Hooper, Lora V.2022-06-222022-06-222020-052020-05-01May 2020https://hdl.handle.net/2152.5/9862Humans are populated by an extensive community of microorganisms, primarily in organs such as the skin, mucosal membranes in the mouth, reproductive organs, and the gut. This complex community, termed the microbiota, is in part comprised of bacteria, many of which have intimate associations with their hosts to promote physiological homeostasis. These organisms, commonly termed commensal bacteria, have a rich and long history with their human hosts and accomplish important functions such as providing the host with nutrients, developing the immune system, and preventing colonization by pathogenic organisms in a process known as colonization resistance. These functions are especially apparent within the gastrointestinal (GI) tract, which contains the richest and most densely populated community of microbes in the body. Healthy gut function relies on the proper structure and balance of this microbial community. Disruption of the community, termed dysbiosis, has been associated with a plethora of diseases such as increased susceptibility to GI infections, neurological disorders, intestinal inflammation, and cancer progression. Dysbiosis is most commonly caused by pharmacological interventions with antibiotics or infection with a GI pathogen. The microbiota is regarded as a barrier against intestinal pathogens, partly due to intense competition for a limited supply of nutrients and space. This suggests that GI pathogens have evolved mechanisms to overcome colonization resistance and outcompete the resident microbiota for resources within the GI tract. Microbiota and host-derived metabolites have a significant impact on the abilities of GI pathogens to successfully establish intestinal infection and the subsequent development of disease. However, the precise mechanism by which microbiota or host metabolites affect the pathogenesis of GI pathogens is not well understood. Many of these nutrients, whether host-, diet-, or microbiota-derived, serve as chemical cues for incoming pathogens. These signals are used by pathogens to gauge resource availability, microbiota composition, host physiology, and location within the intestines to properly deploy virulence strategies that allow for colonization. Microbiota-derived small molecules include toxins, antimicrobials, oligopeptides, hormones, and products of microbial metabolism of host-derived and dietary molecules. Pathogens can directly sense many of these host- and microbiota-derived small molecules, which in turn can regulate their virulence mechanisms. Taken together, developing therapeutics that target the signaling pathways that control virulence-associated functions in pathogens represent an attractive alternative or secondary strategy to tackle bacterial infections. In a previous study, our group conducted a candidate-based screen of 372 independent mutants to look for novel regulators of the T3SS [1]. The candidates of this screen consisted primarily of transcription factors, two-component regulatory systems, anti-terminators and anti-toxins. This work generated a great number of hits that potentially regulate the T3SS of EHEC. Our work sought to characterize novel signaling pathways that directly affect the virulence of enterohemorrhagic Escherichia coli (EHEC) through characterization of some of the hits of said screen in particular the transcriptional regulators ExuR and FadR. Understanding of these signaling pathway could lead us to develop novel strategies to drive down the virulence of enteric pathogens and improve colonization resistance as an alternative approach to control bacterial infections. Here, we found that EHEC senses and utilizes galacturonic-acid (GalA) as a nutrient during infection and moonlights as a signal to downregulate the expression of virulence associated genes. Furthermore, we demonstrated that a pectin-rich diet, which is a source of GalA, increased mice tolerance towards a Citrobacter rodentium infection, a surrogate mice model for EHEC infection. AE pathogens like EHEC and C. rodentium thrive in an inflamed environment. During the onset phase of inflammation, the host-derived polyunsaturated omega-6 long-chain fatty acid (LCFA), arachidonic acid (AA) becomes elevated to produce endogenous lipid signaling molecules like prostaglandins and leukotrienes that act as inducers of inflammation. EHEC can sense long-chain fatty acids through the FadR response regulator. We found that AA is processed by EHEC using canonical LCFA signaling pathways involving the FadL LCFA transporter, the FadD acyl-CoA synthase and the FadR transcriptional regulator. In conclusion, we characterized the signaling pathways that mediate the sensing of galacturonic-acid and arachidonic-acid. We demonstrated that a diet high in pectin can effectively be used to control an infection by C. rodentium by effectively modulating the levels of GalA and affecting virulence in an ExuR dependent manner. We also showed that EHEC is capable of sensing a host produced long chain fatty acid like arachidonic acid to regulate its virulence. These studies highlight the complexity that underlies regulation of the locus of enterocyte effacement and perhaps will serve as a starting point for the development of new strategies to control enteric infections.application/pdfenBacteriaEnterohemorrhagic Escherichia coliMicrobial InteractionsType III Secretion SystemsVirulence FactorsThesis2022-06-221333220198