Indiana University-Purdue University Indianapolis (IUPUI) / The Gram-negative bacterium Pseudomonas aeruginosa causes numerous acute and chronic opportunistic infections in humans. One of its most formidable weapons is a type III secretion system (T3SS), a multi-protein molecular syringe that injects powerful toxins directly into host cells. The toxins lead to cell dysfunction and, ultimately, cell death. Identification of regulatory pathways that control T3SS gene expression may lead to the discovery of novel therapeutics to treat P. aeruginosa infections. In a previous study, it was found that expression of the magnesium transporter gene mgtE inhibits T3SS gene transcription. MgtE-dependent inhibition appeared to interfere with the synthesis or function of the master T3SS transcriptional activator ExsA, although the exact mechanism was unclear. In this work, we demonstrate that mgtE expression acts through the GacAS two-component system to activate transcription of the small regulatory RNAs RsmY and RsmZ. This event ultimately leads to inhibition of exsA translation. Moreover, our data reveal that MgtE acts solely through this pathway to regulate T3SS gene transcription. Our study reveals an important mechanism that may allow P. aeruginosa to fine-tune T3SS activity in response to certain environmental stimuli. In addition, a previous study has shown that the P. aeruginosa gene algR abrogates mgtE mediated regulation of cytotoxicity. AlgR has pleiotropic effects in P. aeruginosa, including regulation of synthesis of the exopolysaccharide alginate. In the second part of my thesis, I show that algR and mgtE genetically crosstalk to inhibit ExsA driven T3SS gene transcription. This genetic interaction between algR and mgtE seems to be specifically directed towards regulation of T3SS gene expression rather than having an indiscriminate effect on multiple virulence attributes in P. aeruginosa. Additionally, we have further demonstrated that AlgR inhibits mgtE transcription. These studies suggest the presence of a T3SS inhibitor that is inhibited by both AlgR and MgtE. Future work will involve transcriptomic and proteomic analysis to identify such an inhibitor. Taken together, this study provides important insight into the molecular mechanisms of mgtE expression and function in P. aeruginosa. We have established that mgtE has pleiotropic effects on cytotoxicity in P. aeruginosa. Thus, given the role that cytotoxicity regulation plays in shaping P. aeruginosa pathogenesis and associated clinical outcomes, mgtE might be an interesting drug target, though extensive future studies are required to validate this proposition. Nevertheless, this research, provides clues for identification of novel therapeutic targets in P. aeruginosa. Hence this work, in the long run, serve to ameliorate the morbidity and mortality in patients infected with P. aeruginosa.

Indiana University-Purdue University Indianapolis (IUPUI) / Pseudomonas aeruginosa is a gram negative opportunistic pathogen with the capacity to cause serious disease by forming biofilms, most notably in the lungs of cystic fibrosis (CF) patients. Biofilms are communities of microorganisms that adhere to a solid surface, undergo global regulatory changes, secrete exopolysaccharides, and are innately antibiotic resistant. Virulence modulation is an important tool utilized by P. aeruginosa to propagate infection and biofilm formation in the CF airway. Many different virulence modulatory pathways and proteins have been identified including the protein, MgtE. MgtE has recently been discovered and has been implicated in virulence modulation, as an isogeneic mutation of mgtE leads to increased cytotoxicity. To further elucidate the role of MgtE in P. aerugionsa infections, transcriptional and translational regulation of this protein following antibiotic treatment has been explored. I have demonstrated that mgtE is transcriptionally upregulated following antibiotic treatment of most of the twelve antibiotics tested utilizing RT-PCR and QRT-PCR. A novel model system was employed, which utilizes cystic fibrosis bronchial epithelial (CFBE) cells homozygous for the ΔF508 mutation for these studies. This model system allows P. aeruginosa biofilms to form on CFBE cells modeling the P. aeruginosa in the CF airway. Translational effects of antibiotic treatment on MgtE have been attempted via Western blotting and cytotoxicity assays. Furthermore, to explore the possibility that mgtE is interacting with a known regulatory pathway, a transposon-mutant library was utilized and the regulatory proteins, AlgR and NarX, among others have been identified as possibly interacting with MgtE. Lastly, an MgtE homologue from Staphylococcus aureus was utilized to further demonstrate the virulence modulatory effects of MgtE by demonstrating the expression of the homologue results in decreased cytotoxicity, exactly like expression of the native P. aeruginosa MgtE. This research explores a newly discovered protein that impacts cytotoxicity and biofilm formation and provides valuable information about P. aeruginosa virulence.