REVIEW ARTICLE Role of post-translational modifications in the acquisition of drug resistance in Mycobacterium tuberculosis Gunjan Arora1 , Ankur Bothra2 , Gareth Prosser3, Kriti Arora4 and Andaleeb Sajid1

1 Yale School of Medicine, Yale University, New Haven, CT, USA 2 National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA 3 Cancer Research UK Manchester Institute, The University of Manchester, Alderley Park, UK 4 Proteus Digital Health, Inc., Redwood City, CA, USA

Keywords Tuberculosis (TB) is one of the primary causes of deaths due to infectious acylation; biotinylation; drug resistance; diseases. The current TB regimen is long and complex, failing of which glycosylation; methylation; Mycobacterium leads to relapse and/or the emergence of drug resistance. There is a critical tuberculosis; phosphopantetheinylation; need to understand the mechanisms of resistance development. With pupylation; Ser/Thr phosphorylation; succinylation; two-component system increasing drug pressure, Mycobacterium tuberculosis (Mtb) activates vari- ous pathways to counter drug-related toxicity. Signaling modules steer the Correspondence evolution of Mtb to a variant that can survive, persist, adapt, and emerge G. Arora and A. Sajid, Yale School of as a form that is resistant to one or more drugs. Recent studies reveal that Medicine, Yale University, New Haven CT about 1/3rd of the annotated Mtb proteome is modified post-translation- 06519 USA ally, with a large number of these proteins being essential for mycobacterial Tel: +1 13019174643 (GA) survival. Post-translational modifications (PTMs) such as phosphorylation, E-mails: [email protected] (GA); [email protected] (AS) acetylation, and pupylation play a salient role in mycobacterial virulence, pathogenesis, and metabolism. The role of many other PTMs is still emerg- Gunjan Arora and Ankur Bothra contributed ing. Understanding the signaling pathways and PTMs may assist clinical equally to the manuscript strategies and drug development for Mtb. In this review, we explore the contribution of PTMs to mycobacterial physiology, describe the related cel- (Received 20 June 2020, revised 16 lular processes, and discuss how these processes are linked to drug resis- September 2020, accepted 30 September tance. A significant number of drug targets, InhA, RpoB, EmbR, and 2020) KatG, are modified at multiple residues via PTMs. A better understanding doi:10.1111/febs.15582 of drug-resistance regulons and associated PTMs will aid in developing effective drugs against TB.

Introduction According to the 2019 global tuberculosis (TB) report there are five forms of drug-resistant TB: Monodrug- by the World Health Organization (WHO), data from resistant TB is usually due to resistance to one of the 202 countries estimated 10 million new cases with front-line anti-TB drugs, and poly-resistant TB has 1.5 million deaths and 1.7 billion latent infections in resistance to more than one first-line anti-TB drug, 2018 (WHO TB report 2019). With pan-TB treatment other than isoniazid (INH) and rifampicin (RIF). Mul- approach, the incidence of drug-resistant TB has tidrug-resistant (MDR) TB is resistant to at least both reduced from 7.4% (558 000 cases) to 6.4% (484 000 INH and RIF, and extremely drug-resistant (XDR) cases) globally [1]. According to WHO classification, TB indicates resistance to INH, RIF, and any of the

Abbreviations EMB, ethambutol; ETH, ethionamide; FQs, fluoroquinolones; INH, isoniazid; LZD, linezolid; MDR, multidrug resistance; Msmeg, Mycobacterium smegmatis; Mtb, Mycobacterium tuberculosis; Ppant, phosphopantetheine; PTM, post-translational modifications; RIF, rifampicin; STPK, Ser/Thr protein kinase; TB, tuberculosis; TCS, two-component system.

The FEBS Journal (2020) ª 2020 Federation of European Biochemical Societies 1 PTMs and drug resistance in Mycobacterium G. Arora et al. injectable second-line anti-TB drugs (such as kanamy- nutrient insults and drug pressure, and initiate a series cin, amikacin, and capreomycin) and a fluoro- of events causing modifications of several important quinolone (FQ). Rifampicin-resistant (RR) TB is proteins. This review addresses the role of PTMs that defined as resistance to RIF, with or without resistance aid in mycobacterial survival and drug resistance of to other anti-TB drugs (WHO classification) [2]. The Mtb. treatment of MDR-TB patients is protracted and diffi- cult, due to several side effects of drugs and fewer Post-translational modifications of alternative treatment options [1,3]. proteins that interact with drugs Control of drug-resistant TB requires the develop- ment of a potent, faster-acting, and less toxic regimen With a genome encoding ~ 4000 genes, Mtb hijacks coupled with the use of better diagnostic and transmis- the host system during infection, manipulates its sion-control practices. Pretomanid (PA-824) in combi- immune response, and adapts to various stresses. This nation with bedaquiline (BDQ) and linezolid (LZD) is possible with spatiotemporal regulation of the pro- was recently approved for the treatment of XDR-TB teome by PTMs. Recent literature suggests that there and treatment-intolerant or nonresponsive MDR-TB. are at least 200 different kinds of PTMs known [13]. The treatment was FDA-approved in August 2019 as Mtb uses different PTMs such as phosphorylation an all-oral, 6-month regimen (BPaL, FDA drug-resis- (Ser/Thr/Tyr and His/Asp), glycosylation, acylation, tance TB regimen). The treatment success rate of biotinylation, methylation, pupylation, and phospho- XDR-TB has increased from 20% before 2014 to 65% pantetheinylation to regulate different aspects of cellu- after 2014 due to the inclusion of BDQ and LZD (pre- lar physiology (Fig. 2). Therefore, this review looks tomanid and BPaL regimen, [4]). However, evidence into the importance of these modifications and their suggests that these treatments are associated with toxi- role in resistance to different drugs (Table S1). cities such as adverse cardiac effects and peripheral – neuropathies [5 7]. Additionally, clinical resistance is Ser/Thr/Tyr phosphorylation already documented to both BDQ and LZD [8,9]. Therefore, there is a need to keep the discovery pipe- The relevance of Ser/Thr/Tyr phosphorylation (STY) line enriched with new chemical entities as well as to comes from the exclusive existence of these elucidate the basis of drug resistance in existing strains. Considerable research has been done to understand the mechanisms by which Mycobacterium tuberculosis (Mtb) circumvents drug selection and acquires resis- tance, such as genetic mutations, altered drug perme- ability due to membrane architecture, or uptake/efflux pumps, and drug inactivation (Fig. 1). A better under- standing of acquired drug resistance is essential to design better antimycobacterial compounds [10–12]. The most common cause of developing drug resistance at the molecular level is the occurrence of mutation(s) in the target genes. These mutations either lead to the evolution of an altered protein or complete loss of expression. Besides mutations, unregulated functions of target proteins or altered expression of transcription factors can also result in the development of resistance or a drug-tolerant phenotype. Several proteins and Fig. 1. Mechanisms of drug resistance in mycobacteria: Recent other macromolecules are known to interact with literature has provided a glimpse of the role of PTMs in acquired drugs, and any alteration in these proteins may affect resistance to various drugs. The schematic diagram highlights the drug sensitization. One of the most important alter- major mechanisms that can lead to the generation of resistance in ations in any protein function could be due to post- bacteria. These include genetic mutations in drug targets, activation of efflux pumps, drug inactivation by cellular enzymes, translational modifications (PTMs). These PTMs such altered uptake and permeability, intrinsic bacterial resistance, as phosphorylation, acetylation, and glycosylation general persistence and tolerance, and PTMs of the proteins that affect protein function, stability, and localization. interact with the drugs. Mycobacteria can sense environmental signals such as

