Year of Award

2024

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Degree Name

Cellular, Molecular and Microbial Biology

Department or School/College

Division of Biological Sciences

Committee Chair

Patrick Secor

Commitee Members

Ekaterina Voronina, Brandon S. Cooper, Brent Ryckman, Paul L. Bollyky

Keywords

bacteriophage, lysogen, pf phage, prophage, pseudomonas, superifection

Abstract

Pseudomonas aeruginosa is a gram-negative opportunistic bacterial pathogen which is as ubiquitous in the natural environment as it is within hospital settings, where it is a readily acquired nosocomial pathogen. Bacteriophages, viruses which infect bacteria, are a central component of the arsenal P. aeruginosa leverages to combat the stresses of infecting mammalian hosts. Over 60% of P. aeruginosa clinical isolates are chronically infected with temperate phages within the family of Inoviridae. These phages, referred to as Pf, have a filamentous virion structure which accommodates genome sizes between 10-20kbp. Pf is capable of integration into the bacterial chromosome where they can exist in a dormant prophage state, or excise and produce viral progeny which extrude from the bacterial host. Overproduction of Pf resulting from infection of a cell that already harbors a prophage; called superinfection, can lead to membrane stress and ultimately cell lysis. Temperate phages, such as Pf, typically encode superinfection exclusion mechanisms. Twitch motility is mediated by the classical bacterial virulence factor, Type IV Pili (T4P) which mediates bacterial adhesion allowing for biofilm formation. Importantly, T4P serve as the cell surface receptor for Pf, among other phages. We describe inhibition of Pf plaquing and twitch motility by a highly conserved Pf superinfection exclusion gene, pfsE (PA0721). Furthermore, we determine that highly conserved aromatic residues facilitate PfsE localization to the inner membrane where PfsE binds to T4P structural subunit PilC, suppressing twitch motility. As most work on Pf phages focuses on Pf4 in the model P. aeruginosa strain PAO1, we sought to interrogate how Pf phage modulate virulence-associated phenotypes within diverse clinical isolate hosts. We describe a technique which targets the Pf lysogeny maintenance gene pflM (PA0718) and demonstrate that pflM deletion results in prophage excision but not replication, leading to total prophage loss. We identify conservation of a DUF5447 domain within all examined pflM alleles, implicating this domain in Pf lysis/lysogeny decision making. Next, we assess the effects different Pf phages have on virulence associated phenotypes and determine that although impacts on quorum sensing and biofilm formation appear to be strain-specific, nearly all Pf phages suppress pigment production and increase bacterial virulence against bacterivorous nematode Caenorhabditis elegans.

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© Copyright 2024 Amelia Kass Schmidt