Protein acetylation is a conserved evolutionary modification that occurs in eukaryotic and prokaryotic proteins and was first discovered in histones (Phillips, 1963; Allfrey et al., 1964). Reversible protein acetylation was studied in the context of the histones until the late 1990s. Recent advancements in high-resolution mass spectrometry and high-affinity purification technology for acetylated lysine peptides revealed that protein acetylation/deacetylation is not restricted to histones, which resulted in detailed studies of the acetylated proteome and its function (Schilling et al., 2019).

Previous studies and database searches revealed that protein acetylation is widespread in fungi. Zhou et al. detected 477 acetylated proteins (5.28%) among all 9,038 proteins of Candida albicans, which was the first study on acetylome in human pathogenic fungi, providing an important initiating point for further study of the functional analysis of acetylated proteins in such fungal pathogens (Zhou X. et al., 2016). The comparative analysis of fungal acetylomes plays an important role in determining the essential role of acetylation in the virulence of human fungal pathogens (Li et al., 2019). Significant differences in the number and sites of acetylated proteins were found according to the stage of human fungal pathogen growth. For example, 2,335 proteins in the mycelium growing stage were identified to be acetylated in Trichophyton rubrum, which was >10 times higher than that in the conidia stage, and may be explained by conidia being in a quiescent state with low metabolic activity (Xu et al., 2018). Further evidence of protein acetylation in human fungal pathogens was observed in Histoplasma capsulatum, Cryptococcus neoformans, and Aspergillus fumigatus (Xie et al., 2016; Brandão et al., 2018; Lin et al., 2020).

In plant pathogenic fungi, Yang et al. identified 1,313 high-confidence acetylation sites in 727 acetylated proteins in Aspergillus flavus, while 577 acetylated sites were reported in 364 different proteins in Fusarium graminearum (Zhou S. et al., 2016; Yang et al., 2019). Several published studies have described the acetylome of different fungal species, including plant pathogenic fungi Phytophthora sojae, Botrytis cinerea, and Magnaporthe oryzae; fungal insect pathogens, such as Beauveria bassiana and Metarhizium anisopliae; and nonpathogenic fungi species, such as Saccharomyces cerevisiae and Yarrowia lipolytica; which are considered as important resources to explore the physiological role of this modification in eukaryotes (Henriksen et al., 2012; Mukherjee et al., 2012; Li et al., 2016; Lv et al., 2016; Wang et al., 2017; Cai et al., 2018c; Liang et al., 2018).

Interestingly, most of the identified acetylated proteins were involved in the regulation of glucose, lipid, and amino acid metabolism (Wang et al., 2017). Important findings regarding the control of metabolism via protein acetylation were reported in prokaryotes (Wang et al., 2010). A large number of metabolic enzymes are also acetylated in S. cerevisiae, which is consistent with the enzymes that regulate central metabolism through reversible acetylation, ensuring that cells respond to environmental changes by rapidly sensing the cellular energy state and flexibly changing rates or direction (Wang et al., 2010; Henriksen et al., 2012). This result can be explained by the central role of acetyl-CoA in intermediary metabolism because acetyl-CoA acts as the acetyl-donor for both enzymatic and non-enzymatic acetylation (Trefely et al., 2019). In other words, the dynamic interplay between cellular metabolism and acetylation plays a key role in epigenetics; however, this is not the focus of this review.

In summary, protein acetylation is widely distributed in fungi. Aside from modifying many key cellular processes, such as enzymatic activity, signal transduction, cell division, and metabolism, it also controls morphological transformation, biofilm formation, acetic acid stress tolerance, and other processes, thereby affecting the entire fungal life cycle (Kim et al., 2015; Cheng et al., 2016; Narita et al., 2019; Lin et al., 2020).

Generally, pathogens are subjected to various environmental challenges, such as temperature variations, an acidic pH, and oxidative stress. Reversible acetylation has emerged as one of the processes critical to maintaining cellular homeostasis and shaping responses to environmental stimuli (O’Meara et al., 2010; Chang et al., 2015; Wang J.-J. et al., 2018). In C. neoformans, the loss of acetylation gene gcn5 caused a reduction in toxicity in a murine intranasal infection model and growth defects at high temperatures (O’Meara et al., 2010). In B. bassiana, the deletion of gcn5 led to a 97% reduction in the conidiation capacity as well as severe defects in the growth of fungal colonies and conidial thermotolerance (Cai et al., 2018b). Moreover, Mst2, which can specifically acetylate histone H3K14 through cooperation with Gcn5 to regulate global acetylation events in B. bassiana, was found to play an important role in sustaining multiple stress tolerances such as osmotic and oxidative stress tolerance, cell wall perturbing stress tolerance, thermotolerance, and UV-B resistance (Wang J.-J. et al., 2018). Furthermore, the Δgcn5 mutants of S. cerevisiae and Schizosaccharomyces pombe showed defects in the cellular response to many stressors, including elevated temperatures, high salt concentrations, and nutrient deprivation (Chang et al., 2015).

