B Brett Finlay

Enteropathogenic Escherichia coli (EPEC) is a bacterial pathogen that causes significant morbidity and mortality worldwide. During infection, EPEC attaches to the intestinal epithelium and uses a molecular syringe-like nanomachine called a Type 3 Secretion system (T3SS) to secrete virulence ‘effector’ proteins directly into human cells, where they manipulate host cell signalling and mediate the characteristic features of infection, including intimate attachment to host cells, formation of actin-rich pedestals, disruption of the epithelial barrier, and dysregulated immune function and cell survival. Some effectors possess enzymatic activities that allow them to directly modify host proteins through post-translational modifications, including ubiquitination, phosphorylation, and proteolysis. However, the host targets of many effectors are unknown and their collective impact on global host post-translational modification has not been widely explored. This work utilizes proteomics tools to characterize the impact of EPEC infection and T3SS effectors on the host proteome. Here, ubiquitin proteomics were used to investigate the EPEC effector ubiquitin ligase NleG, exploring its impact on the host ubiquitome, and identifying and characterizing the relationship of NleG with a novel host substrate: Ataxin-3. We also developed a novel EPEC infection-proteomics workflow to characterize global changes in host protein post-translational modifications during important stages of infection. This method simultaneously profiled changes in protein abundance, ubiquitination, and phosphorylation due to EPEC infection, T3SS effector influences, and host response during early, mid-, and late infection. Infection-proteomics exhibited the highest coverage of the host proteome, ubiquitome, and phosphoproteome of any EPEC proteomics investigation to date, identifying thousands of EPEC infection-, exposure-, and T3SS-associated changes in proteins involved in cell processes regulated by EPEC during infection. These include innate immune signalling, actin remodelling, and cell survival pathways, while also identifying novel pathways targeted by EPEC. This work demonstrates the power of proteomics techniques to characterize impacts of EPEC infection on the host proteome by developing a novel approach to study how EPEC infection and effectors affect host post-translational modification and cell signalling throughout infection. Proteomics tools serve as valuable methods to study host-pathogen interactions and identifying molecular level changes underpinning characteristic phenotypes mediated by EPEC during infection.

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The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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Infectious diarrheal diseases are the third leading cause of mortality in young children, many of which are driven by Gram-negative bacterial pathogens, including enteropathogenic Escherichia coli (EPEC). This thesis employed an amalgamation of omics, in silico prediction, and conventional molecular biology tools to investigate the host-pathogen interactions during EPEC infection, primarily focusing on the systems-level and protein interactions. We employed a dual RNA-sequencing approach to investigate the host and microbe physiology during EPEC infection of an intestinal epithelial cell line (Caco-2/TC-7) with a focus on the role of the Type III Secretion System (T3SS). Our findings showed that T3SS was used by EPEC to suppress various host responses, including immune signaling and apoptosis. We hypothesized that microRNAs (miRNAs) were important for mediating host and pathogen behaviors and identified differentially expressed miRNAs that played a role in suppressing cell death and altering cytokine secretion in response to infection. With regards to bacterial transcriptome, we observed drastic upregulation of the Type II Secretion System (T2SS) genes in EPEC upon host cell contact. To explore the role of the T2SS during natural host infection, we used Citrobacter rodentium, a murine enteric pathogen, as a model of EPEC-caused disease. We demonstrated that this system was functional in vitro with potential roles in intestinal mucin degradation. During host infection, loss of the T2SS or predicted effectors led to a significant colonization defect and lack of systemic spread. Finally, apoptosis was among the pathways with a T3SS-associated pattern of dysregulation during infection. This prompted us to investigate the function of a pro-apoptotic T3SS effector, Map, due to the lack of understanding of its mechanism of action. Through a combination of proximity labelling mass spectrometry and deep learning interaction prediction algorithm, we identified several Map partners, several of which were involved in the electron transport chain (ETC). Collectively, this thesis provides the first survey of the host and bacterial transcriptomes during EPEC infection, the first confirmation of the importance of the C. rodentium T2SS for robust infection in vivo, and identification of novel host mitochondrial proteins interacting with EPEC effector Map.