2 The FEBS Journal (2020) ª 2020 Federation of European Biochemical Societies G. Arora et al. PTMs and drug resistance in Mycobacterium

Fig. 2. Role of PTMs in M. tuberculosis: PTMs such as His-Asp phosphorylation (TCS), STY phosphorylation, Lys modifications (e.g., N- acylation), biotinylation, pupylation, and glycosylation help Mtb in various cellular processes. The surface-exposed STPKs and TCS sense specific signals via extracellular sensory region and relay the information inside the cells. Most of the other PTM reactions are initiated within the cytosol and involve multiple sets of reactions. The basic reaction of each process is depicted.

modifications in eukaryotes, but the last two decades repressor is one of the major DNA damage response of research on signaling mechanisms have contem- (DDR) regulators in Mtb. A DNA-dependent ATPase, plated their significance in Mtb virulence and overall RecA, forms polymeric filaments on DNA with LexA physiology. In Mtb, there are 11 Ser/Thr protein and regulates DNA damage repair. During DDR, kinases (STPKs) and one cognate phosphatase, along RecA gets phosphorylated on Ser207, which inacti- with one Tyr kinase and two Tyr phosphatases. These vates the protein and inhibits its binding to LexA. This kinases and phosphatases reversibly phosphorylate a inhibition causes enhanced DNA damage and the repertoire of overlapping protein substrates upon sens- development of RIF-resistant strains [23]. RecA/LexA ing an environmental signal [14–20]. The phospho-pro- are also involved in drug tolerance and persistence, as teins are involved in critical processes such as cell identified in CRISPRi-based screenings [24]. growth and division, gene expression, protein synthe- Besides DNA-binding proteins, several transcrip- sis, pathogenicity, and drug resistance [20,21]. The tional factors are also involved in drug-resistance phosphorylation of proteins can alter their functional mechanisms. A very well-known example is EmbR, a capacity, stability, and interaction with other partners. SARP family transcription factor, which regulates the Here we discuss some important examples of mycobac- embCAB operon. EmbCAB proteins are arabinosyl- terial phospho-proteins involved in drug binding and transferases that control lipoarabinomannan (LAM)/ resistance (Table S1), that can regulate nucleotide lipomannan (LM) ratio and are principally targeted by metabolism, protein synthesis, metabolism, and mem- the first-line drug ethambutol (EMB) [25,26]. Muta- brane proteins (Fig. 3). tions or changes in expression of these genes affect the A major mechanism for the development of antibi- EMB concentration required to kill the bacteria. Thus, otic resistance is through altered DNA metabolism, EmbR has an important role in EMB function and genetic mutations, and DNA damage [22]. LexA resistance. Interestingly, the activity of EmbR is

The FEBS Journal (2020) ª 2020 Federation of European Biochemical Societies 3 PTMs and drug resistance in Mycobacterium G. Arora et al.

Fig. 3. Molecular mechanisms of different TB drugs and connection with PTMs: Antimycobacterial drugs target various cellular pathways and their associated proteins. A large number of these proteins are also subjected to modifications. This schematic shows antimycobacterial drugs on the left, and their target pathways in the center connected by lines. The major target proteins from each pathway are shown in the shadowed box toward the right. The connection of these pathway proteins with different PTMs is shown at the right end. The diagram depicts that PTMs maintain stringent control of different processes and act as superhubs, establishing a possible connection between the drug targets and their modifications. positively regulated by phosphorylation. Multiple other proteins to carry out its function. Structurally, STPKs such as PknH, PknA, and PknB phosphorylate Ef-Tu consists of three domains, each having a specific EmbR and promote its DNA binding, which leads to role in the process of translation. Phosphorylation of increased transcription of embCAB genes, causing Ef-Tu is shown in diverse bacteria, including Mtb [32– altered resistance and LAM/LM ratio [27–29]. 35]. Mtb Ef-Tu is phosphorylated on several residues, Another transcription factor involved in drug resis- with Thr118 in the GTPase domain being the major tance is EthR, a TetR/CamR-family of the transcrip- site. This phosphorylation decreases its affinity for tional regulator of cotranscribed gene ethA. EthA is a GTP, ultimately decreasing the translation efficiency at monooxygenase required for the activation of second- this step. Interestingly, the phosphorylated form of Ef- line drug ethionamide (ETH). EthR is responsible for Tu is less sensitive to its specific inhibitor, kirromycin decreased EthA expression and ETH drug resistance [32]. This was the first report to show the direct role [30]. EthR is phosphorylated by STPKs, particularly of phosphorylation in altering the affinity of the drug PknF [31]. Phosphorylation by PknF at the N-terminal with a mycobacterial protein. Besides kirromycin, the DNA-binding region decreases the affinity of EthR for expression of Ef-Tu was also found to be decreased DNA, thus causing the de-repression of ethA expres- after treatment with INH, indicating a possibility that sion [31]. An increase in EthA is anticipated to cause INH can affect protein synthesis [36]. Another pro- the hypersensitization of cells for ETH. teomics study showed a twofold higher expression of Protein synthesis machinery in the pathogenic bacte- Ef-Tu in ofloxacin resistant isolate; however, its phos- ria has always been an important target for the drugs. phorylation status is unknown [37]. Almost all the proteins involved in the translation pro- PknG, a thioredoxin-fold containing protein, is one cess are essential for mycobacterial survival. Ef-Tu, a of the 11 STPKs in Mtb which is regulated by translation elongation factor, is a highly conserved autophosphorylation. PknG is secreted during infec- GTPase that interacts with RNA, nucleotides, and tion in macrophages and plays an important role in

