Pseudomonas aeruginosa is an important bacterial pathogen capable of causing life-threatening infections. Unfortunately, successful treatment is threatened by rapid development of multi-drug resistance, and the """"""""pipeline"""""""" of novel anti-pseudomonal drugs is virtually non-existent. One alternative approach is to target resistance mechanisms and to restore the usefulness of key anti-pseudomonal drugs, i.e. the carbapenems. The long-term goal is to identify new therapeutic targets to enhance treatment of multi-drug resistant P. aeruginosa. Although it is well established that downregulation of OprD is the primary mechanism of carbapenem resistance, the molecular mechanisms regulating OprD production remain largely unknown. Understanding these mechanisms can lead to the identification of novel therapeutic targets for which new therapeutics can be directed to restore carbapenem susceptibility. Preliminary studies in our laboratory have identified a unique panel of P. aeruginosa with phenotypes that challenge previous theories of OprD regulation and carbapenem resistance. This panel will serve as the foundation for characterizing the molecular mechanisms of OprD regulation. Using methods that are well established in our laboratory, the specific aims of this grant are  to use Real-Time RT-PCR to evaluate the relationship between oprD expression, oprD transcript stability (mRNA half-life studies) and susceptibility to carbapenems,  to map the start site of oprD transcription (primer extension) and to use promoter deletion clones to identify cis-acting elements within the oprD promoter that are essential for gene expression, and  to use electrophoretic mobility shift assays to confirm the binding of MexT (hypothesized transcriptional regulatory protein) and/or other cellular factors to the oprD promoter, as required for regulation of oprD expression. Data obtained from these experiments will be essential for further delineation of the molecular mechanisms of OprD regulation and resistance to carbapenems. ? ?
STATEMENT Strains of P. aeruginosa that are resistant to all available antibiotics are causing infections with increasing frequency and pose a serious threat to public health. Without the development of novel therapeutic strategies, the successful treatment of life threatening P. aeruginosa infections is seriously compromised. This study will begin to characterize the molecular mehcanisms of OprD-mediated carbapenem resistance, information essential for the identification of novel therapeutic agents that could restore the activity of this important antibacterial class. ? ? ? ?
|Lister, Philip D; Wolter, Daniel J; Hanson, Nancy D (2009) Antibacterial-resistant Pseudomonas aeruginosa: clinical impact and complex regulation of chromosomally encoded resistance mechanisms. Clin Microbiol Rev 22:582-610