The long-term goal of this project is to develop novel therapeutics for the treatment of the Gram-negative pathogens in the Burkholderia genus which includes the Burkholderia cepacia complex (BCC) and Burkholderia pseudomallei. These pathogens cause serious, chronic lung infections and are also major causes of healthcare-associated infections. Treatment of infections caused by these pathogens is often difficult due to the broad-spectrum antibiotic resistance that is commonly seen. The development of novel therapeutics to treat these pathogens is critically needed. Our published work analyzing the genomic diversity of isolates of the BCC species B. dolosa and B. multivorans collected from people with CF has led to the discovery that the fixL gene appears to be under strong positive selection. FixL has been described in Rhizobium and Caulobacter as a sensory histidine kinase of a two-component system that detects oxygen tension and phosphorylates the transcription factor FixJ under low oxygen conditions. Our previous work has found that the BCC FixLJ system is required for pathogenesis and modulation of this pathway has profound effects of virulence. Interestingly, constructs carrying evolved FixL variants associated with periods of clinical decline in patients were more virulent in multiple infection models compared to isogenic constructs carrying ancestral FixL variants. These more virulent FixL sequences had lower FixLJ activity than less virulent constructs carrying ancestral FixL variants demonstrating that increased FixLJ activity is determinantal to virulence. We hypothesize that we can reduce Burkholderia virulence by activating the FixLJ pathway. By targeting virulence, we predict that the bacteria would no longer be able to infect or persist within the host. In the first aim of this project we will identify compounds that activate FixLJ using a high-throughput screen with a Burkholderia fix pathway reporter strain.
In Aim 2 we will evaluate promising lead compounds identified in Aim 1 for their in vitro effectiveness at reducing virulence of multiple Burkholderia species using an in vitro macrophage assay. We also will evaluate the toxicity of lead compounds using cell culture. We plan to identify 3-4 lead compounds with an EC50 in the low micromolar (1-10) range and a toxicity at least 5-fold higher than the EC50. These lead compounds will be further developed into therapies for use to treat Burkholderia infections.
Members of the genus Burkholderia are important pathogens that can cause serious disease often in patients with cystic fibrosis (CF), they also cause healthcare-associated infections and are often highly antibiotic resistant. The long-term goal of this project is to develop new antimicrobial agents targeting a bacterial gene regulation system for modulation of activity which will have dramatic effects on the ability of the bacteria to cause infection. In this proposal we will identify these compounds using a high-throughput screen and evaluate their activity against multiple pathogenic Burkholderia species.