4 The FEBS Journal (2020) ª 2020 Federation of European Biochemical Societies G. Arora et al. PTMs and drug resistance in Mycobacterium blocking the phagosome–lysosome fusion, thus ensur- [53], possibly owing to its affinity for NAD+. Thus, ing the survival of bacteria in the host [38].In the perturbation of the SahH-NAD+ axis due to phos- mycobacteria, PknG regulates the central carbon and phorylation might affect its interaction with INH and nitrogen metabolism through phosphorylation of its contribute to the development of resistance. substrates, primarily GarA [39,40]. Proteins involved The mycobacterial membrane is the most important in metabolism have an important role in drug binding organelle that protects the bacterium from unfavorable and are responsible for the development of resistance environmental conditions, immune system, and stress. [41,42]. Interestingly, besides its role in infection and The drugs that target the membrane lipid synthesis regulating metabolism, PknG was found to be respon- and metabolism, particularly mycolic acids, are very sible for MDR to several drugs including ery- effective against Mtb [21,22]. MabA (beta-ketoacyl- thromycin, imipenem, vancomycin, RIF, EMB, and acyl carrier protein reductase) and InhA (2-trans- INH. The deletion mutant of PknG in M. smegmatis enoyl-acyl carrier protein reductase) are the primary (Msmeg) exhibits increased antibiotic sensitivity and enzymes involved in the second and fourth steps, reduced drug tolerance, which was recovered by com- respectively, of meromycolic backbone elongation plementing the strain with Mtb or Msmeg PknG but cycle during mycolic acid synthesis [54]. The mabA not with kinase-inactive PknGK181M mutant [43,44]. and inhA genes are conserved in mycobacteria and The mechanism of the antibiotic sensitivity is not clear, expressed in the same operon. Interestingly, both of but it is expected that PknG-mediated phosphorylation these proteins are targeted by multiple STPKs with of its substrate(s) may play a role in maintaining this MabA-Thr191 and InhA-Thr266 being the primary phenotype. phosphorylation sites [55,56]. To function as reduc- GarA is a primary substrate of PknG, apparently tases, both MabA and InhA require binding of NAD responsible for metabolic regulation. PknG senses the (P) as a cofactor, which is impaired after phosphoryla- amino acids through GlnH-GlnX, gets activated by tion. Consequently, the activity of MabA and InhA is autophosphorylation, and further phosphorylates reduced after phosphorylation, which causes reduced GarA [45,46]. GarA is an FHA-domain-containing mycolic acid synthesis [55,57,58]. INH and ETH target protein involved in the negative regulation of the TCA InhA and/or MabA and lead to inhibition of their cycle and nitrogen metabolism through interaction expression and functions [59]. This inhibition mimics with a-ketoglutarate dehydrogenase complex (KDH). the phenotype in bacteria as phosphorylation of these Phosphorylation of GarA relieves the inhibition of proteins. It is yet to be studied if phosphorylation of KDH [47]. It is apparent that the effects of PknG dele- MabA and InhA block the binding of drugs or if tion are associated with altered metabolic regulation phosphorylation is induced during drug exposure. by GarA. PknG also regulates the redox homeostatic The next step in mycolic acid synthesis after acyl system by phosphorylation of ribosomal protein L13, carrier proteins (ACP)-reduction is b-ketoacyl-ACP which then binds to RenU, a nucleoside diphosphate- synthesis carried out by KasA and KasB [60]. These linked moiety X (nudix) hydrolase, promoting hydroly- proteins show a similar regulatory profile in terms of sis of FAD, ADP-ribose, and NADH [48]. Since expression (being in the same operon) and PTMs. oxidative stress and antibiotic resistance are closely Multiple STPKs target KasA and KasB as their sub- linked, particularly for INH and ETH, it provides a strates [61]. As in the case with MabA and InhA, possible link of PknG being involved in drug resistance phosphorylated forms of KasA and KasB are less [48–50]. active than their apo-form, indicating the requirement In mycobacteria, the levels of critical metabolites of fine balance for mycolic acid synthesis [61,62]. Both such as homocysteine, adenosine, S-adenosyl methion- KasA and KasB have been implicated in the develop- ine, and S-adenosyl homocysteine (SAM/SAH) are ment of resistance against INH [63], particularly KasB regulated by S-adenosyl homocysteine hydrolase was found to be upregulated in the chemical-genetic (SahH). With its substrate-binding and cofactor bind- interaction screen for the antibiotics, RIF, ETH, INH, ing domains, SahH forms a tetramer and catalyzes the vancomycin, and meropenem [63], indicating an reversible hydrolysis of SAH with the help of the important role in the development of MDR. The inhi- NAD+ cofactor [51]. Interestingly, the residues bition of KasB has been directly associated with acid involved in NAD+ binding are also modified by phos- fastness and virulence of Mtb. Thus, inhibition of phorylation [52]. This modification decreases the affin- these enzymes by phosphorylation might affect their ity of SahH for NAD+ binding, thus affecting overall drug susceptibility. homocysteine levels. SahH was also identified as one PknB is the most important STPK in Mtb due to its of the proteins that bind the INH-NAD(P) complex essential role in survival and involvement in several