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Parkinson’s disease (PD) is the second most common neurodegenerative disease in the world. Gastrointestinal comorbidities, especially constipation and slow colonic transit time, affect most patients and can manifest decades before motor symptom onset, leading to speculation that the gut microbiota may be involved in disease etiology and pathophysiology.Leveraging clinical cohorts and in vitro models, I investigated the microbiota’s role in PD. Bacterial (16S) and fungal (ITS2) amplicon sequencing elucidated the composition of the microbiota in PD patients and controls, and paired serum metabolomics helped infer microbial function. I showed that the PD microbiota had a reduced abundance of several co-abundant short-chain fatty acid producing bacteria, including well-known butyrate producing genera such as Faecalibacterium and Roseburia. The butyrate production capacity of the PD microbiota was inversely associated with colonic transit time as estimated by Bristol Stool Scale score. Conversely, the PD microbiota had a higher abundance of several co-abundant bacteria with diverse metabolic capacities, including the mucin-degrading Akkermansia muciniphila. PD-enriched microbes were strongly associated with increased colonic transit time and serum concentrations of the proteolytic microbial metabolites p-cresol and phenylacetylglutamine. These findings generated mechanistic hypotheses that were carried into in vitro experiments. Regarding PD-relevant microbial metabolites, I showed that butyrate dampened cytokine production and immune cell attachment in a blood brain barrier endothelial cell line, while p-cresol caused inflammation and downregulated genes involved in gut peptide and serotonin biosynthesis in an enteroendocrine cell line. A final clinically-informed mechanistic insight came from the observation that the abundance of the PD-enriched genus Bifidobacterium was correlated with levodopa (L-DOPA) dose in patients. L-DOPA is the primary treatment for PD. It is taken orally, absorbed in the intestines, and must reach the brain intact to exert its clinical effect. Peripheral breakdown of L-DOPA is an ongoing clinical concern, including metabolism by gut microbes. Through in vitro assays, a pathway was discovered whereby certain Bifidobacterium species can metabolize L-DOPA into its equivalent lactic acid using existing tyrosine metabolism genes. In conclusion, in this thesis I demonstrate compositional and functional alterations in the PD microbiota with important downstream consequences on gut function and therapeutic management.

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Parkinson’s disease (PD) is a neurodegenerative disorder characterized by motor dysfunction. Non-motor symptoms including gastrointestinal (GI) dysfunction and mood disorders (such as depression) are also particularly common. GI symptoms include constipation, and PD patients display altered gut microbiota composition. Evidence in animal models points towards a potential causal role for the microbiota in mediating PD pathology. However, the constipation, medication use, and lifestyle habits of PD patients can also be expected to change microbiota composition. In this thesis, I explore the role of the gut microbiota in PD, in terms of the degree that it is shaped by the disease state and its ability to mediate disease symptoms. Using a transgenic mouse model of PD that displays motor deficits, GI dysfunction, and behavioral alterations, I assessed how broad alterations to the gut microbiota impacted the motor and non-motor phenotype. I found that both depletion of the microbiota through antibiotics, and a shift towards a healthy wild-type mouse microbiota, had a minimal impact on the PD-like symptoms. This suggested that the PD-like transgenic state of this model may drive the disease phenotype to a greater extent than the microbiota. Similarly, I demonstrated that the decreased abundance of Lachnospiraceae and decreased abundance of Ruminococcaceae and Oscillospiraobserved in PD patients may be a result of constipation by treating PD mice with laxatives that reversed these shifts. Lachnospiraceae abundance was also found to be decreased by treatment of this model with the PD medications L-DOPA and carbidopa. Conversely, different antibiotic treatment regimens were able to shift the microbial community and alter GI transit time in PD mice. Specific bacterial taxa, such as Lachnospiraceae (Ruminococcus), were associated with transit time – indicating a potential to treat PD constipation via the microbiota. Furthermore, treatment of PD mice with PD medications had a beneficial effect on constipation and depression-like behavior, potentially through increasing the abundance of Turicibacter and promoting butyrate production. This thesis demonstrates that certain PD-associated microbiota alterations may be a result of slowed GI transit or the presence of medications. However, specific shifts to the gut microbiota may in turn mediate non-motor symptoms in PD.

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The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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Worldwide, approximately 340 million children, 50% of children
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Global asthma prevalence has reached epidemic proportions, emphasizing an urgent need for research into the causes of this burdensome and incurable early-onset disease. A growing body of evidence implicates the early life gut microbiota in immunomodulation relevant to asthma, and suggests that an imbalanced (dysbiotic) gut microbiota may precede disease onset. Notably, fungal communities of the infant gut microbiota (the mycobiota) are understudied, but have recently been shown in two birth cohorts to differ strikingly according to atopy/asthma risk in association with bacterial dysbiosis. I hypothesized that fungal dysbiosis in the infant gut contributes to asthma development through interactions with the host and immunomodulatory bacteria to alter key early-life immune mechanisms involved in this disease. To address this hypothesis, I first generated additional evidence supporting associations between bacterial dysbiosis at three months of age with atopic outcomes in Canadian children from the CHILD Cohort Study. I then examined the fungal communities of the gut microbiota in these infants at three months and one year of age and characterized features of early life gut fungal dysbiosis associated with atopic disease outcomes at age five years. Replicating our lab’s findings in Ecuadorian infants, these analyses revealed that overgrowth of the yeast Pichia kudriavzevii in the infant gut was associated with atopic outcomes at age five years. Using a mouse model of allergic airway disease, I then established a causal role for overgrowth of P. kudriavzevii within the infant gut in increasing allergic airway disease severity later in life. Finally, I used in vitro microbiology techniques to determine how bacterial-fungal interactions shape asthma-associated gut microbiota community structures. I found that bacterial-derived short-chain fatty acids (SCFAs), which we found to be reduced in abundance in stool from infants at risk of atopic disease, inhibit the growth of P. kudriavzevii and the ability of this yeast to adhere to gut epithelial cells. Using biologically relevant experimental systems that broadly address the role of early life fungal dysbiosis in asthma, I provide mechanistic insights into gut microbiota bacterial-fungal interactions, mycobiota-immune interactions, and the gut-lung axis. This work may ultimately inform the development of novel microbiota-based therapeutics.

