Linezolid is one of the newest antibiotics used for treatment of infections caused by Gram-positive bacterial pathogens. The first clinically-relevant representative of the oxazolidinone class of antibiotics, it acts upon the ribosome and inhibits protein synthesis in sensitive bacteria. Linezolid shows activity against many drug- resistant Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and may serve as the last line of defense against infections caused by MRSA with decreased susceptibility to vancomycin. So far, only a few linezolid-resistant S. aureus strains have been described worldwide. In almost all of the cases, the resistance was associated with mutations in domain V of 23S ribosomal RNA. This type of resistance appears rather rarely and develops slowly because of the presence of multiple copies of rRNA genes in the S. aureus genome. Horizontal transmission of this type of resistance between Gram- positive pathogens is unlikely. This proposal is aimed at investigation of a new mechanism of linezolid resistance in S. aureus. We discovered this mechanism while analyzing a linezolid-resistant MRSA strain isolated in a hospital in Colombia. Our preliminary studies indicate that resistance of the MRSA isolate to linezolid is mediated by an unusual posttranscriptional modification of a specific nucleotide, A2503 of 23S rRNA, which is located in the linezolid binding site in the ribosome. Modification of A2503 by Cfr methyltransferase, whose gene is present on the chromosome of the Colombian MRSA isolate, renders cells resistant to linezolid. This is the first case of appearance of the cfr gene in a human pathogen. The nucleotide sequence analysis suggests association of cfr with a mobile genetic element. Therefore, this case represents the first example of potentially horizontally transmittable oxazolidinone resistance. Cfr-dependent modification of rRNA is likely to confer resistance not only to linezolid but to other antibiotics acting upon the ribosomal peptidyl transferase center. The main goal of this proposal is to understand the fundamental principles of regulation, function and transmission of this new mechanism of oxazolidinone resistance. The newly acquired knowledge will be essential for combating the Cfr-type of drug resistance. The proposed research plan includes examination of transcriptional and translational control of cfr expression, examination of structural changes in the ribosome that are required to render cells resistant to linezolid, characterization of the structure of the Cfr enzyme, study of the molecular mechanisms of its function and analysis of its genetic environment in the S. aureus chromosome. The results of this study should provide critical insights into the operation of a new, medically- significant, mechanism of resistance to one of the newest antibiotics and may pave the way to developing better oxazolidinone antibiotics as well as newer ways for evading antibiotic resistance.
