Erythromycin is a macrolide antibiotic that inhibits not only mRNA translation but also 50S ribosomal subunit assembly in bacterial cells. An important mechanism of erythromycin resistance is the methylation of 23S rRNA by erm methyl transferase enzymes. We are interested in investigating the true substrate for methylation because it is known from our work and the work of others that fully assembled 50S subunits are not substrates for methylation. We have published a model for 50S ribosomal subunit formation where, the precursor particle that accumulates in erythromycin treated cells is a target for methyl transferase activity. Current studies are aimed at investigating the role of the precursor particle as substrate for ermE methyltransferase activity and the competition between this enzyme and erythromycin for the 50S precursor particle.
Slot-blot hybridization experiments have identified the presence of 23S rRNA in the 50S precursor region. Quantitation of the 23S rRNA in these blots also revealed that the percentage of the precursor increased as the concentration of erythromycin was increased in the growth media. Ribosomal proteins of S. aureus were studied by two-dimensional gel electrophoresis. Protein content of the 50S precursor particle was analyzed by MALDI-TOF. These studies have identified 16 50S ribosomal proteins in the precursor region.
Methyltransferase assays showed that 50S precursor particle was a substrate for ermE methyltransferase. Importantly, RNA that is already assembled into 50S subunits was not a substrate for the enzyme. Inhibition curves showed that macrolide, lincosamide, and streptogramin B (MLSB) drugs bound to the precursor particle with similar affinity and inhibited the ermE methyltransferase activity. Competition experiments suggested that the enzyme can displace erythromycin from the 50S precursor particle and that erm methyltransferase has a lower association constant for the precursor particle compared to that of the erythromycin. This suggests that higher concentrations of erythromycin are needed to combat erm induced resistance.
These studies shed light on the interaction of ermE methyltransferase and erythromycin in the clinically important pathogen S. aureus.
| Identifer | oai:union.ndltd.org:ETSU/oai:dc.etsu.edu:etd-3453 |
| Date | 05 May 2007 |
| Creators | Pokkunuri, Indira |
| Publisher | Digital Commons @ East Tennessee State University |
| Source Sets | East Tennessee State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Electronic Theses and Dissertations |
| Rights | Copyright by the authors. |
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