Date of Award

2020

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Biology

Abstract

Bacteriophages (phages) are viruses that infect bacteria. They are the most abundant life forms on earth. It is estimated that there are approximately 10³⁰ phages on the planet (Chibani-Chennoufi et al. 2004). They outnumber their bacterial hosts approximately 10 to 1. Bacteria inhabit most niches on the planet including the human body, where they play critical roles in health and disease. Since phages shape bacterial populations the same way that viruses, such as smallpox and measles, shaped human populations, it is important to understand how they interact with their bacterial hosts. The majority of the phages that we know of replicate through either the lytic or lysogenic life cycle. Some phages are obligately lytic, while some are temperate. An obligate lytic phage injects its genome into the cytoplasm of its host, where it is replicated, transcribed and translated. The expressed coat proteins are then assembled, and the replicated genome packaged within the coat. The progeny phages then lyse their host (for a review, see Young 2014). In contrast, a temperate phage can choose between two lifecycles: lytic or lysogenic (Hobbs & Abedon 2016). Immediately following injection of its genome into the host cytoplasm, this choice is made. It can directly enter into the lytic cycle as described above or it can enter the lysogenic lifecycle, whereby it is incorporated into the host's genome, either by integration into the chromosome or by becoming an autonomous plasmid (Little 2005). In either case, it replicates in coordination with its host's genome. Such bacteria are known as lysogens and the phage genome is called a prophage. A lysogen can be induced to produce progeny phage. Upon receipt of an inducing signal the prophage will enter the lytic life cycle (Little 2005; Abedon 2008). The prophage genome will then be transcribed, translated, and replicated, generating progeny phage. The cell host then lyses, releasing the newly assembled phage particles into the extracellular environment to infect new hosts. Prophages that reside within lysogens have the capability to block the lytic life cycle of other phages. This is known as superinfection immunity. Superinfection immunity affects the permissivity of the host intracellular environment, and not the actual infection process itself. Infection is defined as the process of adsorption (the phage can either adsorb to the host, or it cannot). The permissivity of the host is defined by events or mechanisms that occur once the phage has already adsorbed to its host, and has injected its DNA inside of the host cytosol. Some host environments are more permissive than others, as they more readily allow viral genomes to go through the lytic life cycle. Superinfection immunity can occur through more than one mechanism. The most well-known mechanism is known as classical immunity. Classical immunity is when the prophage expresses a repressor that binds to operators known to induce the lytic cycle. This same repressor will bind to superinfecting phage genomes and will disallow progression of infection. Other mechanisms of superinfection immunity include changing the receptors that phage bind to or by altering the replication machinery. Until recently, the bladder was considered to be sterile, a long-standing belief that was forged in the mid 1800's by Louis Pasteur, Joseph Lister and William Roberts. These pioneering microbiologists observed that a vial of urine in a sealed container did not become cloudy, in contrast to a vial that was exposed to air or with added tap water. This dogma persisted until recently. Using 16S rRNA gene sequencing, two groups provided evidence that the bladder is not sterile. Both teams detected bacterial DNA evidence in catheterized urine samples deemed 'no growth' by the standard clinical microbiology urine culture technique (Wolfe et al. 2012, Fouts et al. 2012). Two other reports used enhanced culture methods to provide evidence that the sequenced taxa were alive (Khasriya et al. 2013; Hilt et al. 2014). In his more recent dissertation work, Travis Price showed that urinary microbiota can exhibit temporal dynamics. The mechanisms underlying these dynamics remain unknown. One possible mechanism is phage infection. Obligate lytic and temperate phages have been found throughout the human body (see review Barr 2017). They have also been found in the urinary tract. Indeed, one of the first lytic phages identified was isolated directly from urine (d'Herelle 1917). More recently, lytic phages have been detected in the urinary tract (Garetto et al., 2019). However, they are only one part of the urinary phage community; temperate phages have also been identified and some have been isolated (Garetto et al. 2019). Since phages reside within the urinary tract and phages are known to influence bacterial communities, I have chosen to study urinary phage interactions with urinary bacteria. I determined the ability of several urinary and laboratory phages to infect a set of urinary and laboratory strains of E. coli. I then attempted to understand why one E. coli lysogen isolated from the bladder is not immune from infection by one of its prophages. The knowledge obtained with E. coli phages and their urinary hosts will be used as a template for the study of other bladder species and their phage.

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