Fungal KATs/KDACs: A New Highway to Better Antifungal Drugs?

According to the World Health Organization, infectious diseases stand out as the major cause of death worldwide. Although bacterial, viral, and parasitic infections appear to constitute the major threat, the clinical relevance of fungal infections has not been adequately recognized. In fact, invasive fungal infections constitute a biomedical problem of epic proportions, because a handful of human fungal pathogens claim an estimated 1.5 million lives per year [1]. Importantly, invasive fungal diseases represent leading causes of morbidity and mortality in immunocompromised individuals, particularly in patients with hematological malignancies, bonemarrow and organ transplant recipients, intensive care unit patients, preterm neonates, and patients with inborn or acquired immune deficiencies such as AIDS [2]. The vast majority of fungal infections are caused primarily by Candida albicans, Aspergillus fumigatus, and Cryptococcus spp. [2]. The overall mortality rate of 35%–40% for candidemia alone exceeds all gram-negative acute bacterial septicemia [3]. Importantly, pronounced inherent clinical antifungal drug resistance, especially in species like Candida glabrata [4], promotes a dramatic increase of infections [5, 6]. The unsolved challenge of getting fast, reliable, and accurate pathogen-specific clinical diagnosis of fungi has remained as another major impediment to successful and efficient antifungal therapy [7]. A mere four chemical entities (polyenes, azoles, echinocandins, and flucytosine) constitute the armory of clinically relevant drugs [1]. A few variant azoles and echinocandins received recent United States Food and Drug Administration (FDA) approval, but new chemical entities are either missing or mainly experimental in nature [8]. Of note, vaccination against fungal infections is currently unavailable and heavily debated, although recent clinical trials may hold new promises as well as challenges ahead [9–11]. Interestingly enough, compelling evidence indicates that chromatin tightly controls fungal virulence and/or pathogen fitness in the host. Nucleosome remodeling and assembly pathways impact the dynamic interplay with host immune surveillance, facilitate immune evasion, as well as drive antifungal drug resistance [12]. For example, several lysine acetyltransferases (KATs) and lysine deacetylases (KDACs) control fungal virulence [13]. This suggests that KATs/KDACs modifying both histones and non-histone targets could aid in antifungal drug discovery [13, 14]. Here, we provide a comprehensive overview of chromatin modifications in human fungal pathogens, particularly those altering virulence (Table 1, Fig 1). However, owing to space constraints, we will focus our discussion on KDACs/KATs in Candida spp. In addition, we discuss how the modulation of KATs/KDACs in Candida spp. could pave the way for novel therapeutic strategies to combat fungal infections [13].


Introduction
According to the World Health Organization, infectious diseases stand out as the major cause of death worldwide. Although bacterial, viral, and parasitic infections appear to constitute the major threat, the clinical relevance of fungal infections has not been adequately recognized. In fact, invasive fungal infections constitute a biomedical problem of epic proportions, because a handful of human fungal pathogens claim an estimated 1.5 million lives per year [1]. Importantly, invasive fungal diseases represent leading causes of morbidity and mortality in immunocompromised individuals, particularly in patients with hematological malignancies, bonemarrow and organ transplant recipients, intensive care unit patients, preterm neonates, and patients with inborn or acquired immune deficiencies such as AIDS [2].
The vast majority of fungal infections are caused primarily by Candida albicans, Aspergillus fumigatus, and Cryptococcus spp. [2]. The overall mortality rate of 35%-40% for candidemia alone exceeds all gram-negative acute bacterial septicemia [3]. Importantly, pronounced inherent clinical antifungal drug resistance, especially in species like Candida glabrata [4], promotes a dramatic increase of infections [5,6]. The unsolved challenge of getting fast, reliable, and accurate pathogen-specific clinical diagnosis of fungi has remained as another major impediment to successful and efficient antifungal therapy [7].
A mere four chemical entities (polyenes, azoles, echinocandins, and flucytosine) constitute the armory of clinically relevant drugs [1]. A few variant azoles and echinocandins received recent United States Food and Drug Administration (FDA) approval, but new chemical entities are either missing or mainly experimental in nature [8]. Of note, vaccination against fungal infections is currently unavailable and heavily debated, although recent clinical trials may hold new promises as well as challenges ahead [9][10][11]. Interestingly enough, compelling evidence indicates that chromatin tightly controls fungal virulence and/or pathogen fitness in the host. Nucleosome remodeling and assembly pathways impact the dynamic interplay with host immune surveillance, facilitate immune evasion, as well as drive antifungal drug resistance [12]. For example, several lysine acetyltransferases (KATs) and lysine deacetylases (KDACs) control fungal virulence [13]. This suggests that KATs/KDACs modifying both histones and non-histone targets could aid in antifungal drug discovery [13,14]. Here, we provide a comprehensive overview of chromatin modifications in human fungal pathogens, particularly those altering virulence ( Table 1, Fig 1). However, owing to space constraints, we will focus our discussion on KDACs/KATs in Candida spp. In addition, we discuss how the modulation of KATs/KDACs in Candida spp. could pave the way for novel therapeutic strategies to combat fungal infections [13].

