Grant: $196,250 - National Institutes of Health - Aug. 11, 2009
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Award Description: The clinical success of macrolide antibiotics has been curbed due to the spread of resistance. One of the major resistance mechanisms found in pathogenic strains is the modification of the drug-target, the ribosome, by Erm methyltransferase enzymes. The expression of many of the erm resistance genes, including ermC, is inducible by macrolides by a mechanism of induction based on programmed stalling of the ribosome at the regulatory cistron, ermCL. The newer generation of macrolides, ketolide antibiotics, have previously been considered to be non-inducers of inducible erm genes. However, recent data have shown that ketolides do indeed induce expression of ermC. Given the similarity of the structures and binding sites of macrolides and ketolides it has been presumed that the mechanism of erm induction by both antibiotic classes would be the same. However, our preliminary data show that in contrast to erythromycin and macrolides of the second generation, ketolides do not induce ribosome stalling at ermCL. This and several other observations strongly suggest that the mechanism of erm induction by ketolides is principally different from that of macrolides. Unraveling this mechanism is the primary goal of this proposal. We propose three possible models to potentially explain how ketolides induce erm expression. ln the ‘slide-through’ model, ketolides allow the ribosome translating ermCL to reach the initiation codon of ermC via frameshifting or stop codon read-through. In the ‘hopping’ model, ketolides promote the bypass of the ermCL-ermC intergenic region and allow the ribosome to resume translation at the erm cistron. The third, ‘riboswitch’ model suggests that the direct interaction of ketolide antibiotics with mRNA lead to a conformational switch that facilitates initiation of translation of the erm gene. We propose a series of genetic and biochemical experiments to test and discriminate between these models. Finally, we will analyze the possible induction of other clinically relevant erm genes by ketolide antibiotics. Understanding the mechanism of ketolide-dependent induction of the erm genes offers the opportunity to decipher a new molecular mechanism of regulation of expression of antibiotic resistance genes and will contribute to the design of new non-inducing antibiotics.
Project Description: Unlike macrolides, ketolides do not support ribosome stalling at the ermCL regulatory ORF, necessary for activation of the resistance gene ermC. Upon induction with macrolides, translation of ermC should initiate at the canonical initiator codon of the ermC cistron. In contrast, the N-terminal end of the translation product resulting from ketolide induction might include additional amino acid residues due to translation of a portion of the ermC upstream region. We have focused on preparation of plasmid constructs that will help us compare and identify the nature of the translation products of the ermC gene after inducing its expression with erythromycin (a macrolide) or telithromycin (a ketolide). Previously, we have used the plasmid pZ? containing a reporter construct in which the ermC methylase gene was substituted by the lacZ? reporter gene. The original construct included the ermCL regulatory region, the intergenic switch region, and the first two codons of the ermC ORF fused in frame to the reporter gene. We have now modified this cassette to include a larger portion of the 5’ region of the ermC methylase gene (the first 22 codons) since it is possible that the ketolide-dependent induction mechanism involves this area of the mRNA. In addition, to facilitate detection and purification of the antibiotic-induced translation products, we have attached two modules to the 3’ end of the lacZ? gene: a FLAG epitope and a His6-tag tail. The resulting cassette was introduced into the pPOT cloning vector resulting in the plasmid pZ?22-tail. We have tested the inducibility of the reporter in the new cassette by antibiotics using the disc diffusion test. The newly engineered reporter cassette retains its inducibility. Furthermore, although at a lower level compared to erythromycin, telithromycin readily induces expression of the lacZ? gene. In the next experimental step we will test whether the induced translation product can be detected on a gel using anti-FLAG antibody.
Jobs Summary: Employment of the Principal Investigator (Nora Vazquez-Laslop) will be retained, at a 0.75 FTE, using this fund. (Total jobs reported: 0)
Project Status: Not Started
This award's data was last updated on Aug. 11, 2009. Help expand these official descriptions using the wiki below.