Mining the Pseudomonas virulence factor pathway for novel small molecules
Ashley M. Kretsch · Carolina Digital Repository (University of North Carolina at Chapel Hill) · 2021
Bioactive small molecules often play an important role in bacterial virulence. Identification and exploration of these molecules will develop an understanding of new mechanisms for infection and potentially identify novel targets to inhibit virulence. Analysis of bacterial genomes identifies a large number of gene clusters that encode for small molecule-synthesizing enzymes (biosynthetic gene clusters), many of which are unexplored. Through genetic manipulation of these clusters, we can discover their small molecule products and determine their biological roles. We have identified a biosynthetic gene cluster conserved in over 300 strains of Pseudomonads. Many of these strains engage in pathogenic or symbiotic relationships with a range of human, animal, and plant hosts. We have shown that deletions within this gene cluster, named the Pseudomonas virulence factor (pvf), reduce fly infection by the pathogen Pseudomonas entomophila. In particular, the small molecule products of the pvf pathway are suggested to play a role in bacterial signaling and activation of virulence. Through overexpression of the pvf cluster in its native P. entomophila strain, we have discovered and characterized a number of small molecules that the pvf gene cluster is responsible for. These molecules include a class of pyrazine compounds, some of which are new to biology. We use fly infection and promoter-reporter assays to determine the biological roles of these molecules in virulence and cell-to-cell signaling of P. entomophila. Through proteomic analysis of the secretome, we have identified over 500 proteins that are differentially expressed between wildtype and pvf deletion strains, many which are known to be important to virulence and competition. To identify the active molecule responsible for pvf signaling activity we developed an optimized bioactivity-guided extraction and purification method. We can compare active purified fraction from spent media extracts of wildtype and pvf deletion strains using nuclear magnetic resonance (NMR) and liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) to identify the active pvf molecule. By characterizing the structures and functions of bioactive small molecules from pathogens, our work has the potential to develop an understanding for the production of these compounds and reveal novel and useful antimicrobial targets.