Year of Award

2023

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Degree Name

Cellular, Molecular and Microbial Biology

Other Degree Name/Area of Focus

Microbiology and Immunology

Department or School/College

Division of Biological Sciences

Committee Chair

Patrick R. Secor

Commitee Members

Stephen Lodmell, Michael F. Minnick, Scott Samuels, Brandon S. Cooper, Angela D. Luis

Keywords

aeruginosa, Bacteriophage, Filamentous, Pf phage, Pseudomonas, Virulence

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen that often plagues hospitals. More than half of P. aeruginosa isolates are infected by temperate Pf bacteriophage. Pf virions protect bacteria from antibiotics, promote biofilm formation, and modulate animal immune responses in ways that promote chronic infection. These virions can be produced without lysing P. aeruginosa, but lysis may occur during superinfection. Superinfection is the process whereby virions of the same or similar phage infect an already infected host. Temperate phages typically encode superinfection exclusion mechanisms. Here we elucidate one such mechanism in Pf phage. We observed that superinfection-surviving P. aeruginosa were transiently resistant to Pf-infection and deficient in twitch motility. Twitch motility requires type IV pili (T4P), a bacterial cell surface receptor used by Pf to gain entry. We tested the hypothesis that T4P are suppressed by a Pf-encoded protein. We observed that the Pf protein PA0721, which we termed Pf superinfection exclusion (PfsE), suppressed twitch motility, and promoted resistance to Pf infection by binding the T4P protein PilC. Beyond this superinfection exclusion mechanism, we elucidated a second function for PfsE. Upon overexpression, PfsE reduced the production of the virulence factor pyocyanin and transcription of pqs quorum sensing genes. Quorum sensing is density-dependent bacterial communication, whereby bacteria can coordinate complex processes, including pathogenesis and phage defense. We found that PfsE interacts with PqsA by folding into an alternate kinked conformation. We subsequently sought to understand how these quorum system effects affect P. aeruginosa virulence. P. aeruginosa cured of their Pf infection (ΔPf) unsurprisingly show greater pqs activation and pyocyanin production. However, we report that this resulted in a loss of virulence against Caenorhabditis elegans. This seemingly contradictory finding may be explained by our observation that C. elegans mutants were unable to sense bacterial pigments, such as pyocyanin, through their aryl hydrocarbon receptor and are more susceptible to ΔPf infection compared to wild-type C. elegans. Collectively, we describe a dual-function Pf-encoded protein PfsE, which conveys superinfection exclusion and suppresses quorum sensing. Furthermore, the suppression of quorum sensing and downstream virulence factors by Pf allows P. aeruginosa to evade detection by innate host immune responses.

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© Copyright 2023 Caleb Michael Schwartzkopf