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The success of Salmonella Typhimurium as a pathogen relies on its ability to invade and survive within intestinal epithelial cells. S. Typhimurium thrives as an intracellular pathogen through the activity of a type III secretion system (T3SS2) whose secreted effectors create a unique intracellular replicative niche for S. Typhimurium through regulation of host immune pathways, host cell cytoskeleton rearrangement, regulating host cell ubiquitination events, and hijacking the host endosomal system. Significant efforts to elucidate T3SS2 effector molecular mechanisms through single effector studies have thus far been unable to fully explain the events leading to establishment of the S. Typhimurium intracellular niche. We undertook a systematic study to delineate the contribution of each effector associated with manipulation of the host endosomal system—namely SifA, SopD2, PipB2, SteA, SseJ, and SseFG by creating a deletion mutant library containing single-effector and multiple effector deletion mutants. It was shown that each of Salmonella-induced filament biogenesis, intracellular localization of the Salmonella-containing vacuole, intramacrophage replication, colonization, and virulence depends on the activities of multiple effectors. We demonstrate the complex interplay between effectors and highlight the necessity to study T3SS2-secreted effectors as groups, rather than as individual effectorsA T3SS2 overexpression mutant (T3SS2⁺) was created to increase the abundance of secreted T3SS2 proteins and tagged effectors implicated to manipulate the host endosomal system. To identify novel host targets for these effectors while maintaining natural effector delivery throughout infection, a global mass spectrometry screen was performed using the SILAC (stable isotopic labelling with amino acids in cell culture)-labelling method to label host cells and infected them with T3SS2⁺ mutants. Using this method, the host protein annexin A2 was identified as a target for both SopD2 and PipB2. A shared host target for two effectors may explain the complex and nuanced phenotypes observed during S. Typhimurium infection. Together, the work presented here demonstrates that T3SS2-secreted effectors are dependant on each other’s activities during infection and shows binding of two effectors to a single host protein. This work contributes to our understanding of the intracellular lifestyle of S. Typhimurium and sets a new paradigm for future research.

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Child undernutrition is a global health issue that is exacerbated by poor sanitation and infectious disease; however, the microbial and immune contributions to child growth remain poorly understood. To understand how nutrition impacts immune-microbe interactions, I assessed interactions between intestinal bacteria and immunoglobulin A (IgA), the antibody responsible for mucosal homeostasis, in mouse and human undernutrition. In contrast to healthy control mice, undernourished mice failed to develop IgA recognition of commensal Lactobacillus. Glycan-mediated interactions between Lactobacillus and IgA were lost in undernourished mice; this was driven by bacterial adaptation to the nutritional environment, independently of host antibody, and was associated with bacterial mutations in carbohydrate processing genes. Together these data indicate that diet-driven bacterial adaptations shape IgA recognition in the gut, which may have implications for the use of probiotics and oral vaccines in undernutrition. To extend these findings to human populations, I measured IgA-microbiota interactions in the fecal microbiota of 200 children with or without linear growth stunting from Madagascar and the Central African Republic. Stunted children had increased abundance of several pathobionts; two of these, Haemophilus and Campylobacter, were strongly recognized by IgA regardless of nutritional status, while Lactobacillus was broadly IgA-negative. Stunted children also had a greater number of IgA-positive fecal bacteria overall, a phenotype previously seen in inflammatory bowel disease patients. Together, IgA-binding patterns in mice and humans suggest that undernutrition alters intestinal homeostasis. To understand how pathobiotic communities respond to nutrient limitation, I further examined metabolic interactions between human Bacteroidales and E. coli, strains which exacerbate inflammation and growth stunting in undernourished mice. These bacteria experienced a mutual growth advantage which was enhanced by protein-limited, carbohydrate-rich conditions, and which led to outgrowth of B. fragilis and E. coli at the expense of other strains. Thus, cross-feeding between pathobionts might contribute to community dysbiosis in the undernourished gut. Taken together, I show that undernutrition drives not only the composition of the intestinal microbiota but also its metabolic and immune functionality. A better understanding of intestinal microbial function in undernutrition may lead to improved intervention strategies.

