Methyltransferase Erm(37) Slips on rRNA to Confer Atypical Resistance in Mycobacterium tuberculosis

Abstract

Members of the Mycobacterium tuberculosis complex possess a resistance determinant, erm(37) (also termed ermMT), which is a truncated homologue of the erm genes found in a diverse range of drug-producing and pathogenic bacteria. All erm genes examined thus far encode N6-monomethyltransferases or N6,N6-dimethyltransferases that show absolute specificity for nucleotide A2058 in 23 S rRNA. Monomethylation at A2058 confers resistance to a subset of the macrolide, lincosamide, and streptogramin B (MLSB) group of antibiotics and no resistance to the latest macrolide derivatives, the ketolides. Dimethylation at A2058 confers high resistance to all MLSB and ketolide drugs. The erm(37) phenotype fits into neither category. We show here by tandem mass spectrometry that Erm(37) initially adds a single methyl group to its primary target at A2058 but then proceeds to attach additional methyl groups to the neighboring nucleotides A2057 and A2059. Other methyltransferases, Erm(E) and Erm(O), maintain their specificity for A2058 on mycobacterial rRNA. Erm(E) and Erm(O) have a full-length C-terminal domain, which appears to be important for stabilizing the methyltransferases at their rRNA target, and this domain is truncated in Erm(37). The lax interaction of the M. tuberculosis Erm(37) with its rRNA produces a unique methylation pattern and confers resistance to the ketolide telithromycin. Members of the Mycobacterium tuberculosis complex possess a resistance determinant, erm(37) (also termed ermMT), which is a truncated homologue of the erm genes found in a diverse range of drug-producing and pathogenic bacteria. All erm genes examined thus far encode N6-monomethyltransferases or N6,N6-dimethyltransferases that show absolute specificity for nucleotide A2058 in 23 S rRNA. Monomethylation at A2058 confers resistance to a subset of the macrolide, lincosamide, and streptogramin B (MLSB) group of antibiotics and no resistance to the latest macrolide derivatives, the ketolides. Dimethylation at A2058 confers high resistance to all MLSB and ketolide drugs. The erm(37) phenotype fits into neither category. We show here by tandem mass spectrometry that Erm(37) initially adds a single methyl group to its primary target at A2058 but then proceeds to attach additional methyl groups to the neighboring nucleotides A2057 and A2059. Other methyltransferases, Erm(E) and Erm(O), maintain their specificity for A2058 on mycobacterial rRNA. Erm(E) and Erm(O) have a full-length C-terminal domain, which appears to be important for stabilizing the methyltransferases at their rRNA target, and this domain is truncated in Erm(37). The lax interaction of the M. tuberculosis Erm(37) with its rRNA produces a unique methylation pattern and confers resistance to the ketolide telithromycin. Tuberculosis is caused by infection with bacteria belonging to the Mycobacterium tuberculosis complex (MTC) 4The abbreviations used are:MTCMycobacterium tuberculosis complexBCGbacillus Calmette-GuérinMLSBmacrolide, lincosamide and streptogramin BMALDImatrix-assisted laser desorption/ionizationTOFtime-of-flight 4The abbreviations used are:MTCMycobacterium tuberculosis complexBCGbacillus Calmette-GuérinMLSBmacrolide, lincosamide and streptogramin BMALDImatrix-assisted laser desorption/ionizationTOFtime-of-flight and is responsible for 2 million deaths per annum worldwide with almost one-third of the world's population harboring asymptomatic infections (1Butler D. Nature. 2000; 406: 670-672Crossref PubMed Scopus (72) Google Scholar). Treatment for tuberculosis often requires extended courses with several antibiotics, and with resistant strains becoming more prevalent (2Dye C. Williams B.G. Espinal M.A. Raviglione M.C. Science. 