The FEBS Journal (2020) ª 2020 Federation of European Biochemical Societies 5 PTMs and drug resistance in Mycobacterium G. Arora et al. critical processes, such as regulation of proteins that There are two key components of TCS, a mem- mediate cell wall synthesis and maintenance. One such brane-bound sensor His kinase (HK), responsible for example is PonA1, a penicillin-binding protein sensing the environmental stimuli, and a cytosolic involved in peptidoglycan synthesis and maintaining transcriptional response regulator (RR), which gets cell shape [64]. PonA1 possesses two activities required phosphorylated by the HK, thereby regulating down- for peptidoglycan synthesis—transglycosylation and stream gene expression (Fig. 2) [73]. Mtb genome transpeptidation. PknB phosphorylates Thr34 of encodes 11 paired TCSs and some orphan HKs and PonA1 and possibly regulates its transglycosylation RR proteins [74]. Most of the TCSs that regulate Mtb activity [65]. Phosphorylation of PonA1 decreases cell virulence are classified under the OmpR family (includ- elongation due to imbalanced peptidoglycan synthesis. ing PhoPR, SenX3/RegX3, PrrAB, MprAB, and Besides these phenotypic alterations, lack of PonA1 MtrAB). phosphorylation causes fourfold hypersensitization of MtrA (RR) is known to regulate the gene expression the mycobacterial cells to the glycopeptide-based inhi- of virulence-associated genes like dnaA (replicator ini- bitor teicoplanin [65]. Thus, PknB-mediated regulation tiator gene, involved in cell division) and fbpB (en- of PonA1 activity has implications in antibiotic sensi- codes mycolyl transferase FbpB) [75,76]. The structure tivity. of inactive MtrA suggests an extensive intramolecular CwlM is another interesting PknB substrate that is interaction between its regulatory and effector involved in the regulation of peptidoglycan synthesis domains, which gets resolved by phosphorylation through MurA, the first enzyme in peptidoglycan through MtrB (HK) upon activation [77]. MtrA-de- biosynthesis [66,67]. CwlM is also associated with pendent target gene analysis identified iniB, an INH nutrient availability and environmental stress, such as inducible gene, among several other potential targets. the presence of antibiotics. PknB phosphorylates Furthermore, reduced expression of mtrA hypersensi- CwlM under nutrient-enriched conditions favorable tized Msmeg cells for INH and streptomycin, indicat- for mycobacterial growth, leading to hyperactivation ing a direct role of MtrA/B in drug resistance [78].On of MurA. This causes the mycobacterial cells to divide similar lines, other studies showed the critical role of actively, which are more susceptible to antibiotics such MtrA/B in antibiotic susceptibility (Fig. 3). Allelic as INH and RIF, as compared to the cells that are deletion of mtrA/B caused hypersensitization of the starving and nonreplicating [66]. cells to ampicillin, RIF, vancomycin, ciprofloxacin, clarithromycin, and penicillin [79,80]. Two-component systems A recent study used the genetic and molecular dock- ing approaches to develop thiazolidine-based inhibitors His/Asp phosphorylation comprising the two-compo- that bind to a conserved LRXK-motif in the DNA- nent system (TCS) is the classical type of prokaryotic binding domain of MtrA, RegX3, and MprA together signaling mechanism. TCSs are quintessential in sens- [81]. Interestingly, the Lys residue that undergoes ing and responding to environmental stimuli through pupylation is in this same pocket as LRXK-motif of coordinated changes in gene expression [68]. Mycobac- MtrA and regulates the protein turnover [82]. How- teria exploit these TCSs to adapt, survive, and grow ever, it is not known if the pupylation of MtrA is under extreme stimuli including hypoxia, nutrient limi- blocked by thiazolidine-based inhibitors or vice versa. tation, presence of reactive oxygen and nitrogen inter- Similar importance of PTM was reported for inor- mediates, pH alterations, and cell envelope stress [69]. ganic phosphate (Pi)-sensing signal transduction system The response of TCSs to a variety of environmental in the development of drug resistance in Mtb. The sig- signals can result in the development of antibiotic naling molecule PhoY (phoY1 and phoY2) can pro- resistance by four major ways: by modifying the com- mote RIF resistance and persister formation by position of the cell surface, altering the kinetics of mediating Pst/SenX3-RegX3 phosphate sensing drug influx, upregulation of antibiotic-degrading [74,83,84]. Both PhoY1 and PhoY2 proteins play a enzymes and alternative forms of antibiotic resistance, redundant role in preventing activation of SenX3- including persister formation or biofilm production RegX3 TCS when Pi is abundant. Considering that [70]. The role of TCSs to circumvent host-derived drug resistance and persister defects of DphoY1/Y2 stress is well documented [69,71,72]. Owing to their double mutant are reversed by the deletion of the importance in bacterial physiology, TCSs ascend as a regX3 gene [74], it can be assumed that the altered potential target for antibacterial drug designing or vac- phenotype is an outcome of dysregulated metabolism cine development against TB. because of hyperactive SenX3-RegX3 TCS.

6 The FEBS Journal (2020) ª 2020 Federation of European Biochemical Societies G. Arora et al. PTMs and drug resistance in Mycobacterium

