The application of antibiotics to the treatment of bacterial infections revolutionized modern medical practice. In the decades since, a combination of improperly controlled usage 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 are in active development. Many clinically useful antibiotics target the bacterial ribosome. One increasingly prevalent form of resistance to these drugs is alteration of the modification status of the ribosomal RNA (rRNA) via acquired or intrinsic methyltransferase enzymes. While enzymes responsible for incorporating these antibiotic resistance- associated rRNA modifications are known, we understand far less about their mechanisms of action (such as specific substrate recognition), which might offer viable new targets to counter the resistance. Further, we also currently have a poor understanding of the molecular basis for how rRNA methylation affects ribosome-antibiotic interactions. The experiments proposed in this application will directly address these critical gaps in our fundamental knowledge of rRNA methylation and bacterial antibiotic resistance. In the first two aims we will define the molecular mechanisms of ribosome subunit recognition by two different rRNA modification enzymes, the acquired aminoglycoside-resistance 16S rRNA (m7G1405) methyltransferases (Aim 1) and the intrinsic Mycobacterium tuberculosis methyltransferase TlyA (Aim 2). Next, we will develop a new computational and experimental framework for understanding antibiotic-methylated rRNA interactions (Aim 3). Our goal is to explain at the molecular level how rRNA modifications limit drug efficacy and how these effects can be evaded. Collectively, the results of these three independent but complementary aims will deepen our fundamental understanding of the molecular strategies used by rRNA modification enzymes and the impacts of rRNA methylation on antibiotic resistance in bacteria. Our results will support future innovative strategies to counter the resistance conferred by these enzymes, for example, by facilitating the development of inhibitors of m7G1405 methyltransferase activity or 30S substrate binding, and could also lead to the rational design of novel antimicrobials capable of fully evading the effects of rRNA modification.

Public Health Relevance

Increasing antibiotic resistance is a major problem that threatens to end the clinical usefulness of many antibiotics and fundamentally alter our ability to treat bacterial infections. This proposal will extend our studies of enzymes that chemically modify specific RNA molecules to alter antibiotic activity and will also establish a new paradigm for understanding how these modifications impact antibiotic-target interactions. Detailed understanding of mechanisms of resistance and antibiotic-target interactions are necessary prerequisites for the development of new strategies to extend the clinical life of current antibiotics or to create novel designer drugs to combat resistant bacteria.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Xu, Zuoyu
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Emory University
Schools of Medicine
United States
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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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