Characterization of the Pseudomonas virulence factor pathway

Quorum sensing (QS) is a form of bacterial cell-to-cell communication between members of the same or different species, or between bacteria and eukaryotic hosts. QS allows bacteria to function as a collective and coordinate the expression of genes involved in group behaviors such as biofilm formation and virulence factor production during infection. QS is mediated by signaling molecules called autoinducers.The Pseudomonas virulence factor (pvf) cluster encodes enzymes that synthesize signaling molecules (PVF autoinducers) that mediate a quorum sensing system (Pvf). The pvf cluster was discovered in the entomopathogen Pseudomonas entomophila L48 and is conserved in over 300 strains of proteobacteria including plant and animal pathogens. P. entomophila infection of Drosophila melanogaster is fatal and a model system to study host-microbe interactions and innate immunity. Pvf is the only known QS system in P. entomophila and is required for full virulence.We have previously shown that Pvf in P. entomophila regulates over 200 secreted proteins and two families of small molecules—the insecticidal labradorins and the antimicrobial pyreudiones. Here I further investigate this new QS system in the model strain P. entomophila. Transcriptomic analysis of the wildtype and a pvf deletion mutant (ΔpvfA-D) revealed over 300 genes that are likely regulated by Pvf and involved in virulence, the type VI secretion system, siderophore transport, and branched chainivamino acid biosynthesis. I also identified seven putative biosynthetic gene clusters linked to Pvf regulation. The biosynthesis and identification of the PVF autoinducer(s) are beyond the scope of this dissertation; however, here I show that the proposed PVF autoinducer, valdiazen, restores expression of select Pvf regulated genes. Finally, I provide evidence that the genes PSEEN3202 and PSEEN3203 are located upstream of key Pvf regulated genes and therefore likely involved in the novel PVF signal transduction pathway. My work characterizing this understudied QS system is not only relevant to insect-microbe interactions, but to interactions in other pvf-containing strains that infect a variety of plant and animal hosts. Insights gleaned from the study of Pvf can also aid in the development of anti-virulence strategies and biopesticides.