Acetylation modification of proteins is a critical mechanism of cellular adaptation to changing environmental conditions, and is also implicated in the virulence, pathogenesis and metabolism of M. tb. The recent study has identified 1128 acetylation sites on 658 M. tb proteins, and Gene Ontology (GO) analysis of the acetylome revealed that acetylated proteins are involved in the regulation of diverse cellular processes including metabolism and protein synthesis (Xie et al., 2015). Singh KK. et al. showed that acetylation of response regulator protein MtrA inhibited phosphorylation modifications thereby promoting division of M. tb (Singh et al., 2020). The acetyltransferase of M. tb is involved in the modification of various small molecular substrates, including antibiotics, amino acids, and other molecules, thereby regulating transcription, translation, protein folding, and metabolic pathways. The bioinformatics analysis revealed the existence of 47 potential acetyltransferases in M. tb ( Table 1 ), among which three genes, namely Rv2747, Rv3341, and Rv1653, encode essential acetyltransferases (Xie et al., 2019). Additionally, M. tb acetyltransferase can interact with host immune signaling proteins and modulate the host innate immune response against TB (Burckhardt and Escalante-Semerena, 2020. Shvedunova and Akhtar, 2022). The identification of acetylated proteins and acetyltransferases in M. tb provides a theoretical basis and research ideas for the development of novel anti-TB drugs targeting M. tb acetyltransferase, new mechanisms of drug resistance, and precise treatment.

The success of M. tb as a pathogen is partly attributed to its ability to sense and respond to dynamic host microenvironments. Protein acetylation modification plays a key role in bacterial virulence and pathogenicity (Ren et al., 2017). Various M. tb acetyltransferases have been identified and confirmed to act as virulence factors. The acetyltransferase Pat, encoded by Rv0998, has been shown to have acetylase activity that is directly regulated by cAMP binding in vitro (Nambi et al., 2010; Xu et al., 2011). Studies have demonstrated that the acetylation of a conserved lysine 193 (K193) within the C-terminal DNA-binding domain of the cyclic AMP (cAMP) receptor protein (CRP) reduces its DNA-binding ability and inhibits transcriptional activity. The reversible acetylation status of CRP K193 has been shown to significantly affect mycobacterial growth phenotype, alter the stress response, and regulate the expression of biologically relevant genes (Di et al., 2023). Shi SP. et al. (Shi and Ehrt, 2006) generated a Rv2215/dlaT knockout strain and tested its ability to grow, resist nitrosative stress, and cause disease in mice, which demonstrated that Rv2215/dlaT is required for optimal growth of M. tb. DlaT encodes dihydrolipoamide acyltransferase, which together with the pyruvate dehydrogenase E1 component (AceE) and dihydrolipoamide dehydrogenase (Lpd) constitutes pyruvate dehydrogenase (PDH) in M. tb. PDH catalyzes the oxidation of pyruvate by NAD to acetyl-coenzyme A (acetyl-CoA) and CO2. Acetyl-CoA then feeds into the tricarboxylic acid (TCA) cycle.

Although the existence of M. tb acetyltransferases as virulence factors has been discovered, the specific targets of these acetyltransferases and the exact molecular mechanisms affecting M. tb virulence remain to be studied and clarified.

The continuing spread of drug-resistant TB is one of the most urgent and difficult challenges facing global TB control. Studies have found that the activity of most of the existing ten kinds of anti-TB drugs, such as aminoglycosides, chloramphenicol, streptomycin, fluoroquinolones and other drugs is regulated by acetylation modification (Schwarz et al., 2004; Reygaert, 2018). For instance, in addition to mutations in katG, nhA, ahpC, kasA, and ndh genes, isoniazid (INH) resistance is associated with acetyltransferase Rv2170, which catalyzes the transfer of acetyl group from acetyl CoA to INH to form acetylated INH (Silva et al., 2003). The acetylated INH is then decomposed into isonicotinic acid and acetylhydrazine, overcoming INH toxicity and producing resistance (Arun et al., 2020). Furthermore, acetylation modification can also affect the metabolic rate of INH in vivo, thereby affecting its therapeutic effect in different individuals (Jing et al., 2020).