Fig 1. Histone modification by lysine acetylation through writers (KATs) and erasers (KDACs).
Much of the mechanistic knowledge about the role of chromatin modifications in gene expression regulation comes from the nonpathogenic baker's yeast (for excellent recent reviews, see [65][66][67]). Although the precise mechanisms of the interplay between writers, readers, and erasers remain ill-defined in many cases, it is fair to speculate that histone modifiers may play pivotal roles in the adaption of fungal pathogens to host immune defense.

Non-histone Lysine Acetylation in Host-Pathogen Interactions
Interestingly, lysine acetylation of non-histone target proteins is increasingly recognized as a means to regulate cellular processes. Fungal acetylome data are just emerging [20], and it will be exciting to identify virulence modifiers from these genome-wide datasets. Interestingly, acetylation appears abundant in mitochondria [42]. However, it is not clear whether acetylation of mitochondrial proteins takes place in the cytosol before their mitochondrial import or inside mitochondria? How the acetylation status influences mitochondrial function and nuclear crosstalk or even two-component signaling pathways that regulate fungal virulence [43] remains open. Notably, mitochondria and intrinsic signaling pathways play key roles in fungal pathogenesis [43, 44], but a link of acetylation, mitochondria, and virulence remains to be discovered.
Notably, chromatin-related gene regulation contributes to Candida spp. survival in the human host [45] or even inside innate phagocytes. For example, during invasion of dendritic cells by C. albicans, both host and fungal chromatin experience complex modifications that regulate the magnitude of the inflammatory immune response but also the susceptibility of pathogens to immune defense [46]. Interestingly, prominent bacterial pathogens also exploit histone modifications to promote their intracellular replication or to evade host immune defense [47]. For example, Shigella flexneri induces its own uptake by modifying the host actin cytoskeleton [48]. Borrelia burgdorferi [49] and Mycobacterium tuberculosis [50] employ similar strategies to aid their persistence in human host cells.

Using KATs/KDACs Modulators as Novel Antifungal Drugs
A limited arsenal of antifungals inhibit pathogen growth through fungistatic and/or fungicidal mechanisms [8, 51] by interfering with plasma membrane function (amphotericin B), cell wall glucan biogenesis (echinocandins), DNA synthesis (flucytosine), or ergosterol metabolism (azoles). Antifungal therapies are also limited because of toxicity, increasing drug resistance, as well as adverse drug-drug interactions. The former "gold standard" drug amphotericin B invariably causes severe toxicity in patients, limiting its use and effectiveness. Triazoles remain as preferred drugs because of their excellent toxicity profiles, moderate costs, and ease of oral administration [8]. However, the majority of triazoles are fungistatic rather than fungicidal, promoting the emergence of resistance [6]. Furthermore, some non-C. albicans species, most notably C. glabrata, display marked intrinsic resistance to triazoles and in some cases even crossresistance to echinocandins [5]. Nonetheless, the fungicidal echinocandins have been outstanding drugs, but their use is also limited due to poor oral bioavailability, its ineffectiveness against C. neoformans or invasive aspergillosis [6], as well as high cost. Furthermore, recent reports indicate dramatically increasing prevalence of echinocandin-resistant Candida isolates [5,52]. This is a serious matter of concern, especially because these species are increasingly recovered among bloodstream clinical isolates [5]. Remarkably, the incidence of echinocandin-resistant C. glabrata at certain medical centers in the US increased from 2%-3% in 2001 to more than 13% in 2010 [52]. Furthermore, the identification of multidrug-resistant (azoles and echinocandins) C. glabrata isolates [5] has set off the alarm bells, because treatment options for patients infected with such strains have become limited. Thus, the efficient antifungal therapy is hampered by a deadly combination of limited antifungal drug entities, increasing occurrence of bloodstream fungal infections, and emerging resistance, underscoring the critical need for discovering new types of antifungal drugs. Of