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Fecal-oral contamination promotes the persistence of early-life malnutrition. Systemic consequences of malnutrition include stunting, poor immune function, metabolic shifts, and neurocognitive impairment, but the underlying pathology and precise role of fecal microbes remain largely unknown. To address these knowledge gaps, I have utilized an established murine model (MAL-BG) that combines malnutrition and iterative exposure to fecal commensals. MAL-BG mice exhibit altered behavioural and cognitive deficits—poor spatial memory and learning plasticity—putatively linked to aberrant microglia phagocytosis. Microglial alterations occurred independently from neuroinflammation and blood-brain barrier (BBB) disruption, but were linked to systemic lipoxidative stress. Fecal-oral contamination exacerbated systemic, malnutrition-induced oxidative stress within the gut, brain, and liver. Beyond oxidative damage, malnourished livers exhibit fatty liver features. Largely studied in the context of obesity, undernutrition can also trigger NAFLD (non-alcoholic fatty liver disease). A combination of histology, liver metabolomics, and microbiome analyses were performed to assess the impact of diet and gut microbes in the pathology and reversal of undernutrition-induced fatty liver. Intriguingly, fatty liver histology was only observed in the early-life, but not adult, MAL-BG model despite similar liver metabolomic profiles. These findings indicate a crucial window in early-life development that, when disrupted by nutritional deficits, likely shapes liver health trajectories. Importantly, dietary intervention largely mitigated aberrant metabolomic and microbiome features in MBG mice. Collectively, my doctoral work explores (1) gut-brain and (2) gut-liver interactions in the context of undernutrition and intervention. I anticipate my findings will not only provide valued insight into gut microbiota-systemic interactions, but also identify putative therapeutic targets to halt or reverse consequences of childhood malnutrition.

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Mitochondria are essential for human health. While mitochondria are primarily known for their essential role in generating cellular energy, these organelles play many vital roles in eukaryotic cells, including the cellular stress response, innate immunity, and the regulation of intrinsic apoptosis. Mitochondrial proteases are essential for mitochondrial function, including the import of 99% of the mitochondrial proteome, which often requires the proteolytic removal of a mitochondrial targeting sequence (MTS), creating a new protein amino terminus. However, little is known about how proteases regulate mitochondrial functions during health and disease because of the lack of tools able to examine global mitochondrial proteolysis and how it changes during mitochondrial processes. A novel terminomics workflow called ‘MS-TAILS’ was developed to address this gap by identifying changes in the mitochondrial terminal proteome, reflective of mitochondrial proteome-wide proteolysis. MS-TAILS identified the highest coverage of the human mitochondrial proteome and the most sites of import-associated MTS removal of any terminomics study to date, as well as 97 novel sites of mitochondrial proteolysis, demonstrating its utility to study mitochondrial proteolysis, proteases, and proteome import. MS-TAILS was applied to characterize mitochondrial changes during the induction of intrinsic apoptosis: a critical but poorly characterized mitochondrial process. MS-TAILS identified apoptosis-dependent changes in seven mitochondrial proteins not previously implicated in apoptosis, which may indicate conserved early apoptotic events. We examined the role of mitochondrial proteases in innate immunity by conducting the first terminomics study of microbial infection, identifying infection-specific mitochondrial changes during enteropathogenic E.coli (EPEC) infection: a pathogen that uses a type III secretion system (T3SS) to inject effectors into human cells and mitochondria to modulate apoptosis and immunity. The majority of infection- and T3SS effector-dependent mitochondrial changes were unique from canonical apoptosis events, suggesting that EPEC T3SS effectors mediate an infection-associated mitochondrial apoptosis pathway. These findings were examined in a broader context to demonstrate the impact of this thesis work on the field. Overall, this work provides a novel approach to study global dynamics in mitochondrial proteolysis between conditions and therefore addresses a technical gap to characterize mitochondrial proteases, processes, and pathologies, including and beyond apoptosis.

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Early-life malnutrition results in childhood growth stunting, increased intestinal permeability, along with significant changes in the intestinal microbiota and metabolite composition. Here, we aim to characterize and further understand how malnutrition results in these intestinal consequences, and use this knowledge to develop a model to study environmental enteropathy (EE), a chronic inflammatory disease of the small intestine. Three-week-old C57BL/6 mice administered a protein and fat malnourished diet where compared with mice fed an isocaloric standard diet and found to be moderately growth stunted, with barrier dysfunction as observed decreased abundance of tight-junction proteins and increase in intestinal permeability to dextran. Mice given the malnourished diet were also found to have an altered small intestinal microbiota and metabolome, notably including profound changes in the Bacteroidetes and Proteobacteria species able to colonize the upper small intestine, correlated with large differences in the bile acid composition. The colon of malnourished mice had an altered mucosal microbiota composition, a thinner mucus layer and a greater number of microbes able to cross the mucus barrier was visualized by microscopy. We used these data to inform our development of a model for EE in mice. Features of EE include growth stunting, intestinal permeability, villous blunting and chronic intestinal inflammation. After screening a number of microbial cocktails, we demonstrate that early life consumption of a malnourished diet, in combination with exposure to a cocktail of Bacteroidales and E. coli species, remodels the small intestine to resemble the major features of EE observed in humans. Furthermore, this Bacteroidales and E. coli exposure induces an influx of pro-inflammatory intraepithelial lymphocytes in the small intestine, along with increased prevalence of bacterial species adhering to the epithelium, each of which could be initiating the onset of EE features. Further, we infected the malnourished mice with the enteric pathogens H. polygyrus, and S. Typhimurium, and observed striking differences in number of microbes able to colonize the small intestine. These findings provide new evidence of the intestinal impacts of malnutrition, and describe a novel murine model that can be used to elucidate the pathophysiology of this understudied disease.