Another highly conserved TCS PhoPR is possibly energy levels [96]. Recently, acetylome analysis in mul- involved in sensing acidic pH in mycobacteria, whereas tiple studies reported several TCS proteins to be acety- the genetic defect in phoPR leads to attenuated growth lated in Mtb, including RRs such as RegX3, PhoP, of the strain in low-Mg2+ conditions [85,86]. PhoP PdtaR, MtrA, and TcrX [93,97–99]. The MtrAB and (RR) controls the transcription of several genes includ- TcrXY operon can cross-talk in the cell, which is con- ing pks 2, 3, 4, and mls3 involved in the biosynthesis trolled by the acetylation of TcrX and MtrA [89,100]. of the lipids such as sulfatides, polyacyltrehaloses, and Therefore, understanding of such intermolecular inter- diacyltrehaloses [86]. Deletion of phoP causes hyper- actions is important for drug development. sensitivity to antibiotics like vancomycin and cloxacil- Acetylation directly links acetyl-CoA metabolism to lin, and attenuation of the mycobacteria in mice due cellular signaling by regulating enzyme activity, local- to the absence of the above-mentioned lipids. ization, and protein–protein interactions [94,101].N- Mtb is known to stay dormant for several years acetylation can occur enzymatically by N-acetyltrans- within the host without dividing and causing an active ferase and nonenzymatically by acetyl-CoA [102]. infection. An important group of genes involved in However, once acetylated, the modification can be maintaining mycobacterial dormancy is DosR regulon reversed by deacetylase enzymes [103,104].InMtb, [87]. Even though DosRS is not directly involved in enhanced intracellular survival protein (Eis) is an drug resistance in mycobacteria, DosRS regulon helps acetyltransferase targeting aminoglycosides such as the bacteria to maintain a nonreplicating or low-meta- amikacin, capreomycin, and kanamycin and plays an bolic state [88], under which most of the current anti- important role in resistance to this class of drugs [105– TB drugs fail to work. An important regulation of the 107]. Recent studies suggest that Eis can also acetylate DosR transcription factor is by acetylation at Lys182 proteins in vivo and affect chromatin accessibility which is essential for its DNA-binding activity. Upon [108–110]. Finally, a proteomics study identified that sensing the low-metabolic state of bacteria, like hypox- Eis itself gets acetylated [99,111], but the role of acety- ia, Lys182 in DosR undergoes deacetylation and pro- lation on Eis activity and interaction with aminoglyco- motes gene regulation of hundreds of hypoxia-induced sides remains unknown. genes [89,90].InMtb, acetylation is dependent on HupB (HU) is a DNA-binding protein, which regu- cAMP levels and hypoxia may promote deacetylation lates genome compaction and expression in mycobac- by decreasing the local cAMP level, although the teria. Among others, the expression of HupB is direct link between hypoxia, cAMP levels, and affected during the selection pressure of INH in deacetylation is yet to be established [89,91]. Overall, Msmeg [112]. Like many other DNA-binding histone there are limited studies on the role of TCS in drug proteins, HupB is also regulated by acetylation, in resistance; however, examples such as MtrA, DosR, addition to methylation. Mutation in the acetylation and PhoY1/PhoY2 indicate TCS signaling may play site Lys86 of HupB alters mycobacterial gene expres- an important role in the evolution of drug resistance. sion and results in the emergence of INH-resistant forms of mycobacteria [112]. Interestingly, Eis also Acylation acetylates HupB at various Lys residues including Lys86, affecting HupB: DNA interaction [108]. N-Acylation is the transfer of an acyl group from a An important proteomic study on clinical isolates donor (e.g., acetyl-CoA mediates acetylation) to the e- belonging to Mtb Lineage 7 and 4 strains identified amino group of a Lys residue in the target protein various N- and O-acetylated proteins (acetylation at [92,93]. Recent advances in mass spectrometry identi- the –OH group of Ser and Thr residues by an acetyl- fied several types of acylations, such as formylation, transferase) [111]. The study presented a list of pro- butyrylation, propionylation, succinylation, malonyla- teins involved in drug resistance that are modified by tion, myristoylation, glutarylation, and crotonylation acetylation, including PknH, EmbR, PhoP, MurF, [94]. KatG, InhA, Eis, GyrA/GyrB, RpoB, FabG1, and An interesting aspect is the dual regulation of RRs RpsL, associated with INH, EMB, FQs, kanamycin, by acetylation in addition to phosphorylation. Acetyla- and vancomycin resistance (Fig. 3). tion of proteins in a cell is an energy-coupled process As discussed earlier, STPKs are key regulators of because the intracellular levels of acetyl-CoA, acetyl physiology and metabolic processes in mycobacteria. phosphate, or acetate are associated with metabolism Four of Mtb STPKs- PknD, PknK, PknG, and PknH [95]. Cellular adaptation depends on the acetylation of were also found to be acetylated in different studies signaling proteins as one of the mechanisms, main- [93,99,111]. EmbR, which is phosphorylated by PknH tained through carbon metabolism and intracellular and plays a key role in EMB resistance, is also

