A comprehensive review on epigenetic and epitranscriptomic-mediated regulation of antibiotic resistance

2.1 DNA methylation role in antibiotic resistance

The methyltransferase M. ngoAV, found in Neisseria gonorrhoeae, regulates antibiotic resistance. Examination of the mechanism revealed that the knockout mutant, which is more susceptible to bacitracin, demonstrated decreased sensitivity to imipenem and cefotaxime in comparison to the wild-type strain. Altered gene expression included upregulated rlpA (peptidoglycan lytic transglycosylase) and downregulated envC in the mutant. -peptidoglycan.¹⁴ AamA methyltransferase of Acinetobacter baumannii has been reported to modulate antibiotic resistance. Mechanistic studies have shown that mutations result in decreased trmD operon expression, lower antibiotic minimum inhibitory concentrations (MICs) (Polymyxin B, Erythromycin, and Kanamycin A), and diminished ethidium bromide (EtBr) efflux pump activity.¹⁵ The ModA11_ON 1R variant displayed twice the susceptibility to doxycycline, nalidixic acid, and cloxacillin and four times more susceptibility to ciprofloxacin and ceftazidime compared to the modA11::kan variant. Similarly, the ModA12_ON variant exhibited a two-fold increase in sensitivity to cloxacillin, rifampin, and cephalothin compared with the modA12::kan variant.¹⁶ N. gonorrhoeae regulates antibiotic resistance against triton-X through DNA methyltransferase modA13. The mutant modA13 showed upregulation of mtrF, suggesting that it is regulated through modA13.¹⁷ The deactivation of ModS2 in Streptococcus suis resulted in a two-fold increase in ampicillin resistance compared to the activation of ModS2.¹⁸ In Escherichia coli K-12(E. coli K12), removing the dam gene increased ciprofloxacin sensitivity. Similarly, deleting the dam gene in a clinically obtained highly ciprofloxacin-resistant UPEC strain with several mutations conferring resistance to quinolones substantially reduced the ciprofloxacin MIC by more than 50% and MBC90 by 4.6-fold, albeit not fully restoring sensitivity.¹⁹ The lack of Dam or Dcm results in decreased half-maximal effective concentration (EC50) values for a broad range of antibiotics, including β-lactams, tetracyclines, quinolones, aminoglycosides, and macrolides, against Escherichia coli MG1655(E. coli MG1655).²⁰

The double mutant Δdam ΔrecA showed either no growth or delayed growth after 24 hours in the presence of quinolones, contrasting with the control strain. Spot tests indicated that the Δdam ΔrecA double mutant exhibited increased sensitivity compared to both the ΔrecA single mutant and the wild type in both resistant and susceptible genetic backgrounds.²¹ Escherichia coli XL1-Blue (E. coli XL1-Blue) strains, when exposed to nalidixic acid, exhibited a five-fold enhancement in bacterial survival due to elevated dam expression. This increased resistance was correlated with two-fold upregulation of efflux pumps.²² Cells lacking the dcm gene exhibit increased expression of the drug resistance transporter SugE, which is associated with resistance to ethidium bromide (ETBR). Additional investigation revealed that cells with Dcm knockout exhibited greater resistance to EtBr compared to wild-type cells, and the reintroduction of a plasmid-borne dcm gene reinstates EtBr sensitivity. Conversely, cells without SugE displayed higher sensitivity to EtBr than wild-type cells.²³ Genes trmJ, rlmH, and rlmB exhibited increased expression in ampicillin- and gentamicin-resistant E.coli respectively. Conversely, the gentamicin-resistant line showed downregulation of dcm. This finding suggests that methyltransferases play a role in antibiotic resistance.²⁴