2002; 295: 2042-2046Crossref PubMed Scopus (293) Google Scholar), drug therapy is not always successful. In the absence of new antimycobacterial drugs, a better understanding of the resistance mechanisms to existing drugs is desirable. Mycobacterium tuberculosis complex bacillus Calmette-Guérin macrolide, lincosamide and streptogramin B matrix-assisted laser desorption/ionization time-of-flight Mycobacterium tuberculosis complex bacillus Calmette-Guérin macrolide, lincosamide and streptogramin B matrix-assisted laser desorption/ionization time-of-flight Members of the MTC include Mycobacterium africanum, Mycobacterium microti, Mycobacterium bovis, and M. tuberculosis, all of which are intrinsically resistant to macrolide antibiotics (3Falzari K. Zhu Z. Pan D. Liu H. Hongmanee P. Franzblau S.G. Antimicrob. Agents Chemother. 2005; 49: 1447-1454Crossref PubMed Scopus (177) Google Scholar, 4Rastogi N. Goh K.S. Berchel M. Bryskier A. Antimicrob. Agents Chemother. 2000; 44: 2848-2852Crossref PubMed Scopus (47) Google Scholar, 5Truffot-Pernot C. Lounis N. Grosset J. Ji B. Antimicrob. Agents Chemother. 1995; 39: 2827-2828Crossref PubMed Scopus (44) Google Scholar). Although this is due in part to the imperviousness of the mycobacterial cell wall (6Bosne-David S. Barros V. Verde S.C. Portugal C. David H.L. J. Antimicrob. Chemother. 2000; 46: 391-395Crossref PubMed Scopus (37) Google Scholar), the recently discovered resistance determinant, erm(37) (formerly ermMT), also contributes to the lack of macrolide susceptibility in MTC species (7Buriánková K. Doucet-Populaire F. Dorson O. Gondran A. Ghnassia J.-C. Weiser J. Pernodet J.-L. Antimicrob. Agents Chemother. 2004; 48: 143-150Crossref PubMed Scopus (108) Google Scholar). The erm(37) determinant is a truncated homologue of the erm genes found in a diverse range of pathogenic and drug-producing bacteria (8Roberts M.C. Sutcliffe J. Courvalin P. Jensen L.B. Rood J. Seppala H. Antimicrob. Agents Chemother. 1999; 43: 2823-2830Crossref PubMed Google Scholar). Members of the erm family of genes all encode methyltransferases that specifically methylate the N6 position of nucleotide A2058 in 23 S rRNA (Escherichia coli numbering) but differ as to whether they monomethylate or dimethylate this nucleotide (9Skinner R. Cundliffe E. Schmidt F. J. Biol. Chem. 1983; 258: 12702-12706Abstract Full Text PDF PubMed Google Scholar, 10Weisblum B. Antimicrob. Agents Chemother. 1995; 39: 577-585Crossref PubMed Scopus (813) Google Scholar). Erm monomethyltransferases are found predominantly in drug-producing actinomycetes species and confer the MLSB type I phenotype with high resistance to lincosamides, low to moderate resistance to macrolide and streptogramin B antibiotics (10Weisblum B. Antimicrob. Agents Chemother. 1995; 39: 577-585Crossref PubMed Scopus (813) Google Scholar, 11Zalacain M. Cundliffe E. Eur. J. Biochem. 1990; 189: 67-72Crossref PubMed Scopus (33) Google Scholar), but no resistance to the latest generation of macrolides, the ketolides (12Liu M. Douthwaite S. Antimicrob. Agents Chemother. 2002; 46: 1629-1633Crossref PubMed Scopus (88) Google Scholar). Erm dimethyltransferases confer the MLSB type II phenotype with high resistance to all macrolide, lincosamide, and streptogramin B antibiotics (8Roberts M.C. Sutcliffe J. Courvalin P. Jensen L.B. Rood J. Seppala H. Antimicrob. Agents Chemother. 1999; 43: 2823-2830Crossref PubMed Google Scholar, 10Weisblum B. Antimicrob. Agents Chemother. 1995; 39: 577-585Crossref PubMed Scopus (813) Google Scholar) including ketolides (12Liu M. Douthwaite S. Antimicrob. Agents Chemother. 2002; 46: 1629-1633Crossref PubMed Scopus (88) Google Scholar). Type II MLSB resistance with dimethylation of the rRNA is the more common mechanism in bacterial pathogens. The erm(37) gene is, however, atypical as it confers resistance that falls between the type I and type II categories. Expression of the M. tuberculosis erm(37) gene in the non-tuberculous mycobacterium, Mycobacterium smegmatis, confers a pattern similar to type I resistance (7Buriánková K. Doucet-Populaire F. Dorson O. Gondran A. Ghnassia J.-C. Weiser J. Pernodet J.-L. Antimicrob. Agents Chemother. 