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Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) cause enteric diseases resulting in significant morbidity and mortality worldwide. EPEC is a leading cause of potentially fatal watery diarrhea associated with vomiting, fever and dehydration in young children in developing countries, while EHEC causes severe gastroenteritis in both developing and industrialized nations. The success of these pathogens relies on their ability to inject secreted effectors directly into host cells that manipulate a variety of host cell signaling pathways while the pathogen remains extracellular. Much work has been done to identify EPEC/EHEC secreted effectors but the molecular mechanisms of action of many effectors and their contribution to virulence remain poorly understood. To identify novel host targets for EPEC/EHEC-secreted effectors we performed a global mass spectrometry screen using the SILAC (stable isotope labeling with amino acids in cell culture)-labeling method. Using this technology, we were able to identify host binding partners for a number of EPEC/EHEC effectors. Among them, two conserved effectors, NleB and EspL, were found to interact with the same host factor, ensconsin. Ensconsin is a microtubule associated protein that has been identified as an essential cofactor of kinesin-1. This thesis describes the identification and characterization of the interaction between NleB/EspL and ensconsin and identifies a role for these effectors during infection. We use fluorescent time-lapse imaging to demonstrate the significant effect EPEC have on the kinetics of host receptor trafficking during on-going infections and use the Citrobacter rodentium-mouse model of infection to further characterize how these effectors impact virulence during infection. We hypothesize that NleB and EspL play critical roles during A/E pathogen infections and contribute to pathogenesis through alteration of ensconsin function. Information gathered from this study provides insight into our current understanding of the virulence mechanisms of these pathogens as well as the role of secreted effectors during host cell interactions. A more detailed understanding of how pathogenic bacteria alter host cellular functions as part of the disease process could ultimately lead to development of new therapeutics to help control these significant enteric pathogens.

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Salmonella is one of the most abundant bacterial pathogens infecting humans in developed and developing countries. It is the causative agent of disease and mortality resulting in billions of dollars in associated medical costs and lost productivity every year. In the laboratory, findings regarding the physiology of Salmonella infections are often used to model a wide range of bacterial infections impacting fields far beyond the scope of Salmonella pathogenesis alone. For these reasons, significant resources have been dedicated to gaining a better understanding of mechanisms underlying Salmonella infection and the interaction between the pathogen and the immune system. In this thesis, Salmonella is used to study the interaction between bacterial pathogens and the host’s oxidative and nitrosative burst. Recently, new findings have challenged the conventional perspective of reactive oxygen and nitrogen species as merely antimicrobial agents by revealing redox-sensitive virulence mechanisms that benefit Salmonella infection. These new findings, together with processes that drive Salmonella infection, are highlighted in Chapter 1. To better address questions concerning redox stress inside bacteria we used redox-sensitive GFP which enabled real-time analysis of the intra-bacterial redox environment. In Chapter 2 this redox-biosensor combined with high-throughput microscopy, was used to evaluate oxidative/nitrosative stress evasion strategies inside macrophages. In Chapter 3, the same method was used to explore the bacterial outer membrane permeability to hydrogen peroxide. Real-time measurements of the intra-bacterial redox potential revealed novel regulatory mechanisms that alter outer membrane permeability based on the presence or absence of reactive oxygen and nitrogen species. Chapter 4 describes the identification and characterization of a redox-sensitive regulatory modification in Salmonella effector SteB. This modification was found to be crucial for regulation of tubulin-mediated transport of the Salmonella containing vacuole. Cumulatively these studies describe strategies for oxidative/nitrosative stress evasion while also highlighting several mechanisms by which reactive oxygen and nitrogen species aid Salmonella during infection. In Chapter 5, these findings have been integrated in order to gain a more comprehensive understanding of the complicated relationship between Salmonella and oxidative/nitrosative stress which has the potential to lead to the development of novel antimicrobial therapies.

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Rates of allergic airways disease are steadily rising in developed countries, arguing for an environmental etiology. Epidemiological studies have pointed to a role for the infant gut microbiota in immune system development that could alter allergic disease susceptibility. To investigate whether changes in gut microbiota impact disease severity in murine models of asthma and hypersensitivity pneumonitis (HP), we administered clinical doses of antibiotics to mice during different periods in their development. Classically, allergic asthma is induced by T helper type 2 (Th2) inflammatory responses. In contrast, HP develops via Th1/Th17-mediated mechanisms. Consistent with their polarized immune phenotypes, these two diseases were exacerbated after different antibiotic exposures. Mice receiving perinatal vancomycin developed more severe asthma relative to control animals, as demonstrated by increased Th2-driven airway inflammation, antigen-specific IgE and lung pathology. The data presented here suggest that increased asthma severity in this model of allergic airways disease is mediated by mechanisms involving elevated IgE levels and reduced regulatory T cell populations. This effect was not observed in mice given streptomycin, nor when either antibiotic was administered to adult mice. Conversely, the severity of HP was unaffected by vancomycin, but increased after streptomycin treatment; this was demonstrated by exacerbated airway inflammation of the Th1/Th17-type, as well as increased IFNγ and IL-17A cytokine production and lung pathology. Microbial community analysis reveals that antibiotic treatment has profound effects on the gut microbiota; these effects were highly specific to the type of antibiotic used and the length of administration. Bacteroidetes dominated the intestinal flora after streptomycin treatment, while vancomycin drastically reduced diversity and promoted the overgrowth of a distinct group of Firmicutes.The extensive use of antibiotics in our society warrants a closer look at the effects of different antibiotics on the composition of the microbiota and how this may impact the prevalence of diseases like asthma and HP. The work in this thesis presents an interesting dichotomy, where contrasting shifts in gut flora appear to have opposite consequences depending on the immunological nature of the disease.