The absence of acetyltransferase Rtt109 in S. cerevisiae not only activated the transcription of stress-responsive genes but also improved the resistance to oxidative stress, which ultimately contributed to the improvement in acetic acid tolerance (Cheng et al., 2016). The KDAC sirtuin 2, played a role in starvation stress resistance in yeasts; the deacetylase gene rpd3 was also considered essential for starvation stress resistance (Fulco et al., 2003; Nakajima et al., 2016). A previous study on B. bassiana suggested that Δrpd3 significantly reduced the conidial tolerance to wet-heat stress at 45°C but increased the conidial resistance to UV-B irradiation, and the fungal virulence was greatly attenuated in the absence of rpd3 (Cai et al., 2018c). Studies have found that the downregulation of RPD3-type deacetylase RpdA leads to avirulence of A. fumigatus in a murine model for pulmonary aspergillosis (Bauer et al., 2019). In addition, KDACs also play a decisive role as virulence factors in the pathogenic fungus Cochliobolus carbonum and B. bassiana (Baidyaroy et al., 2001; Zhang et al., 2020). Although the mechanism of virulence attenuation and adverse environmental tolerance remains unclear, previous evidence suggests that acetylation/deacetylation can control the virulence level of pathogens by regulating their stress response.

Hyphae have a strong ability to adhere and invade the host, making it easy to maintain their colonization and escape attacks from the host immune system, probably through the release of cell type-specific virulence factors, such as adhesins (e.g., Hwp1, Als3, Als10, Fav2, and Pga55), tissue-degrading enzymes (e.g., Sap4, Sap5, and Sap6), and antioxidant defense proteins (e.g., Sod5) (Sudbery, 2011; Noble et al., 2017). Acetylation and deacetylation play critical regulatory roles in regulating the initiation and maintenance of hyphal development (Garnaud et al., 2016; Kong et al., 2018; Lee et al., 2019). Tribus et al. (2010) found that the repression of the promoter of rpdA knockdown strains resulted in distorted and hyperbranched hyphae and a tremendous loss of radial growth of fungal colonies. MoHOS2-mediated histone deacetylation is important for the development of M. oryzae. In the absence of this mechanism, M. oryzae exhibits defects in hyphae formation, thereby impairing its growth ability inside the host plant (Lee et al., 2019). In B. bassiana, the hyphal cells of Δhos2 mutants are significantly longer than those of the wild type strains, which was concurrent with its inability to develop intact nuclei in hyphal cells (Cai et al., 2018a). The hyphal growth defects of four acetyltransferase mutants of F. graminearum, namely, ΔFgGCN5, ΔFgRTT109, ΔFgSAS2, and ΔFgSAS3 mutants in solid medium, have also been reported (Kong et al., 2018).

In C. albicans, deacetylase Rpd31 and Set3C (Set3/Hos2 HDAC complex) are crucial repressors of the yeast-to-hyphae transition in C. albicans (Hnisz et al., 2010; Garnaud et al., 2016). The acetyltransferase activity of nucleosome acetyltransferase of H4 (NuA4) and the deacetylase activity of Hda1 have also been reported as essential for hyphal initiation and maintenance (Wang X. et al., 2018). Furthermore, NuA4 dynamically regulates hyphal growth by merging and separating with the SWR1 complex, which was mediated by the acetylation of Eaf1 at K173 (lysine residue 173) (Wang X. et al., 2018). Gcn5 was also required for the invasive and filamentous growth of C. albicans, while gcn5 mutant impaired the hyphal elongation in sensing serum and attenuated the C. albicans virulence in a mouse systemic infection model (Chang et al., 2015). Further evidence of the association between acetylation and hyphal growth was found in the acetyltransferase Esa1, which belongs to the MYST (Moz, YBF2, Sas2p, and Tip) family. Wang et al. found that Esa1 was not important for the general growth of C. albicans but was important for its filamentous growth and that Esa1 deletion could prevent filament formation under all hyphal induction conditions (Wang et al., 2013). Overall, hyphal initiation, development, and maintenance are complex processes regulated by acetylation and deacetylation in both filamentous fungi and budding yeasts.