The FEBS Journal (2020) ª 2020 Federation of European Biochemical Societies 7 PTMs and drug resistance in Mycobacterium G. Arora et al. acetylated. Wag31, the homolog of DivIVA involved phosphorylation and acetylation [129]. O-glycosylation in chromosome segregation and division site selection, in mycobacteria is catalyzed by the periplasmic, mem- is acetylated as well as phosphorylated [99,111]. brane-associated enzyme protein O-mannosyltrans- Wag31 is upregulated in MDR Mtb strains [113], and ferase (Pmt), which transfers mannose units (1–3 per its mutations (Q201R) are associated with resistance to site) to the specific Ser/Thr residue(s) of the substrate. the novel mycobactericidal agent, amino pyrimidine- Pmt activity and hence protein O-glycosylation in sulfonamide (APYS1) [114]. mycobacteria appear to associate with protein secre- Drug exposure induces the expression of chaperone tion [130–132]. While much work has focused on the groEL, which helps in stress response by maintaining role of glycosylated mycobacterial proteins in host– the proper folding of proteins that are affected by drug pathogen interactions and immune detection, relatively toxicity. Several transcriptional and proteomics studies lesser efforts have been dedicated to understanding the identified GroEL2 to be highly expressed in exposure roles of protein glycosylation events in drug sensitivity to LZD, streptomycin, and ofloxacin [37,115–117].In and resistance. mycobacterium, GroEL2 is acetylated on multiple sites Studies employing deletion mutants of Pmt have such as Lys33 that is present next to ATP/Mg2+ bind- demonstrated the importance of protein glycosylation ing site 30LGP32. Other acetylated residues, Lys140, in mycobacterial virulence and persistence [132,133]. and Lys195 are involved in the hinge region and Mycobacterium abscessus showed enhanced sensitivity oligomerization interface [99]. Despite being highly of a Pmt deletion strain toward certain bulky antibi- studied, there is yet no direct correlation established otics (bacitracin, RIF) and those targeting peptidogly- between GroEL PTMs and its involvement with drug- can (b-lactams, vancomycin), a result of increased cell resistant phenotype. Acetylation of PpiA and MurF permeability in this strain due to the lack of cell-sur- also linked the processes of protein folding and cell face glycosylated proteins [133]. While the exact reason wall synthesis to resistance against the drugs like for increased permeability in a Pmt deletion mutant is cyclosporine A and vancomycin [93,118]. not yet known, it points to an association between gly- The second most common form of protein acylation cosylated membrane proteins providing a structural is succinylation, involving the transfer of succinyl barrier and intrinsic resistance to antitubercular com- group to a protein at its Lys residue, such as KatG is pounds. Also, Pmt-deficient Mtb shows increased sen- succinylated at Lys310 [119]. KatG is a catalase-perox- sitivity toward oxidative stress, as evidenced by an idase that activates INH [120,121]. The most common inability to grow on solid media lacking exogenous INH resistance mechanism has been identified as the catalase [132], and increased SigH (sigma factor mutations at Ser315 and Arg463 residues of KatG, involved in the bacterial response to oxidative stress) which makes it unable to activate INH, causing an expression [131]. Optimal protein glycosylation may, increase in MIC by 100-fold [122–126]. The succinyla- therefore, be a resistance determinant for compounds tion of KatG at Lys310 (near Ser315) also reduces the that generate reactive oxygen species as part of their activation of INH and confers resistance by increasing mechanisms of action, such as clofazimine and INH. INH MIC by 200-fold [111,119,124]. Besides succinyla- Several antimicrobial resistance genes are glycosy- tion, KatG also gets acetylated at Lys410 and Lys688 lated, including well-known front-line drug-resistance [99]; however, the role of these sites in drug resistance determinants such as RpoB, KatG, BlaC, EthA, and remains unknown. These studies indicate succinyla- GyrAB, as well as several reported antibiotic efflux tion-mediated control of KatG activity may contribute pumps (Rv0194, Rv2994, Rv1273c) [127]. Interestingly, to INH resistance. glycosylation levels of individual proteins were shown to differ across various clinical isolates, providing a Glycosylation possible mechanism of phenotypic and drug-resistance diversity across these strains. Furthermore, the Mtb Protein glycosylation, the addition of mono- or Beijing lineage, a strain known to have enhanced viru- oligosaccharides on amino acid side chains, is an lence and resistance toward many antitubercular drugs enzyme-catalyzed PTM conserved across all domains (Fig. 3) [134], has previously been observed to express of life. It can occur on either Asn (N-linked) or Ser/ higher Pmt levels relative to other strains, supporting Thr residues (O-linked), although the former is rare in this hypothesis [135]. bacteria and has only recently been identified in Specific roles of glycosylation in mycobacteria are mycobacteria [127,128]. A recent study listed 233 pro- confounded by its high degree of association with pro- teins with known glycosylation sites in Mtb, making it tein S-acylation [131]. S-acylation, the covalent addi- one of the more common PTMs after STY tion of fatty acid chain to conserved Cys residues by

8 The FEBS Journal (2020) ª 2020 Federation of European Biochemical Societies G. Arora et al. PTMs and drug resistance in Mycobacterium thioester linkages, is predominantly catalyzed by the Biotinylation essential enzymes lipoprotein diacylglycerol trans- ferase, lipoprotein signal peptidase (LspA), and The majority of antimycobacterial drugs are designed apolipoprotein N- acyltransferase (Lnt). Loss of the against the proteins involved in fatty acid and lipid lipoprotein signal peptide peptidase LspA leads to synthesis pathway, because of the critical importance reduced periplasmic lipoprotein abundance, and con- of mycolic acid constituting fatty acids in mycobacte- comitantly a greater susceptibility to many front- and rial physiology and survival. Acyl-CoA carboxylases second-line antitubercular drugs [136]. While these (ACCs) are required to generate building blocks of studies have investigated the effects of loss of entire Malonyl-CoA, which decide the fate of fatty acid lipo/glycoproteins on different aspects of mycobacte- chains. To perform this function, ACCs require a bio- rial biology, it will also be interesting to observe if tin cofactor. ACCs undergo biotinylation, which is similar effects are seen with just the loss of the PTM mediated by biotin protein ligase (BirA/BPL). Biotiny- itself, through mutagenesis of the modified amino acid. lation activates the ACCs which can carry out car- boxylation of acyl-CoA leading to Malonyl-CoA synthesis [151,152]. The mycobacterial genome con- Pupylation tains multiple ACCs, all requiring biotinylation-based Pupylation is a PTM on Lys residues that marks the activation, thus regulating overall fatty acid synthesis. protein for degradation by protease complexes, similar Interestingly, another enzyme pyruvate coenzyme A to eukaryotic ubiquitinylation [137,138]. Pupylation is carboxylase also gets biotinylated by BirA and medi- unique to actinobacteria and due to its impact on pro- ates lipid catabolism during gluconeogenesis. Thus, teasomal machinery, pupylation is considered an biotinylation and BirA activity decides the lipid con- attractive target for the development of new drugs tent of mycobacteria. Inhibitor of BirA can also against Mtb [139,140]. Proteasome accessory factor A enhance the potency of current TB drugs. This was (PafA) attaches prokaryotic ubiquitin-like protein suggested in a recent study showing the increased (Pup), to the Lys residues of proteasome substrates potency of INH and RIF when combined with BirA inhibitor Bio-AMS (50-[N-(d-biotinoyl) sulfamoyl] (Fig. 2) [138,141]. PafA, along with PafB and PafC, is 0 encoded by an operon. They were shown to be impor- amino-5 -deoxyadenosine) [153]. Since Bio-AMS is tant in protecting Mtb from reactive nitrogen interme- selective for Mtb and its drug-resistant strains, it does diates (RNI) [142,143]. The role of PafC in resistance not affect other commensal bacteria as well as human to broad-spectrum antibiotics, FQs including moxi- cell lines [152]. These studies show that inhibition of floxacin, norfloxacin, ofloxacin, and nalidixic acid, was biotinylation function can be utilized for increasing the recently described in Msmeg [144]. The PafC mutant potency of existing drugs. was twice more susceptible to moxifloxacin-mediated killing, which was also confirmed by enhanced vulner- Phosphopantetheine ability to reactive oxygen exposure [144,145]. Interest- 0 ingly, RNIs have been implicated in the 4 -Phosphopantetheine (Ppant) is a metabolite in the antimycobacterial activity of the nitroimidazole drug, pantothenate and CoA pathway. In this modification, pretomanid, although the role of pupylation in resis- Ppant moiety gets attached to the active site Ser of tance to pretomanid remains unknown [146]. ACPs, peptidyl carrier proteins, and Aryl carrier pro- – One of the proteins identified to be pupylated in teins [154 156]. Phosphopantetheinyl transferases Msmeg by proteomics study was ribosomal L3 protein (PptT) are responsible for adding the free thiol moiety (RplC), which is responsible for LZD resistance in Mtb of Ppant from CoA to acyl reaction intermediates as [147,148]. Although the modification site identified in thioesters, a reaction mimicking Ser phosphorylation the proteomics study is conserved between Msmeg and [154,157]. Ppant addition converts inactive apo-syn- Mtb, its role in LZD resistance is not known. Another thases to active holo-synthases [155]. Attachment of important set of candidates associated with INH resis- the Ppant serves as a prosthetic arm of carrier protein, tance that are pupylated are KasA and KasB, associated which enables them for biosynthetic catalyses such as withcellwallbiosynthesis[82,149]. Pupylation was also fatty acid synthesis, polyketide synthesis, and peptide identifiedonRpoC,theb0 subunit of RNA polymerase, synthesis. The intrinsic drug resistance in Mtb could where rpoC gene mutations are shown to be associated be partially attributed to the lipid-rich cell wall with a – with increased in vitro fitness and resistance to RIF in low permeability [158 162]. The PptT enzyme in Mtb Mtb, although more work is needed to substantiate the plays an important role in the formation of mycolic contribution of pupylation (Fig. 3) [149,150]. acids and lipids [163]. PptT affects the persistence and