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Inflammatory bowel disease (IBD) is a debilitating disease characterized by chronic inflammation caused by multiple factors involving the immune system, intestinal microbiota and epithelial barrier. Microbial dysbiosis is implicated in disease, as there are significant differences in the microbiota composition between affected and healthy individuals. It is not clear if deterioration of microbial composition results in disease or is a consequence of disease. Mucus production by goblet cells serves as one of the crucial mucosal defenses at the interface between the eukaryotic and prokaryotic cells and yet the immunoregulatory pathways involved remain uncharacterized. The inner mucus layer of the intestine functions as a barrier, which serves to minimize microbial translocation, prevents excessive immune activation, and decrease infection. Here we have described methodology to alter the thickness of the inner mucus layer through treatment with antibiotic or a phytonutrient. We showed that the antibiotic metronidazole caused significant thinning of the inner mucus layer accompanied by a dramatic change in the microbial community structure. In contrast, treatment with the phytochemical eugenol resulted in significant thickening of the inner mucus layer that was accompanied by a change in the microbial community. These changes in community structure were complementary; DNA sequencing showed that groups depleted by metronidazole treatment were more abundant with eugenol treatment. To investigate how changes in the integrity of the inner mucus layer affect intestinal defense, Citrobacter rodentium (Cr) was used to examine susceptibility to enteric-induced colitis. Metronidazole-induced reduction in mucus thickness correlated with exacerbated severity of Cr-induced colitis. Thickening of the inner mucus layer with eugenol treatment resulted in protection from Cr-induced colitis. Further, we identified a novel innate immune pathway involved in regulation of goblet cell function and mucus layer production. The NLRP6 inflammasome was shown to regulate mucus secretion and deficiency in any component of the NLRP6 inflammasome resulted in impaired goblet cell function preventing mucin granule exocytosis and mucus layer formation. Abrogated mucus secretion led to increased invasiveness and pathology of Cr infection. Mechanistically, NLRP6 deficiency led to stalled autophagy in goblet cells, providing a link between inflammasome activity, autophagy, mucus exocytosis, and antimicrobial barrier function.

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Salmonella are Gram-negative facultative intracellular pathogens that cross the intestinal barrier, and are taken up by phagocytes where, they can replicate and spread to systemic sites. Salmonella encode two type III secretion systems, Salmonella pathogenicity island 1 and 2 (SPI1 and SPI2), which mediate the translocation of bacterial effectors into the host cell. SPI1 facilitates bacterial uptake into non-phagocytic cells and is involved in forming a special replicative niche, called the Salmonella containing vacuole (SCV). SPI2 is required for maintenance of the SCV, macrophage replication and systemic disease. A comprehensive study of the contribution of individual SPI2 effectors to virulence had not been previously done, and was therefore performed. Strains deficient in specific SPI2 genes were tested for alterations in virulence in a mouse model of typhoid fever, and in epithelial and macrophage cell infections. These experiments showed that many SPI2 effectors are required for replication in macrophages, and that ΔspvB, ΔssaR, and ΔspiC strains were attenuated in mice. Salmonella infection causes many perturbations to the host, including changes in metabolites, specifically arachidonic acid metabolism, which leads to the production of eicosanoids. The effects of Salmonella infection of macrophages on eicosanoids were examined. Salmonella infection increased the expression of prostaglandin synthases, but decreased thromboxane and leukotriene synthases. The SPI2 deletion strains were tested to determine involvement of SPI2 in arachidonic acid metabolism. The SPI2 effectors SseF and SseG, which are largely uncharacterized in macrophage infections, were mainly responsible for the induction of prostaglandins. The effects of prostaglandins on Salmonella infection were studied. It was found that 15-deoxy-Δ12,14-prostaglandin-J2 (15d-PGJ2) significantly reduced Salmonella colonization of macrophages, but not epithelial cells. Furthermore, this occurs independently of SPI1, SPI2, and PPAR-γ. 15d-PGJ2 reduces cytokines and reactive nitrogen species produced by infected macrophages. A role for 15d-PGJ2 in Salmonella infection has not been previously demonstrated. This thesis examines the role of SPI2 in Salmonella virulence and arachidonic acid metabolism.