2.2 Nucleoid Associated Proteins (NAPs) role in antibiotic resistance

Nucleoid-associated proteins, such as H-NS, have also been found to regulate antibiotic resistance, and NapM regulates various antibiotic resistance genes (see Figure 2, and Table 1). In the A. baumannii AB5075 Δhns strain, there was upregulation of genes related to resistance against aminoglycosides, β-lactams, quinolones, trimethoprim, colistin, sulfonamides, chloramphenicol, and colistin. Conversely, compared to the parental strain, genes associated with tetracycline resistance were downregulated in the Δhns strain. Additionally, there was an upregulation of efflux pump-coding genes in the AB5075 Δhns strain.²⁸ Mutation of H-NS in colistin-resistant A. baumannii led to high colistin resistance. Mechanistic studies revealed that in colistin-resistant A. baumannii with an H-NS mutation, there was an increase in the expression of eptA, which is responsible for a second lipid A-specific pEtN transferase. Simultaneously, expression of pmrC, another relevant gene, remained unchanged.²⁵ H-NS regulates multidrug resistance by affecting gene expression in E. coli. Deleting H-NS in DeltaacrAB increased the resistance to erythromycin, oxacillin, doxorubicin, acriflavine, crystal violet, novobiocin, ethidium bromide, methylene blue, tetraphenylphosphonium bromide, sodium dodecyl sulfate, sodium deoxycholate, rhodamine 6G, and benzalkonium chloride. Dual acrEF and mdtE deletion suppressed Δhns-facilitated resistance, suggesting derepression of drug exporter genes.²⁹ Regulation of the AcrEF multidrug efflux pump by H-NS has also been reported in Salmonella enterica serovar Typhimurium.²⁶ NapM, an NAPs that binds to AT-rich DNA of the major groove to protect DNA from DNase I digestion, was found to confer resistance in Mycobacterium smegmatis against rifampicin and ethambutol. Mechanistic studies have shown that the ABC transporter operon is responsible for the napM-dependent ethambutol resistance. NapM regulates anti-tuberculosis drug resistance in Mycobacterium tuberculosis.²⁷ Various NAPs are still poorly explored and require urgent investigation for a more comprehensive understanding of antibiotic resistance. Exploration of epigenetic modifications will unravel a new key regulatory cascade for the regulation of antibiotic resistance, offering a new opportunity to decrease antibiotic resistance mortality.

3.1 Epitranscriptomic modification of rRNA role in antibiotics resistance

Ribosomes is one of major target of antibiotics, and rRNA methylation are emerging as a crucial epitranscriptomic modification to regulate antibiotics resistance along with dysregulation of non-coding RNA expression which epitranscriptomic modification is poorly explored (see Figure 2 & Table 2).^1,4 Ribosomal RNA (rRNA) is a central target of aminoglycoside antibiotics, such as streptomycin, amikacin and 16 srRNA methylase producing A. baumannii and P. aeruginosa, which were found to regulate aminoglycoside resistance.³⁴ Of the 60 amikacin-resistant isolates, the presence of the armA gene, responsible for 16S rRNA methylation, was the sole prevalent gene identified.³⁵ Aminoglycoside-resistant 16S rRNA (m7G1405) methyltransferase RmtC confers aminoglycoside resistance.³⁶ sgm methyltransferase imparts resistance to aminoglycosides by adding m(7)G1405 to 16S rRNA’s A site. Escherichia coli’s 16S rRNA has methylations, such as m(5)C1407 and m(4)Cm1402. Further studies showed that RsmF faces obstacles modifying m(5)C1407 due to sgm accessing adjacent G1405 on the 30S subunit.³⁷ The methylation of U747 and/or U1939 by RlmCD enhances subsequent G748 methylation by RlmAII, promoting effective binding of telithromycin (TEL) to the ribosomes in S. pneumoniae to modulate TEL resistance.³⁸ Erythromycin-resistant methyltransferases such as ErmC, belonging to methyltransferases with a Rossmann fold dependent on S-adenosylmethionine (SAM), methylate at N6 of A2058 in 23S rRNA, which obstructs the binding of various antibiotic classes to 23S rRNA, to confer an MDR phenotype such as erythromycin resistance in bacteria expressing the enzyme.³⁹ Clostridium bolteae 90B3 and Clostridium difficile T10 with Cfr rRNA methyltransferase confer oxazolidinones, phenicols, lincosamides, streptogramin A (PhLOPSA), and pleuromutilin resistance through the alteration of 23S rRNA by m8A2503 methylation.⁴⁰ Similar results were also noted for both Escherichia coli and Staphylococcus aureus.⁴¹ E. coli protein methyltransferase yfgB (rlmN) was found to modify A2503 of the 23S rRNA to m2A. The yfgB knockout resulted in the absence of an alteration in 23S rRNA at nucleotide A2503. Further study showed that the E. coli rlmN-deficient strain exhibited a consistent two-fold increase in susceptibility to sparsomycin, hygromycin A, and tiamulin in comparison to the wild-type strain. Similarly, inactivation of rlmN in S. aureus resulted in a two-fold heightened vulnerability to linezolid.⁴²