2004; 48: 143-150Crossref PubMed Scopus (108) Google Scholar), whereas authentic MTC hosts are resistant to the ketolide antibiotic telithromycin (3Falzari K. Zhu Z. Pan D. Liu H. Hongmanee P. Franzblau S.G. Antimicrob. Agents Chemother. 2005; 49: 1447-1454Crossref PubMed Scopus (177) Google Scholar, 4Rastogi N. Goh K.S. Berchel M. Bryskier A. Antimicrob. Agents Chemother. 2000; 44: 2848-2852Crossref PubMed Scopus (47) Google Scholar) which is more indicative of type II resistance. In this study, we first established that erm(37) is indeed responsible for telithromycin resistance in MTC strains. This was achieved using an attenuated MTC strain, BCG-Pasteur, utilized in production of the bacillus Calmette-Guérin vaccine; the BCG-Pasteur strain has lost the RD2 chromosome region containing erm(37) (13Behr M.A. Wilson M.A. Gill W.P. Salamon H. Schoolnik G.K. Rane S. Small P.M. Science. 1999; 284: 1520-1523Crossref PubMed Scopus (1309) Google Scholar) and is susceptible to telithromycin (4Rastogi N. Goh K.S. Berchel M. Bryskier A. Antimicrob. Agents Chemother. 2000; 44: 2848-2852Crossref PubMed Scopus (47) Google Scholar). Complementation of BCG-Pasteur with recombinant erm(37) restores telithromycin resistance to the level observed in virulent MTC strains and in a BCG strain that still has an intact RD2 region (BCG-Moreau). Introduction of the dimethyltransferase gene erm(E) conferred BCG-Pasteur with even higher telithromycin resistance, whereas the monomethyltransferase erm(O) conferred no telithromycin resistance. Paradoxically (but consistent with a previous report (7Buriánková K. Doucet-Populaire F. Dorson O. Gondran A. Ghnassia J.-C. Weiser J. Pernodet J.-L. Antimicrob. Agents Chemother. 2004; 48: 143-150Crossref PubMed Scopus (108) Google Scholar)), expression of recombinant erm(37) in M. smegmatis conferred the same phenotype as erm(O), with no significant resistance to telithromycin. A molecular explanation is needed to understand the disparate erm(37) phenotypes in the different mycobacterial strains. We employed matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) tandem mass spectrometry to define precisely the locations and number of methyl groups added by Erm(37) to the rRNAs in its authentic MTC host as well as to the rRNA of recombinant M. smegmatis strains. In its MTC host, Erm(37) initially monomethylates nucleotide A2058 but then proceeds to modify one or both of the neighboring nucleotides to add up to three methyl groups (at A2057, A2058, and A2059, Fig. 1). This unique configuration of methyl groups explains the resistance exhibited by the MTC strains, which is distinct from both the type I and type II MLSB patterns. In M. smegmatis, Erm(37) monomethylates A2058 but adds extra methyl groups only sparingly at A2057 or A2059 and produces an overall methylation pattern that is consistent with the type I MLSB phenotype. Bacterial Strains, Plasmids, and Growth Conditions—Bacterial strains and plasmids used in this study are listed in TABLE ONE. Mycobacteria were grown at 37 °C in Middlebrook 7H9 (Difco) liquid broth containing 0.2% (v/v) glycerol and on solid Middlebrook 7H10 medium (Difco) containing 0.5% (v/v) glycerol. Kanamycin (Sigma) was added at 15 μg/ml to maintain the plasmids in the mycobacterial strains. Erythromycin (Sigma) was added at 20 μg/ml to cultures of the BCG-Moreau strain to stimulate expression of erm(37) prior to rRNA analyses. Minimal inhibitory concentrations of the ketolide antibiotic telithromycin (Sanofi-Aventis) for MTC strains were determined at pH 7.3 using the BACTEC radiometric method (BD Biosciences) as reported previously (14Rastogi N. Goh K.S. Antimicrob. Agents Chemother. 1992; 36: 2841-2842Crossref PubMed Scopus (30) Google Scholar).TABLE ONEMycobacterium strains and plasmids used in the present studyStrainDescriptionSource or ref.M. tuberculosis H37RvWild-type isolateATCC27294M. africanumWild-type isolateATCC25420M. microtiWild-type isolateATCC19422M. bovisWild-type isolateATCC19210M. bovis BCG-MoreauVaccine strain (Brazil), RD2 region intactATCC35736M. bovis BCG-PasteurVaccine strain (France), RD2 region deleted1173 P2 Pasteur InstituteM. bovis BCG-Pasteur/pMIP12BCG-Pasteur/empty pMIP12 vector; Kanr (Ref. 42Le Dantec C. Winter N. Gicquel B. Vincent V. Picardeau M. J. Bacteriol. 