The enhanced intracellular survival (Eis) protein encoded by M. tb is an acetyl transferase that targets aminoglycoside antibiotics. Zaunbrecher et al. and Houghton et al. have found that EIS-mediated acetylation modification can inactivate kanamycin, capreomycin and other drugs (Zaunbrecher et al., 2009; Houghton et al., 2013). Reeves et al. found that transcription regulator WhiB7 promoted kanamycin acetylation by enhancing the transcription of Eis genes, and Eis itself was also regulated by acetylation modification (Reeves et al., 2013). Moreover, small molecule inhibitors targeting Eis have also been developed rapidly in recent years. Garzan et al. found that Eis inhibitors can be effectively applied in kanamycin adjuvant combination therapy, which provides a new solution for drug resistance (Garzan et al., 2016; Garzan et al., 2017; Punetha et al., 2020). However, the effect of Eis acetylation on its own activity and its mechanism in regulating aminoglycoside drug resistance remains unclear (Birhanu et al., 2017). Additionally, it has been reported that Rv0262c encoded aminoglycoside 2’-n-acetyltransferase can also acetylate all known aminoglycoside antibiotics, including ribomycin, neomycin B, gentamicin and tobramycin containing 2’ amino, etc. (Hegde et al., 2001). Correspondingly, Rv3225c-encoded acetyltransferase has a low level of aminoglycoside modification activity on aminoglycoside antibiotics, which can lead to resistance of M. tb to aminoglycoside antibiotics through acetylation modification (Kim et al., 2006). Meanwhile, N-acetyl cysteine can artificially increase respiration and additional ROS accumulation, which enhances moxifloxacin lethality in M. tb-infected cultured macrophages and mice. Addition of ROS stimulators to fluoroquinolone treatment of TB constitutes a new direction for suppressing the transition of MDR-TB to XDR-TB (Singh et al., 2022).

In summary, the regulation of drug acetylation modification by M. tb through acetyltransferase is an important cause of drug resistance, according to the studies mentioned above. These studies suggest that small molecule inhibitors targeting M. tb acetyltransferase activity can be developed directly as new anti-TB drugs and can also promote the anti-TB effect of existing drugs by enhancing their sensitivity or preventing drug tolerance.

In the metabolic pathway, approximately 90% of metabolic enzymes in the metabolic pathway, including tricarboxylic acid cycle, gluconeogenesis, glycolysis, glycogen metabolism, fatty acid metabolism, and urea cycle, undergo acetylation modification (Zhao et al., 2010). Rv2170 has been found to possess lysine acetyltransferase activity, which can affect the glyoxylic acid metabolism or tricarboxylic acid cycle by reducing the lysine residues of Isocitrate lyase or Isocitrate dehydrogenase through acetylation modification (Lee et al., 2017). Moreover, the deacetylation of DosR at K182 promotes the hypoxia response in M. tb and enhances the transcription of DosR-targeted genes. Rv0998 has been identified as an acetyltransferase that mediates the acetylation of DosR at K182. Deletion of Rv0998 also promoted the adaptation of M. tb to hypoxia and the transcription of DosR-targeted genes. Mice infected with an M. tb strain containing acetylation-defective DosR^K182R had much lower bacterial counts and less severe histopathological impairments compared with those infected with the wild-type strain (Yang et al., 2018). Additionally, Rv0998 has been shown to regulate carbon flux, change oxidation reaction, and reduce tricarboxylic acid cycle reaction, which may contribute to M. tb survival in mice (Bi et al., 2018; Rittershaus et al., 2018). The acetylase activity of Rv0998 is regulated by metabolism-related products, including cAMP, acetyl-CoA, and the deacetylase Rv1151c (Bi et al., 2018). These findings suggest that targeting M. tb acetyltransferase in its own metabolic pathway could be a potential pathway for anti-TB therapy.

Furthermore, TB is linked to human metabolism, and individuals with diabetes and other metabolic disorders have a higher risk of M. tb infection (Bernal et al., 2014). Therefore, investigating the effect of M. tb acetyltransferase on host metabolism could be a promising new strategy for developing anti-TB therapy targeting metabolism-related enzymes.

