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Bacterial Colonization Dynamics and Ecology of the Developing Zebrafish IntestineStephens, William 03 October 2013 (has links)
Human intestinal microbiomes exhibit a large degree of interindividual compositional variation. Animal models, such as the zebrafish, facilitate the design of controlled and highly replicated studies that allow us to understand the normal variation in vertebrate intestinal composition and to study the rules guiding normal assembly of these complex communities. The smaller intestinal size and high fecundity of the zebrafish allow us to fully sample the intestinal contents of many animals, while the optical transparency allows direct in vivo observation of fluorescently labeled bacterial species within the intestine. The studies in this dissertation utilize these advantages to investigate the composition, colonization dynamics and functional requirements for colonization in the vertebrate intestine. We first describe the taxonomic composition and diversity of the zebrafish intestinal microbiota from wild-caught and domesticated zebrafish. In the process, we identify a set of core bacterial genera that are consistently present in zebrafish intestines. We then use species from two of these genera in subsequent studies to gain a detailed understanding of the colonization dynamics and genetic requirements of the two species. We initially describe the application of light sheet microscopy to imaging the zebrafish intestine and associated colonizing bacteria. We find that a single species, Aeromonas veronii, does not occupy the entire intestinal space and that competition within the same species appears to prevent further colonization. These results are extended to a zebrafish isolated Vibrio species as well as A. veronii by tagging bacteria with transposon insertions and tracking the changes in colonizing population sizes. These insertion libraries also identify genes in each bacterial species that are important in the process of colonization, highlighting the key role for motility and chemotaxis in this process. The descriptions and methods discussed in this dissertation advance the use of this important model organism towards the understanding of vertebrate host-microbial interactions.
This dissertation includes previously published co-authored material as well as unpublished co-authored material. / 10000-01-01
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The Role of the Microbiota in Prey Capture BehaviorSimonson, Levi 21 November 2016 (has links)
There is a growing body of evidence that normal nervous system activity requires signals from resident microbes. We have yet to discover the mechanisms by which the microbiota influence brain function. However, we know that the enteric nervous system (ENS) serves as an important interface between the developing host and its microbiota. In this dissertation I will introduce a novel computer-assisted method for ENS characterization and a novel, incredibly specific mechanism of host-microbe interactions. With new ENS characterization method I developed, it will be possible to better understand the role of the ENS during development, by more rapidly and algorithmically assessing ENS phenotypes. Furthermore, my discovery of a single microbially-sourced protein that influences vertebrate host prey capture behavior and visual system development, will provide a new appreciation for the role resident microbes, both in model organisms and in ourselves. By both establishing a new, less biased, approach to image analysis and describing a surprising new regulatory host-microbe interaction, the work I describe in this dissertation should provide the foundation for an explosion of exciting discoveries in the near future.
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Dissection of the Type IV Pilus Retraction Motor in Neisseria GonorrhoeaeHockenberry, Alyson Marie, Hockenberry, Alyson Marie January 2016 (has links)
Bacteria of the Neisseria are predominately commensal, though N. gonorrhoeae and N. meningitidis are capable of causing disease. Both of these species often asymptomatically colonize humans, a trait reminiscent of their commensal cousins. The factors that shift the balance between asymptomatic carriage and disease are unknown. Pathogenic Neisseria use retractile surface structures called Type IV pili to coordinate community behavior and to initiate and sustain infection. Previously, the contributions of pilus retraction have been studied by deleting the pilus retraction motor, PilT. Recent findings suggest the speed and force exerted by pilus retraction is responsive to environmental cues. By examining several PilT mutants that maintain the ability to retract pili, I show retraction, per se, is not required for N. gonorrhoeae social interactions with bacteria or with human cells. Furthermore, Type IV pilus retraction by the commensal N. elongata affects the host cell differently than retraction by N. gonorrhoeae. These observations collectively suggest pilus retraction properties shape the host cell response to Neisseria colonization and could tip the balance of asymptomatic colonization to symptomatic disease.
