The Role Of Epigenetics In Mycobacterium Tuberculosis Antibiotic Resistance

Tuberculosis,caused by the intracellular pathogen agent Mycobacterium tuberculosis?Mtb?,an ancient and important infection disease,is still the threat to human health.Nearly one-third of the global population was infected by latent tuberculosis.In 2015,an estimated 10.4 million people developed TB and China alone accounted for 7.7% of total cases;1.4 million died from this disease.As one of most successful pathogens of human being,M.tuberculosis owns three “magic weapons”,asymptomatic persistence,virulence and antibiotic resistance.Post-translational modifications?PTMs?are important foreukaryotic and prokaryotic protein function.Protein lysine acetylation?normally referred to N?-lysine acetylation?,a dynamic andreversible PTM,is the transfer of acetyl moiety from acetyl-CoA to the ?-amino groups of lysine residues in proteins.N?-lysine acetylation not only can alter DNA binding activity and thus gene expression,but also can regulateprotein–protein interactions,protein activity,mRNA stability,coordinating carbon source utilization,and metabolic flux.sRNAs?small RNA?,as the name indicates small transcripts,mostly in the range of 50 to 500 nucleotides.There are two types in bacteria: cis-encoded s RNAs and trans-encoded s RNAs.Both of them can function regulatory role by influencing transcription,translation and mRNA degradation.It had been demonstrated that sRNAs play important roles in iron metabolism,acid resistance,oxidative stress,virulence,intracellular survival and so on.Hence,lysine acetylation,glutarylation and s RNAs may involve in bacterial physiology.Based on the above points,the paper mainly contains the following parts:With a combination of anti-acetyllysine antibody-based immune-affinity enrichment with high-resolution mass spectrometry,we identified 1128 acetylation siteson 658 acetylated M.tuberculosis proteins.GO analysis of the acetylome showed that acetylated proteins are involved in the regulation of diverse cellular processes including metabolism and protein synthesis.Six types of acetylated peptide sequence motif were revealed from the acetylome.Notably,several proteins including isocitrate lyase involved in the persistence,virulence and antibiotic resistance are acetylated,and site-directed mutagenesis of isocitrate lyase acetylation site to glutamine led to a decrease of the enzyme activity,indicating major roles of KAc in these proteins engaged cellular processes.Through bioinformatics method,we identified 47 acetyltransferases in the M.tb proteome,which use diverse substrates–antibiotic,amino acids,and other chemical molecules.A comparative analysis of the proteome of the virulent strain H37 Rv and the avirulent strain H37 Ra identified one acetyltransferase,which displayed significant variations in terms of N-terminal deletion,possibly impacting various physicochemical properties.We also found that several acetyltransferases can be acetylated,succinylated or glutarylated.The genome context and co-transcription analysis showed that many acetyltransferases with their neighboring genes belong to one operon,and three of them were confirmed by RT-PCR.Through analyzing previous published “omics” data,we revealed that most of acetyltransferases were down regulated under late stationary phase or low-oxygen dormancy.In order to further explore the function of lysine acetylation,using M.smegmatis as the model organism,we performed a proteome-wide analysis of lysine acetylation.In total,620 acetylation sites and 345 acetylated proteins were identified.The bioinformatics analysis showed that acetylated proteins are involved in diverse known biological process,and found that there are six acetylation motifs including KACH,KACY,KACF and F*KAC.In particular,many of proteins involved in fatty acids are highly acetylated.Based on these,we constructed MSMEG₅458?encodes an acetyltransferase?gene deletion mutant,and found that ?MS5458 mutant showed remarkable resistance to INH and SDS.Results of GC-MS showed that lysine acetylation could affect fatty acid metabolism.We found that 102 differential proteins could be the substrates of MSMEG₅458,including the enoyl-acyl carrier protein reductase?inhA?involved in mycolic acid synthesis.Results of site-mutation and spot test showed that the acetylation of inhA can not lead to the resistance to INH.A decrease in cellular NAD content and increase in cellular NADH content were found and might underlie the resistance of ?MS5458 mutant strain to INH.To explore the distribution of lysine glutarylation in M.tuberculsosis,by using a comprehensive method combining the immune affinity peptide enrichment by the glutaryl-lysine antibody with LC-MS,we finally identified 41 glutarylation sites in 24 glutarylated proteins.These glutarylated proteins are involved in various cellular functions such as translation and metabolism and exhibit diverse subcellular localizations.Three common glutarylated proteins including 50 S ribosomal protein L7/L12,elongation factor Tu,and dihydrolipoamide succinyltransferase are shared between Escherichia coli and M.tuberculosis.Moreover,comparison with other PTMs characterized in M.tuberculosis,15 glutarylated proteins,are found to be both acetylated and succinylated.Notably,several stress-response-associated proteins including HspX are glutarylated.Finally,this paper explored the upstream regulator sRNA and downstream target s RNA of the master regulator IdeR of iron metabolism.By analyzing previous published RNA sequencing data in M.tuberculosis,a partial RNA sequence encoded in-cis to the IdeR gene?called ncRv2711c?was revealed and validated by Northern blot?DIG-labeled probe?and RT-PCR method.Then we developed a simple and efficient method for identification of transcription start site and stop site of bacterial small RNAs,RJEM and validated the accuracy of this method by using E.coli RyhB and M.tuberculosis Mcr7 as the research object.The transcription start site and stop site of ncRv2711 c was identified by RJEM.RT-PCR analysis showed that the transcription of ncRv2711 c was expressed mostly in stationary phase and also under iron limitation.ncRv2711 c overexpressed M.tuberculosis strain?ST377?showed a growth defect under high iron but no difference under low iron condition.Results of western blot showed that overexpression of ncRv2711 c resulted in a decrease in the IdeR protein level.qPCR results showed that the overexpression of ncRv2711 c leads to an increase in the mRNA level of MbtB?involved in iron acquisition?but a decrease in the mRNA level of BfrB?involved in iron storage?.RNA sequencing technologies?RNA-Seq?was used to explore differentially expressed sRNAs of M.tuberculosis under low iron and high iron conditions,and finally 85 iron responsive sRNAs were identified.Bioinformatics analysis showed that the length of these sRNAs are mostly distributed in 50-500 nt and the GC content ranging from 55% to 70%.Through analyzing the Ide R binding motif of the promoters for these sRNAs and EMSA experiments verification,we found that IdeR protein can bind to the promoter region of ncRv0281 c.The promoter activity of ncRv0281 c in the ?IdeR M.smegmatis was lower than that in the wide type,indicatingthat IdeR may positively regulate the transcription of ncRv0281 c.In short,we mapped the global acetylation file in M.tuberculosis,identified the important role of acetyltransferase in antibiotic resistance and extended the understanding of the regulation of sRNAs in iron metabolism.