Date of Award

Spring 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology

First Advisor

Kazmierczak, Barbara

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen known to cause acute infections in individuals with epithelial damage or medically placed devices, leading to sepsis or death in immunocompetent and immunocompromised individuals. Like many other Gram-negative bacteria, P. aeruginosa uses a Type III Secretion System (T3SS) to intoxicate eukaryotic cells. This T3SS is essential for P. aeruginosa to establish an infection and avoid clearance by immune cells and is also linked to increased morbidity and mortality in infections. Despite this necessity, activation of the T3SS slows P. aeruginosa’s cell growth and permits innate immune system detection and resulting inflammation. To balance these costs, P. aeruginosa cells heterogeneously express T3SS genes when uniformly exposed to T3SS-activating signals. Previous work shows that T3SS-ON cells largely originate from a subpopulation of cells that express higher levels of the T3SS transcriptional activator, ExsA. These ExsA+ cells are ‘primed’ to respond immediately to receiving T3SS activating signals by upregulating T3SS gene expression rapidly secreting T3SS effectors. However, the mechanistic basis by which T3SS-primed cells are generated is currently unknown. All T3SS genes are regulated by the transcriptional activator, ExsA. ExsA is in turn regulated through a complex partner-swapping network and positively autoregulates its own expression, as well as that of its anti-activator ExsD, ExsD’s antiactivator ExsC, and ExsC’s binding partner ExsE. These features create a bistable regulatory network for T3SS expression. There is one ExsA-independent promoter, PexsA, within the T3SS regulon, which is located upstream of ExsA and regulated by a Vfr-cyclic-AMP (cAMP) complex. I hypothesized that a cAMP signal could promote transcription from this promoter and suffice to drive P. aeruginosa cells into a stable primed state. Using flow cytometry, I tested this hypothesis at the single cell level and observed that exogenous cAMP was sufficient to increase the proportion of ExsA+, T3SS-primed cells. Further work using whole cell cryo-electron tomography (cryo-ET) revealed that a subpopulation of P. aeruginosa cells assembled T3SS needles naïve of a T3SS-activating signal, with cAMP treatment increasing both the proportion of ExsA+ ‘primed’ cells and the number of cells assembling a T3SS needle. I further explored the role of endogenous cAMP in T3SS-priming, exploiting strain-specific differences in priming ‘setpoint’ to examine correlations between intracellular cAMP levels and the priming phenotype. I also investigated the influence of environmental and metabolic signals on T3SS-priming at the single cell level. By screening panels of single carbon sources, I identified 17 compounds that significantly altered the proportion of ExsA+ primed cells. These included previously unrecognized modulators of T3SS expression, as well as known inhibitors and inducers, which acted by altering the proportion of T3SS-primed cells. Lastly, I examined whether Type IV Pilus (T4P)-mediated surface sensing, which increases endogenous cAMP levels, would alter the setpoint of T3SS-priming. I observed that only mutations in PilA, the major T4P pilin subunit, and PilJ, the methyl-accepting chemotaxis T4P protein significantly decreased the proportion of T3SS-primed cells. The interaction of these two proteins is postulated to be necessary for adenylate cyclase activation by the Pil/Chp chemotaxis system. As exogenous cAMP could complement this phenotype in trans, endogenous cAMP is likely to be a bona fide signal for T3SS-priming. I propose that the T3SS activation is regulated at two steps. Sufficient ExsA must be made to cross the threshold that separates bistable off and on states of T3SS gene expression. On cells assemble T3SS needles before shutting down further gene expression via the ExsA/D and ExsC/E protein sequestration mechanism, creating a stable and heritable primed phenotype. These primed cells are poised to respond to T3SS-activating signals, which trigger ExsE secretion and a partner swapping cascade that liberates ExsA, by immediately upregulating T3SS gene expression and effector secretion. Signals such as cAMP play a key role in priming, by stimulating ExsA transcription from the PexsA promoter. My findings also suggest that ExsA expression and T3SS-priming respond to a variety of nutrient signals, and further elucidate the link between T3SS expression and T4P-mediated chemosensing, opening new avenues for future research.

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