2001; 183: 2157-2164Crossref PubMed Scopus (79) Google Scholar)7Buriánková K. Doucet-Populaire F. Dorson O. Gondran A. Ghnassia J.-C. Weiser J. Pernodet J.-L. Antimicrob. Agents Chemother. 2004; 48: 143-150Crossref PubMed Scopus (108) Google ScholarM. bovis BCG-Pasteur/pOMV16BCG-Pasteur/pMIP12 plasmid containing erm(37) (ermMT) from M. tuberculosis H37Rv; Kanr7Buriánková K. Doucet-Populaire F. Dorson O. Gondran A. Ghnassia J.-C. Weiser J. Pernodet J.-L. Antimicrob. Agents Chemother. 2004; 48: 143-150Crossref PubMed Scopus (108) Google ScholarM. bovis BCG-Pasteur/pOMV20BCG-Pasteur/pMIP12 plasmid containing erm(E) from Saccharopolyspora erythraea; Kanr7Buriánková K. Doucet-Populaire F. Dorson O. Gondran A. Ghnassia J.-C. Weiser J. Pernodet J.-L. Antimicrob. Agents Chemother. 2004; 48: 143-150Crossref PubMed Scopus (108) Google ScholarM. bovis BCG-Pasteur/pOMV30BCG-Pasteur/pMIP12 plasmid containing erm(O) (srmA) from Streptomyces ambofaciens; KanrThis studyM. smegmatis mc2 155/pMIP12M. smegmatis mc2155 (Ref. 43Snapper S.B. Melton R.E. Mustafa S. Kieser T. Jacobs Jr., W.R. Mol. Microbiol. 1990; 4: 1911-1919Crossref PubMed Scopus (992) Google Scholar)/empty vector7Buriánková K. Doucet-Populaire F. Dorson O. Gondran A. Ghnassia J.-C. Weiser J. Pernodet J.-L. Antimicrob. Agents Chemother. 2004; 48: 143-150Crossref PubMed Scopus (108) Google ScholarM. smegmatis mc2155/pOMV16Strain plus plasmid-encoded erm(37)7Buriánková K. Doucet-Populaire F. Dorson O. Gondran A. Ghnassia J.-C. Weiser J. Pernodet J.-L. Antimicrob. Agents Chemother. 2004; 48: 143-150Crossref PubMed Scopus (108) Google ScholarM. smegmatis mc2155/pOMV20Strain plus plasmid-encoded erm(E)7Buriánková K. Doucet-Populaire F. Dorson O. Gondran A. Ghnassia J.-C. Weiser J. Pernodet J.-L. Antimicrob. Agents Chemother. 2004; 48: 143-150Crossref PubMed Scopus (108) Google ScholarM. smegmatis mc2155/pOMV30Strain plus plasmid-encoded erm(O)7Buriánková K. Doucet-Populaire F. Dorson O. Gondran A. Ghnassia J.-C. Weiser J. Pernodet J.-L. Antimicrob. Agents Chemother. 2004; 48: 143-150Crossref PubMed Scopus (108) Google Scholar Open table in a new tab Preparation of Ribosomes and RNA Purification—M. bovis BCG and M. smegmatis strains (TABLE ONE) were grown to late log phase and harvested by centrifugation. Cells were washed twice by being suspended in 100 ml of buffer A (10 mm Tris-Cl, pH 7.2, 4 mm MgCl2, 10 mm NH4Cl, 100 mm KCl) and pelleted by centrifugation before being resuspended in 10 ml of buffer A and lysed using a French press (M. bovis) or by sonication (M. smegmatis). Cell debris was removed by centrifugation at 30,000 × g for 20 min at 4 °C; ribosomes were pelleted by centrifugation at 100,000 × g for 2 h at 4 °Cand were redissolved in 200 μl of cold buffer A. Ribosomal proteins were removed by phenol/chloroform extraction, and the rRNA was redissolved in 30 μl of H2O. MALDI Mass Spectrometry Analysis of rRNAs—The 23 S rRNA region around A2058 in M. bovis and M. smegmatis was using a of rRNA was to of a to the nucleotide which is in the MTC and M. smegmatis 23 S RNA were with F. RNA 2004; PubMed Scopus Google Scholar). The was and by The rRNA was on a and F. RNA 2004; PubMed Scopus Google Scholar). The rRNA was with A or (Sigma) to of a for MALDI mass spectrometry of rRNA in were with μl of in μl of and μl of and for h at 37 were were with 10 mm with 10 mm The were using a MALDI mass in time-of-flight F. RNA 2004; PubMed Scopus Google Scholar, J. F. 2002; PubMed Scopus (88) Google Scholar, J. F. Douthwaite S. PubMed Scopus (72) Google Scholar). were using the mass spectrometry was on a MALDI mass in The for was to 2 and was were using the by the to the region of Mycobacterium 23 S rRNA was extended with of using a of mm mm and rRNA as were on (12Liu M. Douthwaite S. Antimicrob. Agents Chemother. 