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Developing Methods Based on Light Sheet Fluorescence Microscopy for Biophysical Investigations of Larval ZebrafishTaormina, Michael 29 September 2014 (has links)
Adapting the tools of optical microscopy to the large-scale dynamic systems encountered in the development of multicellular organisms provides a path toward understanding the physical processes necessary for complex life to form and function. Obtaining quantitatively meaningful results from such systems has been challenging due to difficulty spanning the spatial and temporal scales representative of the whole, while also observing the many individual members from which complex and collective behavior emerges.
A three-dimensional imaging technique known as light sheet fluorescence microscopy provides a number of significant benefits for surmounting these challenges and studying developmental systems. A thin plane of fluorescence excitation light is produced such that it coincides with the focal plane of an imaging system, providing rapid acquisition of optically sectioned images that can be used to construct a three-dimensional rendition of a sample. I discuss the implementation of this technique for use in larva of the model vertebrate Danio rerio (zebrafish).
The nature of light sheet imaging makes it especially well suited to the study of large systems while maintaining good spatial resolution and minimizing damage to the specimen from excessive exposure to excitation light. I show the results from a comparative study that demonstrates the ability to image certain developmental processes non-destructively, while in contrast confocal microscopy results in abnormal growth due to phototoxicity. I develop the application of light sheet microscopy to the study of a previously inaccessible system: the bacterial colonization of a host organism. Using the technique, we are able to obtain a survey of the intestinal tract of a larval zebrafish and observe the location of microbes as they grow and establish a stable population in an initially germ free fish. Finally, I describe a new technique to measure the fluid viscosity of this intestinal environment in vivo using magnetically driven particles. By imaging such particles as they are oscillated in a frequency chirped field, it is possible to calculate properties such as the viscosity of the material in which they are embedded. Here I provide the first known measurement of intestinal mucus rheology in vivo.
This dissertation includes previously published co-authored material.
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Imaging Vibrio Cholerae Invasion and Developing New Tools for 3D Microscopy of Live AnimalsLogan, Savannah 30 April 2019 (has links)
All animals harbor microorganisms that interact with each other and with their hosts. These microorganisms play important roles in health, disease, and defense against pathogens. The microbial communities in the intestine are particularly important in preventing colonization by pathogens; however, this defense mechanism and the means by which pathogens overcome it remain largely unknown. Moreover, while the composition of animal-associated microbial communities has been studied in great depth, the spatial and temporal dynamics of these communities has only recently begun to be explored.
Here, we use a transparent model organism, larval zebrafish, to study how a human pathogen, Vibrio cholerae, invades intestinal communities. We pay particular attention to a bacterial competition mechanism, the type VI secrection system (T6SS), in this process. In vivo 3D fluorescence imaging and differential contrast imaging of transparent host tissue allow us to establish that V. cholerae can use the T6SS to modulate the intestinal mechanics of its host to displace established bacterial communities, and we demonstrate that one part of the T6SS apparatus, the actin crosslinking domain, is responsible for this function.
Next, we develop an automated high-throughput light sheet fluorescence microscope to allow rapid imaging of bacterial communities and host cells in live larval zebrafish. Light sheet fluorescence microscopy (LSFM) has been limited in the past by low throughput and tedious sample preparation, and our new microscope features an integrated fluidic circuit and automated positioning and imaging to address these issues and allow faster collection of larger datasets, which will considerably expand the use of LSFM in the life sciences. This microscope could also be used for future experiments related to bacterial communities and the immune system.
The overarching theme of the work in this dissertation is the use and development of advanced imaging techniques to make new biological discoveries, and the conclusions of this work point the way toward understanding pathogenic invasion, maximizing the use of LSFM in the life sciences, and gaining a better grasp of host-associated bacterial community dynamics.
This dissertation includes previously published and unpublished co-authored material.
