ADVANCING MICROBIAL-TARGETED PHAGE THERAPEUTICS USING GNOTOBIOTIC MOUSE MODELS

Abstract

Adherent-invasive Escherichia coli (AIEC) exhibit proinflammatory properties that have been implicated in the pathogenesis of Crohn’s disease (CD), a form of inflammatory bowel disease (IBD), which also includes ulcerative colitis (UC). It is estimated that AIEC are present in one-third of patients with IBD and have been associated with exacerbating intestinal inflammation and disease reactivation. Current therapies, which include topical corticosteroids, immunomodulators, and biologics, actively target the dysregulated immune system and neglect underlying microbial drivers of disease, including AIEC. Antibiotic use in CD lacks specificity and may worsen microbiome disruption, which could lead to further exacerbation of intestinal colitis or increased risk of secondary infections. These shortcomings have prompted interest in bacteriophages (phages) for targeted microbiome editing. In this thesis, I identified HER259, a phage active against the clinical AIEC strain NRG857c. Using newly developed gnotobiotic models of AIEC-exacerbated colitis, I showed that HER259 mitigates NRG857c-excerbated colitis, not solely through bacterial load reduction, but through the attenuation of AIEC virulence factors. This included suppression of the FimH adhesin, which was illustrated through a significant inversion of the fimS promoter to its ‘off’ orientation. Withdrawal of HER259 treatment leads to reversion of the fimS promoter to the ‘on’ orientation and reactivated colitis in mice. FimH suppression was corroborated by immunohistochemistry, which revealed HER259-treated NRG857c having reduced capacity to invade the lamina propria, a key mechanism of pathogenesis that is, in part, mediated by FimH. Clinical monitoring and immunohistochemistry of mice colonized with NRG857c ΔFimH revealed a similarly attenuated colitis phenotype to that of HER259-treated mice. Reisolated HER259-treated NRG857c also had impaired abilities to form biofilms, another virulence mechanism of AIEC supported by FimH. Concurrently, I showed that HER259 intervention can attenuate colitis reactivation in a dual-cycle chemically-induced model of colitis, which supports the use of phage therapy to prevent or more effectively treat flares. Building on this and considering phage therapy would likely be employed as adjunct to conventional drugs, I prophylactically administered HER259 with a subtherapeutic dosage of budesonide; a common first-line intervention used to treat mild-moderate colitis in patients. Mice that received the combined HER259-budseonide therapy responded significantly better than either intervention alone, and was mechanistically independent of the microbiota’s ability to metabolize budesonide. Collectively, these findings support targeted phage therapy as an adjunct treatment approach in IBD, demonstrating modulation of bacterial virulence and improved response to conventional treatments. Practically, this approach of phage therapy has the potential to reduce drug-related side effects frequently associated with existing interventions, thereby improving clinical outcomes and patient lives.