One of the key virulence traits of fungi is morphological plasticity (Li et al., 2017). Although some human fungal pathogens mainly exist in the form of budding yeast cells (such as C. neoformans) or filamentous hyphal structures (such as Aspergillus), C. albicans alternates between these and other forms, usually in response to specific environmental cues (Takagi et al., 2019). In addition to yeast-to-hyphae transition, C. albicans can undergo a reversible switch between two morphologies, known as the white and opaque phases. Although the white and opaque cell types share the same genome, white cells caused more severe virulent in toxicity in mouse models (Kvaal et al., 1997). The class II deacetylase Hda1 selectively inhibits white-to-opaque switches, while the class I deacetylase Rpd31 suppresses transitions in both directions (Srikantha et al., 2001). Moreover, the sirtuins Hst3 and Sir2 were repressors of the white-to-opaque switch, whereas Set3C was the key activator (Figure 1; Pérez-Martín et al., 1999; Hnisz et al., 2009; Stevenson and Liu, 2011). Thus, C. albicans requires interaction with KDACs function for its morphological plasticity, which is central to its pathogenesis.

Biofilm formation on host tissues and indwelling medical devices is highly associated with fungal pathogenicity and drug resistance because the extracellular matrix hinders drug diffusion (Nobile et al., 2014). Fungal adhesion on both biotic and abiotic surfaces is the first phase of biofilm formation, which is closely related to the fungal cell wall and is critical to all later stages of biofilm development (Nett et al., 2011; Lohse et al., 2018). The relationship between biofilm resistance and the cell wall integrity pathway has been confirmed (Nett et al., 2011). In C. albicans, Nobile et al. (2014) found that the deletion of set3 and hos2 in C. albicans reduced biofilm formation and biomass, and these mutants appeared more resistant to yeast dispersion in vivo. Heat shock protein 90 (Hsp90) was a key regulator of biofilm dispersion and drug resistance and could be acetylated on lysine 27 and 270 (Robbins et al., 2012). Compromised Hsp90 function reduced the biofilm formation of C. albicans in vitro and impaired the dispersal of biofilm cells, blocking their capacity to serve as reservoirs of infection (Robbins et al., 2011). Moreover, Hsp90 was involved in the resistance of A. fumigatus biofilms to drugs (Robbins et al., 2011). Another study on A. fumigatus found that acetyltransferase GcnE was also required for biofilm formation (Lin et al., 2020). The list of device-associated infections caused by biofilms is expanding daily. Thus, the urgent determination of the mechanisms whereby acetylation/deacetylation participates in regulating biofilm formation is crucial.

A distinguishing feature of fungi is their ability to produce a variety of small molecules that contribute to their survival and pathogenicity. These substances include compounds such as pigments, which play a role in virulence and protect fungi from environmental damage, and toxins that kill host tissues or hinder competition from other organisms. The absence of deacetylase HdaA in Aspergillus nidulans caused the upregulation of carcinogenic sterigmatocystin (Shwab et al., 2007). In A. fumigatus, ΔhdaA knockout strains had a decreased production of the virulence factor gliotoxin (Lee et al., 2009). In the plant pathogenic fungus Fusarium fujikuroi, the deletion of hda1 or hda2 inhibited the production of red polyketide pigment bikaverin, plant hormone gibberellin, and mycotoxin fumaric acid; however, the deletion of hda1 did not affect the production of mycotoxin fusarins, and the deletion of hda2 did not affect the production of pigment fusarubin. This finding indicated that the impact of acetylation on transcriptional regulation is usually more complex because of the functional complementarity of different KDAC genes (Studt et al., 2013).

AflO, a key enzyme in aflatoxin biosynthesis, was acetylated at lysine 241 and 384 and played a vital role in the pathogenicity of A. flavus (Yang et al., 2019). Six proteins involved in the virulence of B. cinerea were found to be acetylated (BcSak1, Hpt1, Bcchs2, CHSV, PKS, and BOS1) (Lv et al., 2016). In F. graminearum, 10 virulence-related proteins were also acetylated, including Kin4, Sty1, and Gpmk1 (Zhou X. et al., 2016). Deoxynivalenol (DON), a mycotoxin produced by F. graminearum, is a virulence factor that helps fungi colonize and spread within spikes (Kong et al., 2018). The DON production levels of ΔFgSAS3 and ΔFgGCN5 mutants were almost zero compared with that of wild type strain (Kong et al., 2018). Although DON is not a protein, acetylation plays an important role in its metabolism.