The FEBS Journal (2020) ª 2020 Federation of European Biochemical Societies 9 PTMs and drug resistance in Mycobacterium G. Arora et al. is essential for survival and replication of bacilli during to the compounds, are poorly understood. To respond the infection in mice [159]. A recent study identified to environmental cues, Mycobacterium has evolved compound 8918, which targets PptT, as a promising complex signaling schemes that are interconnected. Mtb drug since it impairs the synthesis of trehalose The existence of cross-resistance, where resistance to dimycolates, trehalose monomycolates, and phthio- one antimicrobial compound increases the resistance cerol dimycocerosates. Interestingly, another enzyme to other drugs and, collateral sensitivity that leads to in the pathway PptH can hydrolyze the Ppant moiety enhanced susceptibility to another drug, indicates the and potentiate the mycobactericidal action of the role of signaling networks in antimicrobial resistance amidinourea. Further, mutations in PptH made [174–176]. This suggestes the presence of intercon- mycobacteria resistant to amidinourea. Identification nected signaling nodes, which regulate response to of PptH is exciting as this has shown, how the reversi- drug pressure by fine-tuning the activity of modified ble enzymes, balance each other and also help bacteria proteins (Fig. 3). gain resistance to a drug [164]. To survive in the presence of a potent drug, bacteria have to sense promptly and respond in terms of Methylation altered transcription, translation, and enzyme func- tions. PTMs are known to play a key role in regulat- Methylation of Arg, Lys, and His residues of histones ing these processes that may confer drug resistance; is an indispensable modification that impacts various however, this has not been evaluated in detail. In this biological processes [165]. The genome of Mtb encodes review, we discussed the examples of different PTMs several methyltransferases and demethylases, although such as phosphorylation, acetylation, and glycosyla- their role in acquired drug resistance remains largely tion, in directing mycobacterial responses to modulate unexplored. Erythromycin ribosome methylase antimicrobial resistance. Many of these proteins were (Erm37) modifies 23S rRNA and provides Mtb an found to be regulated by multiple PTMs, indicating intrinsic resistance to macrolides [166,167]. This their importance in sensory signaling. One of the secreted enzyme is also shown to reach the host examples is HupB, which is post-translationally regu- nucleus and demethylates histone H3, which represses lated by acetylation, methylation, and phosphorylation the expression of genes involved in ROS production [112,165,169]. Modification of Lys86 is associated with (including NOX1, NOX4, and NOXA1 [168]). As the formation of drug-tolerant colony variants, which mentioned earlier, mycobacterial HupB is known to be show enhanced resistance to INH. Consequently, methylated in addition to acetylation and Ser/Thr mutation of this Lys (abolishing its ability to be modi- phosphorylation [112,169]. It is unclear if Erm37 can fied) was associated with loss of bacterial ability to also methylate HupB. Given the importance of HupB form drug-tolerant colony variants. The result of these in INH resistance, it will be interesting to know how modifications on HupB is to bring about genome reor- methylation impacts acquired drug resistance (Fig. 3). ganization by alteration of chromatin structure. This in turn leads to epigenetic regulation of the drug-resis- Conclusions and Future Directions tant phenotype. The combined effect of different modi- fications on genome arrangements and drug resistance In 1949, the first antibiotic effective against Mtb, strep- remains unknown. We hypothesize that maintaining tomycin, was given to humans to treat TB. Within a dynamic control of different PTMs on a large number few years of usage, resistance to streptomycin was of Mtb proteins provides leverage that does not observed in Mtb. Since then, many chemical com- require excessive genome rearrangements and energy. pounds have been screened and shown to be active Another important target of multiple PTMs is against the Mtb, though very few compounds have MtrA, a two-component RR involved in the transcrip- been developed for human use [170–173]. However, tion of several important genes. Besides His/Asp phos- the bacterium quickly adapts and develops resistance. phorylation, MtrA is modified by Ser/Thr The advances in genomics revealed that acquired resis- phosphorylation, acetylation, and pupylation tance against these drugs is due to specific mutations [74,149,177]. MtrA and its sensor kinase MtrB have in the genes that are related to the drug’s mechanism been implicated in sensitizing the mycobacterial cells of action or by modulation of regulatory processes, to multiple drugs [76,78–80]. PTMs regulate the activ- which help mycobacteria tolerate, persist, and develop ity of MtrA under different environmental conditions, drug resistance. The mechanisms by which bacteria which culminate in altered expression of genes and sense signals because of drug pressure and modulate thus downstream pathways. It would be interesting to their responses, allowing the development of resistance find if there is a direct correlation between MtrA