3.2 Epitranscriptomic modification of tRNA role in antibiotics resistance

Transfer RNA (tRNA) is a fundamental type of non-coding RNA that assists in the translation of mRNA into proteins by attaching the correct amino acids to the growing polypeptide chain.⁴⁴ New tRNA-modifying enzymes have been identified, shedding light on their significance in bacterial physiology. For instance, truD is involved in Ψ13 modification, dusA (also known as yjbN) catalyzes D16 and D17 modifications, Um32 modification is mediated by trmJ (yfhQ), and trmJ (yfhQ) is responsible for Cm32 modifications.⁴⁵ tRNA modification is an emerging critical component of bacterial physiology. Several modifications have been reported to regulate biological processes. For example, a decrease in Mg2+ levels lowers TrmD activity, resulting in a reduced modification of tRNAPro. This reduction prompts the attenuation of the MgtL leader peptide, facilitating the expression of the mgtA transporter gene. The ratio of mcmo5U to cmo5U notably increased during growth. Low iron levels decrease MiaB activity, leading to a decreased modification of tRNASer. This diminishes the translation of uof and fur, which act as negative regulators of the low-iron response.⁴⁶ Recent research has broadened our understanding of how epitranscriptomic modifications of tRNA contribute to antibiotic resistance so we scrutizes the role of epitranscriptomic modification in tRNA in antibiotic resistance in this section (see Figure 2 & Table 2).

Methylation of m1G37-tRNA controls proline codons near the open reading frame, boosting bacterial drug resistance via trmD. Reduced m1G37 levels in Escherichia coli and Salmonella disrupt membrane integrity, hindering the development of ampicillin, carbenicillin, kanamycin, gentamicin, paromomycin, rifampicin, and ciprofloxacin resistance.⁴³ Further studies have shown that TrmD inhibitors could be used to overcome antibiotic resistance.⁴⁷ tRNA modification genes, such as Tgt, DusB, TruA/B/C, TrmA/B/E/H, and RlmN, were found to modulate responses to several antibiotics, such as aminoglycosides, fluoroquinolones, β-lactams, chloramphenicol, and trimethoprim in Vibrio cholerae suggesting that their role needs to be further explored to elucidate the role of tRNA modification in antibiotic resistance.⁴⁸ In addition, several tRNA modifications have been associated with bacterial survival. For example, m7G tRNA methyltransferase (TrmB) catalyzes m7G tRNA modification, which is crucial for stress responses and pathogenesis in Acinetobacter baumannii baumannii,⁴⁹ while the enzyme m1A22-tRNA methyltransferase (TrmK) transfers a methyl group from SAM to adenine 22 in tRNAs, which is crucial for Staphylococcus aureus survival during infection.⁵⁰ This finding suggests that epitranscriptomic modification of tRNA orchestrating key players can be targeted to develop new antibiotics or reverse antibiotic resistance to overcome the global threat of antibiotic resistance.

3.3 Epitranscriptomic modification of sRNA, and riboswitches role in antibiotics resistance

It is imperative to acknowledge that, while rRNA methylation has been a focal point in understanding epitranscriptomic modifications and their role in antibiotic resistance, a vast expanse of uncharted territory remains. The intricate world of non-coding RNA, encompassing cis and trans small non-coding RNA, as well as riboswitches and their respective targets, presents a rich tapestry of regulatory elements that are yet to be explored in terms of their methylation status. As we navigate the complex landscape of epitranscriptomics, it becomes evident that our current understanding is the tip of the iceberg. The myriad roles played by non-coding RNA in controlling antibiotic resistance are tantalizing, with profound implications for therapeutic interventions. Our exploration has led us to compile a comprehensive summary of non-coding RNA entities that orchestrate the intricate dance between RNA modification and antibiotic resistance, as presented in Table 3. However, the absence of detailed insights into the methylation status of these non-coding RNA elements underscores the need for further investigations. Unraveling the specific modifications that these regulatory elements undergo will undoubtedly provide a more nuanced understanding of their function in the context of antibiotic resistance. This crucial information could unveil new targets for intervention, shaping the landscape of antibiotic development and refining our strategies to overcome bacterial resistance. While our current understanding has shed light on the interplay between rRNA methylation and antibiotic resistance, it is a mere prelude to the broader symphony orchestrated by non-coding RNA. The journey into the epitranscriptomic regulation of antibiotic resistance is far from over, and the unexplored realms of non-coding RNA have motivated researchers to delve deeper into the intricacies of their methylation dynamics. Within this uncharted territory, the next chapter of epitranscriptomics in antibiotic resistance regulation awaits the promise of transformative insights for the future of antimicrobial strategies.