TB arises from the interplay between bacterial virulence and host immunity. The virulence factors of M. tb enable it to evade the host immune system and survive within the host (Zhu et al., 2019). For example, Kim et al. (Kim et al., 2012) found that Eis protein inhibits JNK-dependent autophagy, phagosome maturation and Reactive Oxygen Species (ROS) production through acetylation of DUSP16/MKP-7 at K55 site. Duan Liang et al. (Duan et al., 2016) found that Eis protein inhibits macrophage autophagy by increasing histone H3 acetylation, up-regulating IL-10 expression, and then activating AKT/mTOR/P70S6K pathway. Rv3423.1, a novel histone acetyltransferase from M. tb, has been shown to mediate acetylation at the H3K9/K14 positions by co-localizing with the host chromatin in the nucleus. By binding to the host chromatin, Rv3423.1 may manipulate the expression of host genes involved in anti-inflammatory responses, allowing M. tb to evade clearance and survive in the intracellular environment (Jose et al., 2016). Another protein secreted by mycobacteria under hypoxia, FadA (Fatty-acid degradation A), acts as an acetyltransferase that converts host acetyl-CoA to acetoacetyl-CoA. This reduces the acetyl-CoA level and suppresses H3K9Ac-mediated expression of the host proinflammatory cytokine Il-6, thereby promoting granuloma progression (Yang et al., 2021). Eis also acetylates M. tb HU (MtHU), which leads to reduced DNA interactions and altered DNA compaction capacity of NAP (Ghosh et al., 2016). Overexpression of Eis can result in excessive acetylation of HU and genomic decompression. Given the importance of HU for M. tb survival, it is possible that its acetylation by Eis is also linked to drug resistance and survival.

Thus, understanding the role of acetyltransferases in host immunity against TB may offer a new therapeutic approach to TB infection.

Protein acetylation and deacetylation is catalyzed by protein acetyltransferases and deacetylases, respectively, of which several families exist. There are two types of protein acetylation: the acetylation of proteins at the ε-amino group and the acetylation of the a-amino group of the N-terminal amino acid (Bernal et al., 2014; Khadela et al., 2022). While the acetylation of the α-amino group of the N-terminal amino acid of proteins is very rare in bacteria, it is frequent in eukaryotes (30 - 80% of proteins) and archaea (14 - 29% of proteins) (Polevoda and Sherman, 2003; Soppa, 2010). On the other hand, the acetylation of proteins at the ε-amino group of internal lysine residues is a widely distributed PTMs, frequent in all domains of life. In eukaryotes, the physiological relevance of N-ε-lysine protein acetylation is well demonstrated. It has been demonstrated that the 8-amino group of multiple lysine side chains in histones can be acetylated to manipulate gene expression by regulating chromatin tightness or influencing transcription factor binding in promoters and distal enhancers, as well as histone DNA interactions (Roth et al., 2001; Yuan et al., 2009; Bannister and Kouzarides, 2011; Barnes et al., 2019). For the first time, Choudhary et al. identified the existence of acetylation modification at 3600 lysine sites on 1750 proteins, suggesting that lysine acetylation has a wide range of regulatory effects (Choudhary et al., 2009). Non-histone acetylation plays a key role in physiological and pathological processes, including the regulation of enzyme activity, protein degradation, protein interaction, subcellular localization, chromatin regulation and metabolism (Drazic et al., 2016; Narita et al., 2019). Abnormal protein acetylation or deacetylation is closely related to many diseases, such as leukemia, cancer, diabetes, infectious diseases, cardiovascular and nervous system related diseases and so on (Timmermann et al., 2001; Morales-Tarré et al., 2021).

During M. tb infection, the host anti-TB immune response was regulated by M. tb-induced gene expression, which is one of the strategies for its intracellular survival and progression of TB. Research reports that M. tb-infected macrophages inhibit histone H3 acetylation (H3Ac) in the interleukin-12B (IL-12B) promoter region, leading to down-regulation of IL-12B expression and inhibition of Th1 type immune response. This promotes M. tb survival in the host (Chandran et al., 2015). Wang et al. found that M. tb infection inhibit HLA-DR gene expression by regulating the recruitment of HDAC complex in the HLA-DR promoter to enable its intracellular survival (Wang et al., 2005). Chen et al. found that the expression of H3K14Ac in peripheral blood lymphocytes of TB patients was reduced, especially the specific enrichment in the promoter region of TNF-α and IL-12B was decreased, which was related to the survival rate of TB patients (Chen et al., 2017). In addition, the up-regulated expression of HDAC1 inhibits the expression of H3K14Ac and plays a role in the outcome of active pulmonary TB and its clinical treatment. Moores et al. found that M. tb regulates the expression of matrix metalloproteinases (MMP-1 and MMP-3) via HDAC and histone acetyltransferase (HAT) activity and the manipulation of histone acetylation modification, which is a key factor in TB immune pathogenesis (Moores et al., 2017). These studies suggest that acetylation of histones, or acetylation of specific lysine sites, is associated with intracellular survival of M. tb and the development of TB.