Melanin is a pigmented polymer that protects fungal cells against oxidative stress, phagocytosis, and antifungal drugs. It also modifies the host immune responses by reducing the susceptibility of melanized microbes to the host defense mechanisms (Brandão et al., 2015, 2018). Brandão et al. (2018) found that the change in virulence of Δhda1 mutants of C. neoformans might be due to its markedly reduced formation of capsule, melanin, and extracellular proteases, all of which are specifically required for the survival of microbes in the host. Maeda et al. (2017) found that deletion of the HdaA homolog in Magnaporthe orzyae increased the expression of melanin biosynthesis genes. Although the effect of KDAC inhibitors (KDACis) on melanin synthesis has been confirmed, the specific mechanism of acetylation in melanin formation remains unclear (Brandão et al., 2015). The abovementioned studies suggest that protein acetylation/deacetylation can affect the virulence of fungi by participating in the regulation of secondary metabolite biosynthesis.

TSA is the best-known broad-spectrum KDACi. It was first isolated from a culture medium of Streptomyces platensis and initially appeared as an antifungal drug to inhibit the growth of Trichophyton and Aspergillus (Tsuji et al., 1976). The inhibition of RpdA activity by TSA resulted in a significant delay in the growth and germination of fungal species, such as A. fumigatus, A. nidulans, Aspergillus terreus, Penicillium chrysogenum, and Neurospora crassa (Bauer et al., 2016). Hnisz et al. (2010) found that TSA was involved in triggering the yeast-to-hyphae conversion of C. albicans by inhibiting Set3C, which controls protein kinase A signaling through Efg1. TSA also increased the susceptibility of Candida sp. to azole antifungals by inhibiting the biosynthesis of ergosterol (Smith and Edlind, 2002). Lamoth et al. (2014) found that the combination of TSA and azole drugs in the treatment of A. fumigatus also showed a promising possibility. Considering instability and rapid metabolism of TSA, the development of highly selective inhibitors is very important for mitigating potential toxicities caused by high doses (Tan and Liu, 2015). Sodium butyrate, apicidin, and suberoylanilide hydroxamic acid are also effective broad-spectrum KDACis; however, their use in antifungal therapy requires further investigation.

MGCD290, a fungal-specific Hos2 inhibitor in Candida sp., displayed moderate activity when used alone (Pfaller et al., 2009, 2015). However, the use of MGCD290 in combination with fluconazole, voriconazole, and posaconazole significantly increased the susceptibility of fungal species in vitro such as azole-resistant Candida, Mucor, and Fusarium sp. (Pfaller et al., 2009; Lamoth et al., 2015). When fluconazole, which had inactive activity against filamentous fungi, was used in combination with MGCD290, there was a distinctly favorable influence of the fluconazole MICs of Aspergillus strains, resulting in a conversion from resistance to susceptibility (Pfaller et al., 2009). Interestingly, Hos2 was a homologous gene of HosA in A. nidulans (Pidroni et al., 2018). However, the deletion of HosA did not affect the efficacy of any antifungal drugs, which contradicted the specificity of MGCD290 (Pidroni et al., 2018). These contradictory results may be explained by the different biological functions of HosA-type proteins in different Aspergillus species, or more likely by the fact that MGCD290 does not specifically act on HosA-type enzymes in filamentous fungi (Pidroni et al., 2018). Therefore, although the specificity of MGCD290 is a debatable issue, MGCD290 has great application prospects in antifungal therapies.

Nicotinamide (NAM), a form of vitamin B3, is a typical non-competitive inhibitor of sirtuins (Orlandi et al., 2017). NAM possesses an antibacterial activity, inhibits cell proliferation and enhances the antiproliferative effect of cytostatic drugs (Xing et al., 2019). NAM’s potential to inhibit the growth of Mycobacterium tuberculosis, Plasmodium falciparum, and HIV has been demonstrated in clinical trials (Murray, 2003; Tcherniuk et al., 2017). Furthermore, NAM displayed broad-spectrum activity against multiple clinical isolates, including C. parapsilosis, C. tropicalis, C. glabrata, C. krusei, C. neoformans, and fluconazole-resistant C. albicans (Xing et al., 2019). NAM also reduced the kidney burden in a mouse model of disseminated candidiasis (Wurtele et al., 2010). Moreover, two different Aspergillus species, A. fumigatus, and A. nidulans, were very sensitive to NAM (Wurtele et al., 2010). NAM also reduced the activity of some enzymes produced by fungi, such as C. albicans, T. rubrum, and Trichophyton mentagrophytes, which supports the use of NAM as an antifungal drug (Ciebiada-Adamiec et al., 2010). In addition to NAM, sirtuin inhibitors include the specific SIRT1 and SIRT2 inhibitors sirtinol, cambinol and EX-527, but their value in the treatment of infection is unclear (Eckschlager et al., 2017).