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Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively) are attaching and effacing (A/E) bacterial pathogens that cause diarrheal disease. EPEC causes severe infantile diarrhea in developing countries whereas EHEC infection results in severe bloody diarrhea worldwide. These pathogens employ a type-III secretion system (T3SS) encoded on the locus of enterocyte effacement (LEE) pathogenicity island (PAI) to inject a panel of effector proteins directly into infected host cells where they subvert host cell functions. The roles of many type-III secreted (T3S) effectors remain to be elucidated. Here, we have elucidated functions for the T3S effectors EspZ and NleC.EPEC infection causes host cell cytotoxicity and death. We demonstrated that EspZ enhances host cell survival during EPEC infection. Removal of espZ from the EPEC genome (∆espZ) exacerbated host cell cytotoxicity. We found that EspZ interacts with host CD98 and contributes to protection against EPEC-mediated cytotoxicity by enhancing phosphorylation of focal adhesion kinase (FAK). Further investigation revealed that EPEC ∆espZ infection caused a severe decrease in host mitochondrial inner membrane potential (∆ψm) concurrently with host cell lysis. We also found that EspZ localizes to host cell mitochondria and interacts with the translocase of inner mitochondrial membrane (TIM) 17B. These studies are the first to demonstrate EspZ function.Many non-LEE encoded (Nle) effector proteins impact innate immune signaling and we thus examined the contribution of the T3S zinc-metalloprotease NleC to this phenotype. We identified the host acetyltransferase p300 as a target of NleC and show that NleC causes decreased abundance of p300 in cellular nuclei. We further demonstrate that over-expression of p300 antagonizes repression of interleukin (IL)-8 secretion by EPEC and that small interfering ribonucleic acid (siRNA) knock-down of p300 dampens IL-8 secretion by EPEC ∆nleC-infected cells. Thesis work has identified a target of NleC and provided the first example of a bacterial virulence factor targeting the acetyltransferase p300.The work presented in this thesis provides novel insight into the function of two T3S effector proteins, EspZ and NleC. The mechanistic insight gained by these studies has thoroughly enhanced the understanding of these important virulence factors and their contribution to A/E pathogen infection.

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The gallbladder has long been recognized as a site of infection during systemic salmonellosis; yet little is known regarding bacterial pathogenesis in this organ. My PhD research constitutes the first detailed characterization of a bacterial infection of the gallbladder, focusing on the local biology, pathology, and immunology of Salmonella infection. Using a murine model of acute typhoid fever, it was found that Salmonella in the gallbladder show a unique behavior, as they remained confined to gallbladder epithelial cells without translocating to the mucosa. Moreover, they replicated within these cells instead of phagocytes. These findings add yet another functional significance to the invasion phenotype in vivo, and together with the presence of high numbers of extracellular, luminal bacteria, put forward the concept that acute infection of the gallbladder may be important for later events in the bacterial life cycle. A murine model of persistent typhoid infection revealed that gallbladder colonization occurs intermittently during chronic infection and that colonization may result in pathological damage. The in vivo work described here validates some of the paradigms of Salmonella infection, but also shows that Salmonella accumulation in vivo does no exclusively occur in the canonical intra-macrophage niche. This research also established a new system for the study of Salmonella Typhimurium’s biology, and a way to probe the biological function of individual gene products in a meaningful in vivo infection model. The model was validated in a screen of Salmonella mutants of known virulence factors involved in intracellular survival and replication within host cells. Novel phenotypes were described within this more natural host:pathogen environment, which highlighted potentially new biological functions for several Salmonella genes. This is the first study of its kind, which reveals the usefulness of the in vivo gallbladder epithelial cell infection model. It is hoped that future studies using this system shall continue to impact the field of Salmonella pathogenesis.

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Intestinal microbiota comprise microbial communities that reside in the gastrointestinal tract and are critical to normal host physiology. Understanding the microbiota’s role in host response to invading pathogens will further expand our knowledge of host-microbe interactions, as well as foster advances in the design of novel therapeutic and prophylactic methods. In this dissertation I used clinically relevant doses of antibiotics to disturb the intestinal microbiota balance in a murine infection model. Pre-infection perturbations in the microbiota with two antibiotics resulted in increased mouse susceptibility to Salmonella enterica serovar Typhimurium intestinal colonization, greater post-infection alterations in the microbiota, and more severe intestinal pathology. This demonstrates the importance of a balanced microbiota community in host response to an enteric pathogen. This infection model also allowed further characterization of the host-pathogen-microbiota interactions during enteric salmonellosis. It was shown that in the presence of high numbers of indigenous microbes S. Typhimurium deficient in Salmonella pathogenicity island 2 (SPI2) is unable to trigger intestinal inflammation, while a SPI1 mutant strain promotes late typhlitis. Additionally, it was demonstrated that pathogen-induced intestinal inflammation does not always translate into extensive alterations to the host microbiota, as inflammation during a SPI1 mutant infection did not promote the same changes in host microbiota composition and numbers as inflammation induced by wild-type S. Typhimurium. Differential neutrophil recruitment by the three S. Typhimurium strains was implicated as one possible agent of microbiota perturbations. A thorough understanding of the tripartite host-microbiota-pathogen relationship in the progression of the enteric infections is needed to fully appreciate the disease process, as well as to suggest new avenues through which to interfere with the infection progression. These studies enhance our understanding of the microbiota’s role in the progression of S. Typhimurium infection and the effects of inflammation upon the microbiota, thus broadening our knowledge of S. Typhimurium pathogenesis and associated host response.