Recently, the studies of histone acetylome-wide associations (HAWAS) showed that there were at least 2000 differences in acetylation sites associated with differential gene expression in the whole genome of peripheral granulocytes and monocytes of TB patients and healthy people. Histone acetylation quantitative trait locus (haQTL) analysis revealed candidate causal immunophenotypic changes in different populations of granulocyte and monocyte haQTL. M. tb infection regulates the differential enrichment of the inward rectifier potassium channel subfamily promoter J member 15 (CNJ15) of H3K27Ac, which enhances cell apoptosis and promotes M. tb clearance in vitro (Del Rosario et al., 2022). On the other hand, trained immunity, proposed by Netea et al. and Joosten et al., has become an important new evaluation index system for host immune protection induced by TB vaccines (Netea et al., 2016; Joosten et al., 2018). Post-immunization mediated trained immunity (mainly affecting H3K27Ac) of BCG or MTBVAC, an active M. tb candidate vaccine, can enhance the production of cytokines by monocytes and thus provide immune protection (Tarancón et al., 2020; Sheng and Cristea, 2021). Li et al. found that BCG infection can up-regulate the expression level of p300 in mature THP-1 cell lines and regulate the acetylation level of histone H3 and AP-2α. It was further demonstrated that trichostatin A (TSA), a broad-spectrum histone deacetylase inhibitor, enhances the enrichment of the toll-like receptor2 (TLR2) promoter by regulating the acetylation of AP-2α. Furthermore, promoter transcriptional activity was increased to up-regulate TLR2 gene expression (Li et al., 2013). Pennini et al. found that activation of TLR2 inhibits IFN-induced acetylation of histones H3 and H4 (Pennini et al., 2006). Therefore, targeted regulation of acetylation of specific lysine sites in histones may be an important way to enhance the host’s effective resistance to M. tb infection and/or promote immune clearance.

In addition to histone acetylation, non-histone acetylation also plays a crucial role in regulating cellular processes. Like histone proteins, non-histone proteins are also modified by histone acetyltransferases and HDACs. Various studies have reported that non-histone acetylation plays an essential role in the occurrence, development, and outcome of infectious diseases caused by viruses, bacteria, and other pathogens such as DNA virus, influenza virus, rabies virus, and Salmonella typhi infection (Wu et al., 2010; Green et al., 2018; Song et al., 2020). Furthermore, non-histone acetylation modification has been found to regulate autophagy, apoptosis, and inflammasome activation in innate immune responses (Wan et al., 2017; Son et al., 2020; Pagán et al., 2022) Autophagy-related proteins such as ATG5, ATG7, ATC8, and ATG12 can be acetylated by p300, leading to inhibition of autophagy (Lee and Finkel, 2009; Battaglioni et al., 2022; Wang D. et al., 2022). On the other hand, NAD⁺-dependent histone deacetylase Sirt1 can deacetylate ATG5, ATG7, and LC3, promoting autophagy occurrence (Lee et al., 2008).

In the process of M. tb infection, studies have shown that Sirt1 activation induced by M. tb infection can activate autophagy by directly mediating MAP1LC3B/LC3B deacetylation, which may limit the growth of intracellular M. tb (Iqbal et al., 2021). These findings suggest that acetylated autophagy-related proteins play a key role in regulating autophagy activation and inhibition, and the role of autophagy in host anti-M. tb infection has been established (Pellegrini et al., 2021). Non-histone deacetylation mediated by Sirt family proteins is suggested to be significant in understanding M. tb-mediated inflammatory response and discovering new drug targets (Cheng et al., 2017; Bhaskar et al., 2020; Yang et al., 2021; Yang et al., 2022). In another recent experiment, Brandenburg J. et al. showed that Wnt family member 6 (WNT6) promotes foam cell formation during TB by regulating key lipid metabolism genes including Acetyl Coenzyme A Carboxylase (ACC2). These findings open new perspectives for host-directed adjunctive treatment of pulmonary TB (Brandenburg et al., 2021).

Based on the above studies, non-histone acetylation modification is shown to be effective in host anti-TB immunity. Additionally, non-histone modifications involved in cell signal transduction, protein interaction, protein aggregation, protein degradation, and subcellular localization may also play a critical role in regulating host anti-TB immunity during M. tb infection.

