The application of antibiotics to the treatment of bacterial infections revolutionized modern medical practice. In the decades since, a combination of improperly controlled use and the remarkable ability of bacterial populations to develop resistance to these drugs has severely restricted the clinical usefulness of many antibiotics. We are now at a critical juncture where the majority of useful antibiotics have known and sometimes extensive resistance, and few novel replacements or strategies to combat the resistance problem exist. The aminoglycoside antibiotics are one important example of a large group of drugs with retained activity against key human pathogens but where the problem of resistance continues to increase. Of particular concern is the increasing identification over the last decade of pathogenic bacteria harboring exceptionally broad- spectrum and high-level aminoglycoside resistance determinants. These 16S ribosomal RNA (rRNA) methyltransferases chemically modify the aminoglycoside binding site on the 16S rRNA of the small (30S) bacterial ribosomal subunit. Two distinct families of aminoglycoside-resistance methyltransferases have been identified that modify 16S rRNA on nucleoside guanosine 1405 or adenosine 1408, to create m7G1405 and m1A1408 modifications, respectively. Functionally analogous self-protection resistance enzymes of both types were earlier identified in aminoglycoside-producing bacteria, and it is thought that these genes have laterally transferred to the pathogenic bacteria. Recent studies from our lab and others revealed the molecular structures of each enzyme, of both drug producer and pathogenic origin, and provided a first view of an m1A1408 methyltransferase bound to its substrate. However, additional evidence suggests that this enzyme family may employ diverse mechanisms to accomplish substrate specificity and modification. Further, comparatively little is known about how m7G1405 achieve the equivalent molecular functions. This proposal describes experiments in two connected aims that will directly address these deficiencies.
In Aim 1 we will define the molecular mechanisms employed by the m1A1408 methyltransferases using a combination of biochemical, molecular biological and structural biology approaches to determine the molecular bases for control of m1A1408 methyltransferase activity by the S-adenosyl-L-methionine cosubstrate, and by the 30S subunit substrate.
Aim 2 will provide the first detailed molecular insights into 30S recognition and modification by the m7G1405 methyltransferases using an innovative, interdisciplinary combination of approaches including synthetic chemistry to produce novel SAM analogs that will enable for the first time essential biochemical and structural studies of these clinical important resistance enzymes. Together these studies will provide novel and fundamentally important insights into the structures, activities and mechanisms of action of the aminoglycoside-resistance methyltransferases and will directly lay a secure foundation for future development of new strategies to counter the resistance they confer.
Increasing resistance is a major problem that threatens to end the clinical usefulness of many antibiotics and fundamentally alter our ability to treat bacterial infections. Our goal is to define the molecular mechanisms and activities of resistance enzymes that chemically alter specific RNA molecules to prevent antibiotic binding and thereby confer resistance in serious human pathogenic bacteria. Detailed understanding of such fundamental mechanisms of antibiotic resistance is a necessary prerequisite for the development of new strategies to extend the clinically useful life of current antibiotics or to create novel designer drugs to combat resistant bacteria.
|Witek, Marta A; Kuiper, Emily G; Minten, Elizabeth et al. (2017) A Novel Motif for S-Adenosyl-l-methionine Binding by the Ribosomal RNA Methyltransferase TlyA from Mycobacterium tuberculosis. J Biol Chem 292:1977-1987|
|Vinal, Kellie; Conn, Graeme L (2017) Substrate Recognition and Modification by a Pathogen-Associated Aminoglycoside Resistance 16S rRNA Methyltransferase. Antimicrob Agents Chemother 61:|
|Corrêa, Laís L; Witek, Marta A; Zelinskaya, Natalia et al. (2016) Heterologous Expression and Functional Characterization of the Exogenously Acquired Aminoglycoside Resistance Methyltransferases RmtD, RmtD2, and RmtG. Antimicrob Agents Chemother 60:699-702|
|Witek, Marta A; Conn, Graeme L (2016) Functional dichotomy in the 16S rRNA (m1A1408) methyltransferase family and control of catalytic activity via a novel tryptophan mediated loop reorganization. Nucleic Acids Res 44:342-53|
|Zelinskaya, Natalia; Witek, Marta A; Conn, Graeme L (2015) The Pathogen-Derived Aminoglycoside Resistance 16S rRNA Methyltransferase NpmA Possesses Dual m1A1408/m1G1408 Specificity. Antimicrob Agents Chemother 59:7862-5|
|Myers, Cullen L; Kuiper, Emily G; Grant, Pei C et al. (2015) Functional roles in S-adenosyl-L-methionine binding and catalysis for active site residues of the thiostrepton resistance methyltransferase. FEBS Lett 589:3263-70|
|Savic, Miloje; Sunita, S; Zelinskaya, Natalia et al. (2015) 30S Subunit-dependent activation of the Sorangium cellulosum So ce56 aminoglycoside resistance-conferring 16S rRNA methyltransferase Kmr. Antimicrob Agents Chemother 59:2807-16|
|Kuiper, Emily G; Conn, Graeme L (2014) Binding induced RNA conformational changes control substrate recognition and catalysis by the thiostrepton resistance methyltransferase (Tsr). J Biol Chem 289:26189-200|
|Dunkle, Jack A; Vinal, Kellie; Desai, Pooja M et al. (2014) Molecular recognition and modification of the 30S ribosome by the aminoglycoside-resistance methyltransferase NpmA. Proc Natl Acad Sci U S A 111:6275-80|
|Witek, Marta A; Conn, Graeme L (2014) Expansion of the aminoglycoside-resistance 16S rRNA (m(1)A1408) methyltransferase family: expression and functional characterization of four hypothetical enzymes of diverse bacterial origin. Biochim Biophys Acta 1844:1648-55|
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