptibility and virulence. The project will take advantage of timely genome-wide approaches, including next generation sequencing such as RNA-Seq, Chip-Seq and extensive bioinformatics to identify novel fungal genetic networks whose transcriptional efficiency is subject to control by the histone modifiers Set3C and Hat1/Hat2. The phenotypic consequences of interfering with these regulatory events will be tested in appropriate homozygous C. albicans deletion strains lacking certain HDAC and HAT target genes to be generated by reverse genetics. Virulence properties of HAT/HDAC target genes will be studied ex vivo using primary innate immune cells such as macrophages and dendritic cells, as well as in vivo, using appropriate mouse models of fungal virulence.

The central hypothesis of this project was that histone modification by lysine acetylation and thus the chromatin state critically controls pathogenicity, drug resistance and stress response of Candida species, the major human fungal pathogens. Hence, the main aim was to decipher the mechanisms by which paradigm fungal histone acetyltransferases such as Hat1 control biological processes such as DNA repair, and how Hat1-mediated chromatin remodeling at target genes or other genomic regions control antifungal drug resistance, virulence and stress response. Moreover, we addressed unsolved questions how prototypic histone deacetylases (HDACs), including the Set3C complex and Rpd31 and Rpd3, regulate genes that integrate environmental stimuli from signalling and sensing pathways to control filamentation, morphogenesis, biofilm formation and virulence. We used genetic and next generation genome sequencing approaches such as RNA-seq and Chip-seq paired with bioinformatics to identify both the genome-wide target sets of both HAT/HDAC genes in Candida albicans. Furthermore, biochemical and molecular cell biology approaches, as well as the use of mouse models of virulence led to the discovery of novel mechanisms through which HATs/HDACs control fungal virulence and host-pathogen interactions. In brief, the major discoveries and outcomes of this project can be summarized as follows:We showed for the first time that the histone acetyl transferase Hat1 has multiple roles in controling efficient DNA repair, oxidative stress response, as well as antifungal drug resistance in the most frequent human fungal pathogen Candida albicansThe Set3C histone deacetylase complex controls fungal virulence by adjusting expression levels and activities of transcription factors required for driving morphogenetic changes such as filamentation and biofilm formation, as well as morphogenetic switching. The genetic ablation of Set3C and Hat1 attenuates fungal virulence, demonstrating that HDACs(HATs constitute novel antifungal drug target genes.We established a proof of principle demonstrating the suitablility of both HDACs and HATs as suitable targets for antifungal drug discovery.We showed that the Hat1 acts in concert with the histone chaperones HIR and CAF-1 to modulate antifungal drug susceptibility, as well as stress response and immune evasion. Finally, we showed that the HDACs Rpd3 / Rpd31 play opposing roles in morphogenetic switching in C. albicans by forming Rpd3- / Rpd31-specific regulatory complexes acting on the same regulator (a new collaborative effort of this project with a FWF-Lise Meitner Postdoc)Taken together, this project successfully validated and established that fungal histon modifiers represent potential targets for antifungal drug discovery, but also opened a new field in infectious disease research on human fungal pathogens related to role of chromatin in regulating cell-fate decisions and fungal virulence.