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 point where the majority of these drugs 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 wide application in clinical practice but where the problem of resistance continues to increase. Of great concern is the identification over the last several years of pathogenic bacteria with broad-spectrum high-level resistance conferred by enzymes that modify the aminoglycoside binding site on the RNA of the small ribosomal subunit. Two distinct families of aminoglycoside-resistance methyltransferases have been identified that modify 16S ribosomal RNA on nucleotide G1405 or A1408. 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. However, in both cases the resistance enzymes involved are very poorly structurally and biochemically characterized. This proposal describes experiments grouped into two connected aims that will directly address this deficiency.
In Aim 1, we will determine the high-resolution X-ray crystal structures of members of both families of aminoglycoside-resistance methyltransferases (i.e. those targeting G1405 and A1408) from both aminoglycoside-producers and pathogenic bacteria. We will also characterize newly identified potential members of this resistance methyltransferase family that may provide further detailed insights into the origins of these enzymes in pathogenic bacterial populations. Finally, for each enzyme family, we will characterize and compare the binding of the essential methyl group donor molecule S-adenosyl-L-methionine (SAM) using mutagenesis and isothermal titration calorimetry (ITC).
In Aim 2, we will fully dissect the mechanism of methyltransferase- small ribosome subunit recognition, which represents the best target for future specific enzyme inhibitors. We will determine critical amino acids within each enzyme for target specificity using site-directed mutagenesis in combination with binding and functional assays, map the docking site on the small ribosome subunit using structure probing and cross-linking experiments, and provide the first high-resolution view of an antibiotic resistance methyltransferase enzyme bound to the small ribosome subunit using X-ray crystallography. Together these studies will provide novel and fundamentally important insights into the structure and function of aminoglycoside-resistance methyltransferases and will lay a secure foundation for any 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 structures and activities of enzymes that chemically alter specific RNA molecules to prevent antibiotic binding and thereby confer resistance. Such studies are essential to understand basic mechanisms of antibiotic resistance in bacteria and may one day underpin the development of new strategies to extend the clinically useful life of current antibiotics or to create novel designer drugs to combat resistant bacteria.
|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|
|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|
|Zelinskaya, Natalia; Rankin, C Robert; Macmaster, Rachel et al. (2011) Expression, purification and crystallization of adenosine 1408 aminoglycoside-resistance rRNA methyltransferases for structural studies. Protein Expr Purif 75:89-94|
|Macmaster, Rachel; Zelinskaya, Natalia; Savic, Miloje et al. (2010) Structural insights into the function of aminoglycoside-resistance A1408 16S rRNA methyltransferases from antibiotic-producing and human pathogenic bacteria. Nucleic Acids Res 38:7791-9|