Conclusions and Outlook
Fungal infections are associated with astronomical annual Medicare costs, exceeding billions in Europe or the US, thus causing enormous economic burdens to already strained healthcare systems. Hence, current efforts in drug discovery are obviously lagging behind the need for improved antifungals. Unfortunately, the fundamental roles of KATs/KDACs in fungal pathophysiology, gene regulation, and/or adaptive genetic/epigenetic changes have not yet attracted enough attention in antifungal drug discovery. Moreover, among other roadblocks on the antifungal innovation highway, the academic setting has been struggling with insufficient funding from public and private bodies, thus further impairing the translation from basic science to application. For instance, grant support for fungal pathogen research falls several orders of magnitude below the levels of prominent bacterial or parasitic pathogens (http://www.gaffi. org/ and https://gfinder.policycures.org/PublicSearchTool/). Importantly, major pharma companies no longer entertain large-scale targeted antifungal discovery, partly because of high costs, limited number of validated targets, and high propensity of adverse toxicity owing to the eukaryotic nature of fungal pathogens. Importantly, the long-standing hesitation to exploit nonessential fungal genes as antifungal targets needs a careful reevaluation. Actually, a genetic argument predicts that essential genes may in fact even be poorer targets due to risks of drug resistance development, particularly in prophylactic settings or when overused. In fact, any gene affecting fungal fitness or adaptive changes in the host, irrespective of whether a fungal or a host gene could serve as a proper antifungal target [59]. Of note, all antifungal drugs target fungal growth in the host. However, there is increasing and compelling evidence that modulating the amplitude and magnitude of the host inflammatory immune response can be beneficial for the outcome of invasive fungal diseases [60][61][62]. Thus, chromatin-mediated adaptive changes during fungal pathogen host interplay opens new windows of opportunities and may hold great promises for future antifungal drug discovery.
Targeting fungal KATs/KDACs as a therapeutic strategy could also offer decisive advantages. First, fungal KATs/KDACs are structurally less well conserved, and some of the modifications are exclusively found in fungi, minimizing the risk of immune toxicity (Table 1, Fig 1). Second, the expansion of genome-scale genetic technologies, especially CRISPR/Cas9 approaches [63], makes it feasible to use dual-systems biology approaches to decipher the dynamic underlying host-pathogen relations [64] but also to better understand molecular mechanisms of KDAC/KAT functions under host immune surveillance. Of course, potential risks exist as well, because drug-mediated KDAC/KAT modulation may also lead to hyper-virulence phenotypes. For instance, blocking fungal KATs/KDACs can debilitate drug resistance but could otherwise lead to hypervirulence, owing to fitness gain in vivo due to inefficient recognition by immune surveillance [38]. Of note, virulence data on the role of other important chromatin or histone regulators mediating reversible phosphorylation and/or methylation of histones are unavailable for most fungal pathogens (Table 1). Thus, it is tempting to speculate that these genes will most likely expand the potential pool of suitable antifungal drug targets. Finally, another underexplored area is the role of non-chromatin, non-histome proteins modified by KDACs/KATs or other chromatin modifiers (Table 1). Interestingly, recent evidence indicates that non-histone targets of KATs may also play fundamental roles in fungal virulence and drug resistance [14], opening yet another new window of opportunity in antifungal drug discovery.