2002; 46: 1629-1633Crossref PubMed Scopus (88) Google Scholar). and on of the M. tuberculosis complex (TABLE ONE) that erm(37) an important in resistance to MLSB antibiotics (7Buriánková K. Doucet-Populaire F. Dorson O. Gondran A. Ghnassia J.-C. Weiser J. Pernodet J.-L. Antimicrob. Agents Chemother. 2004; 48: 143-150Crossref PubMed Scopus (108) Google Scholar) as well as to the ketolide antibiotic telithromycin (TABLE M. tuberculosis, M. africanum, M. microti, M. bovis, and the BCG-Moreau strain all possess an of erm(37) and high resistance to telithromycin. The BCG-Pasteur strain only in its lack of the erm(37) determinant and its susceptibility to telithromycin. Complementation of BCG-Pasteur with a plasmid-encoded of erm(37) restores the phenotype. Expression of the monomethyltransferase erm(O) confers no telithromycin resistance in BCG-Pasteur, whereas high resistance is conferred by the dimethyltransferase erm(E) (TABLE of species from the Mycobacterium tuberculosis complex the ketolide antibiotic telithromycin All strains, with the of M. bovis BCG-Pasteur, possess the erm(37) The three BCG-Pasteur strains are with erm genes from a plasmid (TABLE inhibitory bovis bovis bovis BCG-Pasteur bovis BCG-Pasteur bovis BCG-Pasteur inhibitory Open table in a new tab A2058 in of Erm(37) was determined by the rRNA in the BCG-Pasteur and strains. The of rRNA containing nucleotide A2058 were by MALDI mass of rRNA with the A and of and have similar or and often a that is to we an rRNA by the mycobacterial 23 S from to 1). of this to containing A2058 A of or an of that unique and in the mass erm(37) the BCG-Pasteur and strains were grown the a single at that no methylation at Growth of the BCG-Moreau strain with in of the and the of new at and to the of and three methyl This methylation pattern was by A of the a containing A2058 that at and to and are with not of the rRNA and the to in were on erm(37) The BCG-Pasteur strain not in the of at 20 rRNA of of Erm(37) methylation in the rRNA were by the and using MALDI tandem mass In this the RNA are and then to the mass of nucleotide The in the of the was to in nucleotide A2058 and to the of a single methyl group The with methyl groups was in a similar and was a of which were at A2057, A2058, and A2059. This pattern is by the of one being at A2057 and A2058 and the being at A2058 and A2059. In a with single methyl groups at A2057 and A2059 is also but is as no only at A2057 or A2059 was observed in the that one of the was was in using The at was by tandem mass spectrometry to be a unique with one methyl group on of the three at and erm(37) Expression in M. erm(37) gene from M. tuberculosis was a in a plasmid and in M. smegmatis (TABLE were with erm(O) and which encode well A2058 and M. smegmatis its gene expression of which requires by or similar Antimicrob. Agents Chemother. PubMed Scopus Google Scholar, P. N. S. C. R. A. S. A. 2005; PubMed Scopus Google Scholar, Douthwaite S. Antimicrob. Agents Chemother. 2005; 49: PubMed Scopus Google Scholar). 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Expression of the erm(37) gene in MTC strains requires with an antibiotic as Expression of erm(37) in methylation of the 23 S and this is as a of in the to the of and three methyl groups The was and by tandem mass spectrometry that the methyl group was to the first by Erm(37) is to monomethylate specifically at The then adds a methyl group at A2057 or A2059 Fig. before to add a methyl group that the rRNA with a single methyl group on of the three A2057, A2058, and A2059. its extended of Erm(37) as a monomethyltransferase at of the nucleotides it In bacteria that possess an erm expression requires prior with low concentrations of or a similar mechanisms are found in pathogenic and drug-producing bacteria B. Antimicrob. Agents Chemother. 1995; 39: PubMed Scopus Google Scholar) as well as in the M. smegmatis Douthwaite S. Antimicrob. 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