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Entomopathogenicity to Two Hemipteran Insects Is Common but Variable across Epiphytic Pseudomonas syringae StrainsSmee, Melanie R., Baltrus, David A., Hendry, Tory A. 19 December 2017 (has links)
Strains of the well-studied plant pathogen Pseudomonas syringae show large differences in their ability to colonize plants epiphytically and to inflict damage to hosts. Additionally, P. syringae can infect some sap-sucking insects and at least one P. syringae strain is highly virulent to insects, causing death to most individuals within as few as 4 days and growing to high population densities within insect hosts. The likelihood of agricultural pest insects coming into contact with transient populations of P. syringae while feeding on plants is high, yet the ecological implications of these interactions are currently not well understood as virulence has not been tested across a wide range of strains. To investigate virulence differences across strains we exposed the sweet potato whitefly, Bemisia tabaci, and the pea aphid, Acyrthosiphon pisum, both of which are cosmopolitan agricultural pests, to 12 P. syringae strains. We used oral inoculations with bacteria suspended in artificial diet in order to assay virulence while controlling for other variables such as differences in epiphytic growth ability. Generally, patterns of pathogenicity remain consistent across the two species of hemipteran insects, with bacterial strains from phylogroup II, or genomospecies 1, causing the highest rate of mortality with up to 86% of individuals dead after 72 h post infection. The rate of mortality is highly variable across strains, some significantly different from negative control treatments and others showing no discernable difference. Interestingly, one of the most pathogenic strains to both aphids and whiteflies (Cit7) is thought to be nonpathogenic on plants. We also found Cit7 to establish the highest epiphytic population after 48 h on fava beans. Between the nine P. syringae strains tested for epiphytic ability there is also much variation, but epiphytic ability was positively correlated with pathogenicity to insects, suggesting that the two traits may be linked and that strains likely to be found on plants may often be entomopathogenic. Our study highlights that there may be a use for epiphytic bacteria in the biological control of insect crop pests. It also suggests that interactions with epiphytic bacteria could be evolutionary and ecological drivers for hemipteran insects.
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Molecular Interactions of Salmonella with the Host Epithelium in Presence of CommensalsDesai, Prerak T. 01 December 2011 (has links)
Food-borne infections are a major source of mortality and morbidity. Salmonella causes the highest number of Food-borne bacterial infections in the US. This work contributes towards developing strategies to control Salmonella by (a) defining receptors used by Salmonella to adhere to and invade the host epithelium; (b) developing a host receptor based rapid detection method for the pathogen in food matrix; (C) and defining mechanisms of how probiotics can help alleviate Salmonella-induced cell death in the host epithelium.
We developed a cell-cell crosslinking method to discover host-microbe receptors, and discovered three new receptor-ligand interactions. Interaction of Salmonella Ef-Tu with Hsp90 from epithelial cells mediated adhesion, while interaction of Salmonella Ef-Tu with two host proteins that negatively regulate membrane ruffling (myosin phosphatase and alpha catenin) mediated adhesion and invasion. We also showed the role of host ganglioside GM1 in mediating invasion of epithelial cells by Salmonella.
Further we exploited pathogen affinity for immobilized gangliosides to concentrate them out of solution and from complex food matrices for detection by qPCR. A sensitivity of 4 CFU/ml (3 hours) in samples without competing microflora was achieved. Samples with competing microflora had a sensitivity of 40,000 CFU/ml.
Next we screened several probiotic strains for pathogen exclusion potential and found that Bifidobacterium longum subspp. infantis showed the highest potential for Salmonella enterica subspp. enterica ser. Typhimurium exclusion in a caco-2 cell culture model. B. infantis shared its binding specificity to ganglioside GM1 with S. ser. Typhimurium. Further, B. infantis completely inhibited Salmonella-induced caspase 8 and caspase 9 activity in intestinal epithelial cells. B. infantis also reduced the basal caspase 9 and caspase 3/7 activity in epithelial cells in absence of the pathogen. Western blots and gene expression profiling of epithelial cells revealed that the decreased caspase activation was concomitant with increased phosphorylation of pro-survival protein kinase Akt, increased expression of caspase inhibiting protein cIAP, and decreased expression of genes involved in mitochondrion organization, biogenesis and reactive oxygen species metabolic processes. Hence, B. infantis exerted its protective effects by repression of mitochondrial cell death pathway which was induced in the presence of S. ser. Typhimurium.