Arylamine N-acetyltransferase comprises N-acetyltransferase 1 (NAT1) and N-acetyltransferase 2 (NAT2) in humans (Hein et al., 2022). NAT2 is mainly expressed in the liver and the GI tract (Husain et al., 2007), and is responsible for the N-acetylation polymorphism observed in human populations (Weber and Hein, 1985; McDonagh et al., 2014; Agundez and Garcia-Martin, 2018; Mitchell, 2020). There are several single nucleotide polymorphisms (SNPs) in the coding exon of the NAT2 gene, which are inherited as NAT2 haplotypes and genotypes and confer rapid, intermediate, and slow acetylator phenotypes that modify drug metabolism (Hein, 2009; Hein and Millner, 2021).

Although isoniazid (INH) remains one of the major first-line drugs, the extensive use of INH to treat active and latent TB infections is compromised by INH-induced hepatotoxicity and liver failure (Hall et al., 2009; Sterling et al., 2020). The NAT2 genotype dependent pharmacokinetic parameters measured in human subjects have been confirmed by measurement of INH N-acetylation both in vitro and in situ in cryopreserved human hepatocytes and the TB patient (Doll et al., 2017; Hein and Millner, 2021). A study conducted in Indonesia revealed that patients with TB and the slow-acetylator phenotype caused by NAT2 variants are highly susceptible to drug-induced liver injury caused by anti-TB drugs, confirming the association between slow-acetylator NAT2 variants and susceptibility to drug-induced liver injury in an Indonesian population (Yuliwulandari et al., 2016). Furthermore, recent studies concluded that INH N-acetylation in human subjects differs significantly with respect to rapid, intermediate, and slow acetylator NAT2 genotypes in terms of plasma half-life, bioavailability (area under the curve), plasma metabolic ratio of INH to N-acetyl-INH, and clearance. The meta-analysis studies report that slow acetylators were significantly more likely to experience hepatotoxicity from INH treatment for TB than rapid acetylators (Khan et al., 2019; Richardson et al., 2019).

Personalized therapy, also known as host-directed therapy (HDT), is being developed in many recent studies for conditions such as TB caused by M. tb. Epigenetic processes, including acetylation modification, play a crucial role in the development of personalized HDT (Marimani et al., 2018). One of the most focused themes among these studies is NAT2 polymorphisms. The paradigm for NAT2 phenotype-dependent dosing strategies is presented as a value of pharmacogenomics-guided isoniazid therapy for the prevention and treatment of TB. Béranger Agathe et al. have demonstrated that NAT2 genotype is the most impactful factor of INH metabolism, compared with low-birth-weight (LBW) and preterm infant born (Béranger et al., 2022). Phenotype-dependent dosing strategies aim to reduce the risk of adverse reactions, increase therapeutic efficacy, reduce costs, and improve patient care and disease prevention. Several studies have proposed pharmacogenomics-guided INH therapy for TB (Matsumoto et al., 2014; Jung et al., 2015; Choi et al., 2017; Motta et al., 2018; Suvichapanich et al., 2018; Jing et al., 2020). A recent study conducted in the USA and Brazil found that the clearance rates of INH were lowest in predicted slow acetylators (median 19.3 L/hr), moderate in intermediate acetylators (median 41.0 L/hr), and highest in fast acetylators (median 46.7 L/hr) (Verma et al., 2021). Moreover, there are significant differences in the distribution of NAT2 gene polymorphisms among different nationalities and races, the anti-TB treatment regimens adopted by patients are different, and the tolerance and exclusion standards of the subjects are different (Suarez-Kurtz et al., 2016; Zahra et al., 2020; Zhang et al., 2021). Depending on the NAT2 genotype of the patients, several studies have evaluated isoniazid doses of 2.5 mg/kg (0.5 times standard dose), 5 mg/kg (standard dose), and 7.5 mg/kg (1.5 times standard dose) for slow, intermediate, and fast metabolizers, respectively. As a result, a better treatment success rate was achieved, and the occurrence of liver function injury was reduced (Azuma et al., 2013; Huerta-García et al., 2020).

All these results suggest that understanding the diversity of drug-related genetic markers is critical for individualized drug-gene therapy programs in ethnic minorities in China and populations highly mixed with these ethnic groups. The above studies could make personalized TB treatment dosing available in reality. Pharmacogenomic-guided dosing can help achieve consistent drug levels and improve clinical outcomes.