Pseudomonas aeruginosa is a Gram-negative bacterium that causes acute nosocomial infections as well as chronic infections in cystic fibrosis (CF) patients. P. aeruginosa utilizes a type III secretion system (T3SS) during acute infections to promote host cell cytotoxicity and inhibit phagocytosis. Regulation of T3SS expression can be classified into two distinct categories: intrinsic and extrinsic. T3SS intrinsic regulation involves the well-characterized ExsECDA cascade that controls T3SS gene transcription. Extrinsic regulation involves global regulatory systems that affect T3SS expression. Despite general knowledge of global regulation of T3SS expression, few specific mechanisms have been elucidated in detail. The overall goal of my thesis work was to provide clarity to global regulatory mechanisms controlling T3SS expression. One well-documented observation is that P. aeruginosa isolates from CF patients commonly have reduced T3SS expression. In chapter II, I describe how the MucA/AlgU/AlgZR system, commonly activated in CF isolates through mutation of the mucA gene, inhibits T3SS gene expression. My experiments demonstrate that the AlgZR two-component system inhibits ExsA expression through two separate global regulatory systems. First, as previously described, AlgZR inhibits ExsA expression by reducing activity of the cAMP/Vfr signaling pathway. Vfr, a homolog of Escherichia coli Crp, regulates T3SS gene expression through an unknown mechanism. Second, AlgZR alters the activity of the RsmAYZ system to specifically reduce ExsA expression. The RNA-binding protein RsmA, a homolog of E. coli CsrA, activates ExsA expression at a post-transcriptional level. Previous studies in our laboratory identified several transposon insertion mutants that appeared to be novel extrinsic regulators of T3SS gene expression. One of those candidates, named DeaD, is a putative ATP-dependent RNA helicase. My experiments in chapter III reveal that DeaD regulates T3SS expression by directly stimulating exsA translation. Mutants lacking deaD have reduced exsA translational reporter activity and ExsA expression in trans fails to complement a deaD exsA double mutant for T3SS gene expression. I demonstrate that purified DeaD stimulates ExsA expression in a coupled in vitro transcription/translation assay, confirming our in vivo findings. In chapter II, I observed that RsmA activates the transcription of RsmY and RsmZ, two small non-coding RNAs that act to sequester RsmA from target mRNAs. My experiments in chapter IV begin to dissect the RsmA-activation mechanism of RsmY/Z expression. I show that RsmA activation requires the previously described Gac/Lad/Ret system that controls RsmY/Z expression. RsmA, however, does not alter Gac/Lad/Ret gene transcription or translation. Interestingly, an RsmA variant deficient in RNA-binding, RsmA R44A, was able to complement an rsmA mutant for RsmY/Z expression. I hypothesized that RsmA interacts with an unknown protein to activate RsmY/Z expression and identified several potential interaction partners using co-purification assays. Together, my combined experiments elucidate novel global regulatory pathways controlling T3SS gene expression during acute and chronic P. aeruginosa infections, and provide a foundation towards the goal of developing future treatment options.

Pseudomonas aeruginosa is an important pathogen of the immunocompromised, causing both acute and chronic infections. In cystic fibrosis (CF) patients, P. aeruginosa causes chronic disease. The impressive sensory network of P. aeruginosa allows the bacterium to sense and respond to a variety of stimuli found in diverse environments. Transcriptional regulators, including alternative sigma factors and response regulators, integrate signals changing gene expression, allowing P. aeruginosa to cause infection. The two-component transcriptional regulator AlgR is important in P. aeruginosa pathogenesis in both acute and chronic infections. In chronic infections, AlgR and the alternative sigma factor AlgU activate the genes responsible for alginate production. Previous work demonstrated that AlgU controls rsmA expression. RsmA is a posttranscriptional regulator that is antagonized by two small RNAs, RsmY and RsmZ. In this work, we demonstrate that AlgR directly activates rsmA expression from the same promoter as AlgU. In addition, phosphorylation was not necessary for AlgR activation of rsmA using algR and algZ mutant strains. AlgU and AlgR appear to affect the antagonizing small RNAs rsmY and rsmZ indirectly. RsmA was active in a mucA22 mutant strain using leader fusions of two RsmA targets, tssA1 and hcnA. AlgU and AlgR were necessary for posttranscriptional regulation of tssA1 and hcnA. Altogether, our work demonstrates that the alginate regulators AlgU and AlgR are important in the control of the RsmA posttranscriptional regulatory system. These findings suggest that RsmA plays an unknown role in mucoid strains due to AlgU and AlgR activities.