10 The FEBS Journal (2020) ª 2020 Federation of European Biochemical Societies G. Arora et al. PTMs and drug resistance in Mycobacterium regulation by PTMs under drug pressure. Therefore, 5 Van Deun A, Decroo T, Tahseen S, Trebucq A, drugs targeting key nodes of this interconnected web Schwoebel V, Ortuno-Gutierrez N, de Jong BC, Rieder of PTMs will be highly desirable for these constantly HL, Piubello A & Chiang CY (2019) WHO 2018 evolving bacteria. treatment guidelines for rifampicin-resistant tuberculosis: uncertainty, potential risks and the way forward. Int J Antimicrob Agents 55, 105822. Acknowledgement 6 Olayanju O, Esmail A, Limberis J, Gina P & Dheda K We acknowledge the NIH Fellows editorial board for (2019) Linezolid interruption in patients with help in manuscript editing. AB is supported by the fluoroquinolone-resistant tuberculosis receiving a bedaquiline-based treatment regimen. Int J Infect Dis intramural research program of the National Institute 85,74–79. of Allergy and Infectious Diseases, NIH. 7 Guglielmetti L, Jaspard M, Le Du D, Lachatre M, Marigot-Outtandy D, Bernard C, Veziris N, Robert J, Conflict of interest Yazdanpanah Y, Caumes E et al. (2017) Long-term outcome and safety of prolonged bedaquiline treatment The authors declare no conflict of interest. for multidrug-resistant tuberculosis. Eur Respir J 49, 1601799. Author contributions 8 Veziris N, Bernard C, Guglielmetti L, Le Du D, Marigot-Outtandy D, Jaspard M, Caumes E, Lerat I, All the authors have read and approved this review Rioux C, Yazdanpanah Y et al. (2017) Rapid manuscript. Specific section contributions are as fol- emergence of Mycobacterium tuberculosis bedaquiline lows: GA and AS conceptualized the study. GA coor- resistance: lessons to avoid repeating past errors. Eur dinated and supervised the study. GA, AB, KA, GP, Respir J 49, 1601719. and AS involved in primary draft compilation. AS and 9 Richter E, Rusch-Gerdes S & Hillemann D (2007) GA prepared the figures. GA, AB, KA, GP, and AS First linezolid-resistant clinical isolates of performed literature survey and bibliography. AS and Mycobacterium tuberculosis. Antimicrob Agents AB collected supplementary information. Chemother 51, 1534–1536. 10 Bothra A, Arumugam P, Panchal V, Menon D, Srivastava S, Shankaran D, Nandy A, Jaisinghani N, Peer Review Singh A, Gokhale RS et al. (2018) Phospholipid The peer review history for this article is available at homeostasis, membrane tenacity and survival of Mtb https://publons.com/publon/10.1111/febs.15582. in lipid rich conditions is determined by MmpL11 function. Sci Rep 8, 8317. 11 Arumugam P, Shankaran D, Bothra A, Gandotra S & References Rao V (2019) The MmpS6-MmpL6 operon is an oxidative stress response system providing selective 1 Lange C, Dheda K, Chesov D, Mandalakas AM, advantage to Mycobacterium tuberculosis in stress. Udwadia Z & Horsburgh CR Jr (2019) Management J Infect Dis 219, 459–469. of drug-resistant tuberculosis. Lancet 394, 953–966. 12 Eldholm V & Balloux F (2016) Antimicrobial 2 Xie YL, Chakravorty S, Armstrong DT, Hall SL, Via resistance in Mycobacterium tuberculosis: the odd one LE, Song T, Yuan X, Mo X, Zhu H, Xu P et al.(2017) out. Trends Microbiol 24, 637–648. Evaluation of a rapid molecular drug-susceptibility test 13 Sigrist CJ, de Castro E, Cerutti L, Cuche BA, Hulo N, for tuberculosis. NEnglJMed377,1043–1054. Bridge A, Bougueleret L & Xenarios I (2013) New and 3 Bloom BR, Atun R, Cohen T, Dye C, Fraser H, continuing developments at PROSITE. Nucleic Acids Gomez GB, Knight G, Murray M, Nardell E, Rubin E Res 41, D344–D347. et al. (2017) Tuberculosis. In Major Infectious 14 Sajid A, Arora G, Singhal A, Kalia VC & Singh Y (2015) Diseases, Chapter 11, 3rd edn (Holmes KK, Bertozzi Protein phosphatases of pathogenic bacteria: role in S, Bloom BR & Jha P, eds), pp. 233–313. The physiology and virulence. Annu Rev Microbiol 69,527–547. International Bank for Reconstruction and 15 Sajid A, Arora G, Gupta M, Upadhyay S, Nandicoori Development, Washington, DC. VK & Singh Y (2011) Phosphorylation of 4 Olayanju O, Limberis J, Esmail A, Oelofse S, Gina P, Mycobacterium tuberculosis Ser/Thr phosphatase by Pietersen E, Fadul M, Warren R & Dheda K (2018) PknA and PknB. PLoS One 6, e17871. Long-term bedaquiline-related treatment outcomes in 16 Alber T (2009) Signaling mechanisms of the patients with extensively drug-resistant tuberculosis Mycobacterium tuberculosis receptor Ser/Thr protein from South Africa. Eur Respir J 51, 1800544. kinases. Curr Opin Struct Biol 19, 650–657.

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as regulators of bedaquiline tolerance in Supporting information Mycobacterium tuberculosis. Nat Microbiol 1, 16078. 177 Carette X, Platig J, Young DC, Helmel M, Young Additional supporting information may be found AT, Wang Z, Potluri LP, Moody CS, Zeng J, Prisic S online in the Supporting Information section at the end et al. (2018) Multisystem analysis of Mycobacterium of the article. tuberculosis reveals kinase-dependent remodeling Table S1. List of Mtb proteins associated with antimy- of the pathogen-environment interface. mBio 9, cobacterial agents and their post-translational modifi- e02333-17. cations.

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