Grant: Discovery Grants Program - Individual

Acinetobacter baumannii is a Gram-negative pathogen and an organism of significant interest due to its high resistance to antibiotics. Although A. baumannii's resistance to antibiotics is a result of a combination of various acquired and intrinsic factors, proton-gradient-dependent pumps belonging to the Resistance-Nodulation-division (RND) family are considered the most important factors contributing to the intrinsic antibiotic resistance of A. baumannii. RND pumps are an ancient family of proteins and their homologs have been reported across all three kingdoms. RND pumps have been most studied in Gram-negative bacteria primarily due to their ability to pump out a variety of antimicrobials across the two membranes. Due to their ancient nature, there is a consensus that the efflux of antibiotics is not their primary role, however physiological function(s) of RND pumps remain unknown. In this application, we propose to study two RND pumps of A. baumannii, AdeIJK and AdeFGH to determine their natural functions. Preliminary data from our laboratory suggest that fatty acid perturbations can result in the upregulation of AdeIJK pump. Further, we have also observed that the deletion of AdeN, the transcriptional repressor of AdeIJK pump, results in an altered expression of almost 250 different genes. This suggests that perhaps AdeIJK pump is part of a response in A. baumannii that results from the loss of AdeN. Identifying and characterizing genes that are co-regulated with AdeIJK will provide important clues about the natural function(s) of AdeIJK pump. Similarly, we have isolated mutants of A. baumannii that overexpress AdeFGH pump. These mutants contain a mutation in csrA that encodes for the Carbon Storage Regulator protein. CsrA, an mRNA binding protein, represses several stationary phase processes and activates exponential-phase functions in E. coli. It also modulates the regulation of genes involved in central carbon metabolism, secondary metabolite metabolism, motility, biofilm formation, as well as virulence. We are therefore proposing to study the link between central carbon as well nitrogen metabolism and the expression of AdeFGH in A. baumannii. We will employ a unique approach and carry out the proposed studies in an antibiotic susceptible environmental isolate of A. baumannii. Our approach of carrying out these studies in an environmental isolate of A. baumannii is likely to provide more relevant findings on the physiological functions of AdeIJK and AdeFGH pumps. This work will lead to a better understanding of how the expression of RND efflux pumps may be influenced by metabolic pathways in A. baumannii.