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Salmonellosis poses a global threat to human health. Host resistance against Salmonella enterica serovar Typhimurium (S. Typhimurium) in the murine model is mediated by Natural resistance-associated macrophage protein 1 (Nramp1/Slc11a1). Nramp1 is critical for host defense, as mice lacking Nramp1 fail to control bacterial replication and succumb to low doses of S. Typhimurium. Despite this critical role, the mechanisms underlying Nramp1’s protective effects are unclear. This thesis presents a detailed analysis of Nramp1 expression in the murine gastrointestinal tract and its impact on S. Typhimurium infection following oral infection. Dendritic cells (DCs) that sample the intestinal lumen are among the first cells encountered by S. Typhimurium and play an important role in Salmonella pathogenesis. Intestinal, splenic and bone marrow derived DCs (BMDCs) all expressed Nramp1 protein. In intestinal DCs, Nramp1 expression is restricted to a discrete subset of DCs (CD11c⁺ CD103-) that express elevated levels of pro-inflammatory cytokines in response to bacterial products. While Nramp1 expression did not affect S. Typhimurium replication in DCs, infected Nramp1⁺/⁺ DCs secreted more inflammatory cytokines (IL-6, IL-12 and TNF-α) than Nramp1-/- DCs. This suggests that Nramp1 expression promotes accelerated inflammatory responses to S. Typhimurium. This hypothesis was tested using the Salmonella-induced colitis model, where pre-treatment of mice with antibiotics enhances colonization of the cecum/colon and induces massive inflammation. We found that Nramp1⁺/⁺ mice mounted a faster and more robust inflammatory response characterized by elevated pro-inflammatory cyto/chemokines (IFN-γ, TNF-α and MIP1-α) and recruitment of neutrophils and macrophages, thereby limiting spread of S. Typhimurium to systemic sites and ultimately protecting the host.Nramp1⁺/⁺ mice also developed a chronic Salmonella infection of the gastrointestinal tract that led to severe tissue fibrosis. Intestinal fibrosis is a serious complication of Crohn’s disease, often requiring surgical intervention but the mechanisms underlying its development are poorly understood due to the lack of relevant animal models. A novel model of severe and persistent intestinal fibrosis caused by chronic bacterial induced colitis was developed. Since the pathology closely resembles human fibrosis, we present a valuable tool for investigating host and bacterial contributions to inflammatory bowel diseases, as well as infectious colitis.

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The SH2 domain-containing inositol 5’-phosphatase, SHIP, negatively regulates hematopoietic cell functions and is critical for maintaining immune homeostasis. However, whether SHIP plays a role in controlling bacterial infections in vivo remained unknown. Salmonella enterica causes human salmonellosis, a disease that ranges in severity from mild gastroenteritis to severe systemic illness, resulting in significant morbidity and mortality worldwide. The focus of this work was to determine the role of SHIP in a murine model of systemic Salmonellosis. Susceptibility of ship⁺/⁺and ship⁻/⁻ mice to S. enterica serovar Typhimurium infection was compared. ship⁻/⁻ mice displayed an increased susceptibility to both oral and intraperitoneal S. Typhimurium infection and had significantly higher bacterial loads in intestinal and systemic sites than ship⁺/⁺mice, indicating a role for SHIP in the gut and systemic pathogenesis of S. Typhimurium in vivo. Blood cytokine levels showed that infected ship⁻/⁻ mice produce lower levels of Th1 polarizing cytokines compared to ship⁺/⁺ animals, and analysis of supernatants taken from M2 bone marrow derived macrophages correlated with this data. M2 macrophages were the predominant population in vivo during both oral and intraperitoneal infections. Because M2 macrophages are poor defenders against bacterial infection, these data suggest that M2 macrophage skewing in ship⁻/⁻ mice contributes to ineffective clearance of Salmonella.The role of SHIP in the gut during enteric infections was also explored. ship⁻/⁻ mice were not susceptible to Citrobacter rodentium infection, yet developed severe inflammation of the ileum upon infection with this bacterium, with Salmonella, or when challenged orally with LPS. Increased collagen deposition was also observed at early time points post-infection, suggesting that ship⁻/⁻ mice may be used to study the development of inflammatory bowel diseases characterized by fibrosis, such as Crohn's. Because SHIP is such a critical negative regulator in both innate and adaptive immune cells, it has the potential to significantly alter the outcome of infections. This work highlights the fact that SHIP is important in vivo during Salmonellosis and opens new avenues to explore targeting SHIP in therapies for both systemic infections as well as inflammatory bowel diseases.

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