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Microbial Programming of the Neonatal Pig2013 July 1900 (has links)
Microbial succession, composition and ecological distribution within the gastro-intestinal tract are critical areas of study since commensal bacteria have been shown to affect animal health and development. A series of experiments were conducted to determine whether altered microbial succession in neonatal animals would modulate the development and health of pigs later in life. An initial experiment in conventional pigs was conducted to establish the early postnatal microbial succession profile and to identify early colonizing bacterial species. Culture-independent analysis of digesta and mucosal microbiota showed distinct variation between the proximal and distal gastro-intestinal tract (GIT) indicating that fecal or distal gut profiles cannot be used to predict succession in the upper GIT. Temporally, Clostridium spp. were found to be most prevalent in the GIT microbiota of the neonatal pig up to 0.5 d of age, accompanied by a high abundance of Escherichia and Shigella spp. These genera were transiently displaced by Streptococcus spp. followed by a preponderance of Lactobacillus spp. between 3 and 20 d of age. Subsequently, a “snatch-farrow” model was employed to modulate early postnatal microbial succession and investigate the effects on postweaning microbial composition. Pigs were collected into sterile towels directly from the vaginal canal and transferred to a sterile isolator environment for the first 4 days. Pigs were either inoculated with sow feces or not at 1 d of age resulting in significant differences in fecal microbial profile at 4 days of age, prior to removal from isolators. Analysis using terminal restriction fragment length polymorphisms (TRFLP) of intestinal microbiota at 28 d of age did not show significant clustering or variation in diversity indices for either group during the 4-d postnatal isolator phase. However, enumeration of selected taxa using quantitative PCR did indicate significant treatment differences in postweaning microbiota. Despite these results, this approach was rejected for further use as the protocol provided only moderate control of early postnatal colonization and variation and unpredictability of the timing of natural farrowing contributed to significant litter effects. Finally, a gnotobiotic monoassociation model was used investigate the effects of modulating early postnatal microbial succession on postweaning physiology, microbial composition and mucosal gene expression. Twenty-four cesarean-section derived piglets were monoassociated for the first 4 days of life with either L. mucosae (L), S. infantarius (S), C. perfringens (C) or E. coli (E). Pigs from treatments E and L animals showed the highest growth rate during the conventional rearing period (7-28 d of age). Monoassociation with different bacterial species during the first 4 d of life resulted in significant changes in postweaning microbial composition in small intestine and colon as assessed by quantitative PCR, although TRFLP did not identify unique clustering by treatment or variation in diversity. L. mucosae was the only inoculant species with significant variation, with a reduction in the colonic mucosa at 28 days of age. Monoassociation with L. mucosae was also associated with increased nutrition related gene expression in small intestine. Pigs monoassociated with E. coli had low expression of microbial sensing (TLR2 and 4), NFkappaB complex genes and mucins at 28 d of age. This study clearly showed that controlled early microbial succession in neonatal pigs altered post-weaning commensal microbiota composition, postweaning physiology and host gene expression in small and large intestine. The findings suggest the importance of peri-natal management and feeding strategies in promoting postweaning health and performance.
