The Microbiome and Parkinson's Disease
No abstract available.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
No abstract available.