P. aeruginosa is a Gram negative bacterium that causes opportunistic infections. In particular it can infect the lungs of people with the genetic disease cystic fibrosis. Once the infection is established antibiotics and the host immune system can not clear the bacteria because they overproduce an exopolysaccharides that is referred to as alginate. My work has focused on how does this bacteria sense its environment and secrete alginate to survive. Most of the research in the field has focused on mutations that happen and lock on the alginate overproduction or mucoid phenotype.
I was always curious to learn if P. aeruginosa can activate alginate in response to conditions. To do this I used ammonium metavanadate (AMV) to induce membrane stress. In E. coli it had been shown that AMV would activate the RpoE sigma factor. AlgU is the RpoE ortholog of P. aeruginosa. AMV added to Pseudomonas isolation agar induces alginate overproduction. This was interesting but we really did not have a clear idea of why this stress activates mucoidy. Therefore, we setup a project to look at the effects on the transcriptome and determine the genes required for mucoidy on PIAAMV. Growth on PIAAMV compared to PIA, causes 1/3 of the genome to be differently expressed. See the paper for in depth description of all of the changes. By screening the PA14 mutant library on PIA and PIAAMV we found 117 genes that were required for the PIAAMV mucoidy. These genes can be grouped into classes. However, we don’t know exactly why each gene or class is required. For example, why does knocking out some peptidoglycan genes turn off mucoidy on PIAAMV? There were a number of genes that encode hypothetical proteins with unknown functions. Ultimately there is still much to be learned with PIAAMV and projects in my future lab will look into these pathways and mechanisms.
In addition to the genetic requirements and effects of growing on PIAAMV. We also observed that strain PAO1 grown on PIAAMV is attenuated for virulence. Furthermore, we were able to show that this “live attenuated” strategy could be used as a vaccine. Further research will revisit this project and continue to examine the possibilities of this approach.
Ma is an obligate exoparasitic bacterium that feeds on and kills Pseudomonas aeruginosa that was first described in 1983. Recently, this Gram negative bacterium was sequenced and genetically characterized by Dr. Martin Wu. However, we still do not know exactly how it kills P. aeruginsoa. Figuring out this mechanism may be a key to discovering new antibiotic mechanisms. We have established models and performed studies to test the feasibility of treating P. aeruginsoa infections with Ma. Currently, we are looking for funding for this research to determine if predatory bacteria can be used as therapeutics or biocontrol agents.
The goal of the PIAAMV PA14 mutant library screen was to determine the genes required for mucoidy. However, we also noticed some strains were rugose on PIA. The rugose phenotype is shown in the figure to the left. In panel A an image of a wild type and rugose strain are shown as captured by a BioRad XRS gel doc. In B 3D rendering is shown to illustrate the colony morpholy. And in panel C the same strains are shown grown on PIA with congo red dye.
Work is currently in progress to determine why these mutants display this phenotype, but one mutant in particular caught our attention. We found a mutant that was nonmucoid on PIAAMV, but rugose on PIA. This mutant has an inactivated manganese transporter. We also observed a number of other phenotypes that are relevant to the pathogenesis of P. aeruginosa. Furthermore, we learned that this mutant is attenuated in virulence. These data have caused us to begin characterizing manganese transport and its effects on regulation of virulence as well as survival. If manganese is as essential as our unpublished studies suggest, future studies need to be designed to target manganese transport as a weakness of P. aeruginosa.