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Transcriptome Sequencing Reveals Novel Candidate Genes for Cardinium hertigii-Caused Cytoplasmic Incompatibility and Host-Cell InteractionMann, Evelyne, Stouthamer, Corinne M., Kelly, Suzanne E., Dzieciol, Monika, Hunter, Martha S., Schmitz-Esser, Stephan 21 November 2017 (has links)
Cytoplasmic incompatibility (CI) is an intriguing, widespread, symbiont-induced reproductive failure that decreases offspring production of arthropods through crossing incompatibility of infected males with uninfected females or with females infected with a distinct symbiont genotype. For years, the molecular mechanism of CI remained unknown. Recent genomic, proteomic, biochemical, and cell biological studies have contributed to understanding of CI in the alphaproteobacterium Wolbachia and implicate genes associated with the WO prophage. Besides a recently discovered additional lineage of alphaproteobacterial symbionts only moderately related to Wolbachia, Cardinium (Bacteroidetes) is the only other symbiont known to cause CI, and genomic evidence suggests that it has very little homology with Wolbachia and evolved this phenotype independently. Here, we present the first transcriptomic study of the CI Cardinium strain cEper1, in its natural host, Encarsia suzannae, to detect important CI candidates and genes involved in the insect-Cardinium symbiosis. Highly expressed transcripts included genes involved in manipulating ubiquitination, apoptosis, and host DNA. Female-biased genes encoding ribosomal proteins suggest an increase in general translational activity of Cardinium in female wasps. The results confirm previous genomic analyses that indicated that Wolbachia and Cardinium utilize different genes to induce CI, and transcriptome patterns further highlight expression of some common pathways that these bacteria use to interact with the host and potentially cause this enigmatic and fundamental manipulation of host reproduction. IMPORTANCE The majority of insects carry maternally inherited intracellular bacteria that are important in their hosts' biology, ecology, and evolution. Some of these bacterial symbionts cause a reproductive failure known as cytoplasmic incompatibility (CI). In CI, the mating of symbiont-infected males and uninfected females produces few or no daughters. The CI symbiont then spreads and can have a significant impact on the insect host population. Cardinium, a bacterial endosymbiont of the parasitoid wasp Encarsia in the Bacteroidetes, is the only bacterial lineage known to cause CI outside the Alphaproteobacteria, where Wolbachia and another recently discovered CI symbiont reside. Here, we sought insight into the gene expression of a CI-inducing Cardinium strain in its natural host, Encarsia suzannae. Our study provides the first insights into the Cardinium transcriptome and provides support for the hypothesis that Wolbachia and Cardinium target similar host pathways with distinct and largely unrelated sets of genes.
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The Role of Dysfunctional Na+/H+ Exchange in the Development of Dysbiosis and Subsequent ColitisHarrison, Christy Anne, Harrison, Christy Anne January 2017 (has links)
The last half-century has seen a dramatic and alarming rise in the incidence of autoimmune disease in industrialized nations too rapid to be accounted for by genetics alone. Among those, Inflammatory Bowel Disease (IBD) has risen from a western disease affecting industrialized populations to an emerging global threat affecting diverse populations around the world. IBD is a complex disease that combines genetic susceptibility and environmental exposure, but one aspect appears to be clear: the involvement of the gut microbiome. Current thought holds that IBD is an autoimmune attack on commensal microbiota, causing extensive collateral damage to the host intestinal tissues in the process. However, it has remained unclear in the field whether the changes observed in the IBD microbiome are causative in nature or whether the microbiome is responding to already-underway inflammatory processes within the host. This dissertation investigates one host factor in particular with regard to the microbiome and the development of inflammation: sodium-hydrogen exchange at the brush border, mediated by sodium hydrogen exchanger 3 (NHE3). NHE3 is inhibited during active IBD, but its loss in knockout animals is also enough to promote spontaneous colitis in a microbiome-dependent fashion. This dissertation investigates the specific contribution of the microbiome in NHE3 knockout animals to determine whether loss of NHE3 may be mediating the onset of colitis through pro-inflammatory changes in the microbiome. Our results suggest that the microbiome fostered in an NHE3-deficient environment may accelerate the onset and severity of experimental colitis, though likely in concert with additional host factors.
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