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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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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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Characterization of a Phylogenetically Convergent Nitrogen-Dependent Antimicrobial Mechanism Against Serratia marcescens Utilizing a D. melanogaster Infection ModelNathan J Poling (9768401) 17 December 2020 (has links)
<div>Host-pathogen interactions are the result of long term evolutionary processes due to the conflicting goals of the host and the infections pathogens in their quest for survival, creating an interplay of co-evolution as various adaptation are acquired by one and then in turned adapted to by the other. Selection of the host’s antimicrobial strategies and the resultant adaptations of infectious microorganisms leads to the development of complex and dynamic relationships ranging from symbiotic to commensal to pathogenic. In an effort to understand the selective process and identify unique mechanisms of antimicrobial defense, sera from 18 species (7 invertebrate, 11 vertebrate) were tested for antimicrobial potential against 20 Gram-negative and 11 Gram-positive bacteria. <i>Alligator mississippiensis</i> sera exhibited the strongest inhibitory potential. A transposon mutagenesis screen performed on the resistant bacterium <i>Serratia marcescens</i> identified several genes, including <i>glnL</i>, as necessary for defense. The <i>glnL</i> gene encodes for the sensory histidine kinase/phosphatase NtrB, controlling the expression of regulatory genes in response to nitrogen limitation. Attenuated growth of the Tn::<i>glnL</i> mutant in the presence of alligator serum and minimal media was rescued with nitrogen supplementation, suggesting the existence of a mechanism for nitrogen limitation as an antimicrobial strategy in alligator sera. Utilization of a <i>Drosophila melanogaster</i> oral model of infection showed that <i>glnL</i> is required for <i>S. marcescens</i> virulence, and nitrogen supplementation rescued the phenotype, as measured by fly mortality and bacterial cfu recovery. S. marcescens, an environmentally ubiquitous Gram-negative bacterium, is an opportunistic pathogen in several species, including alligators and Drosophila. Subsequent <i>in vitro</i> testing of the antimicrobial potential of invertebrate hemolymph utilizing the Tn::<i>glnL</i> mutant showed a nitrogen-dependent growth inhibition of species in the order Dipteria. Combined, these results support a model of evolutionary convergence of nitrogen limitation as an antimicrobial mechanism. This work not only identifies a novel antimicrobial strategy that could be used in the development of therapeutics, and a novel virulence factor in <i>S. marcescens</i>, but has broad mplications for bacterial management and can provide insight into the evolutionary history of host-pathogen interactions.</div>
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Host-Microbe Relations: A Phylogenomics-Driven Bioinformatic Approach to the Characterization of Microbial DNA from Heterogeneous Sequence DataDriscoll, Timothy 30 May 2013 (has links)
Plants and animals are characterized by intimate, enduring, often indispensable, and always complex associations with microbes. Therefore, it should come as no surprise that when the genome of a eukaryote is sequenced, a medley of bacterial sequences are produced as well. These sequences can be highly informative about the interactions between the eukaryote and its bacterial cohorts; unfortunately, they often comprise a vanishingly small constituent within a heterogeneous mixture of microbial and host sequences. Genomic analyses typically avoid the bacterial sequences in order to obtain a genome sequence for the host. Metagenomic analysis typically avoid the host sequences in order to analyze community composition and functional diversity of the bacterial component. This dissertation describes the development of a novel approach at the intersection of genomics and metagenomics, aimed at the extraction and characterization of bacterial sequences from heterogeneous sequence data using phylogenomic and bioinformatic tools.
To achieve this objective, three interoperable workflows were constructed as modular computational pipelines, with built-in checkpoints for periodic interpretation and refinement. The MetaMiner workflow uses 16S small subunit rDNA analysis to enable the systematic discovery and classification of bacteria associated with a host genome sequencing project. Using this information, the ReadMiner workflow comprehensively extracts, assembles, and characterizes sequences that belong to a target microbe. Finally, AssemblySifter examines the genes and scaffolds of the eukaryotic genome for sequences associated with the target microbe. The combined information from these three workflows is used to systemically characterize a bacterial target of interest, including robust estimation of its phylogeny, assessment of its signature profile, and determination of its relationship to the associated eukaryote.
This dissertation presents the development of the described methodology and its application to three eukaryotic genome projects. In the first study, the genomic sequences of a single, known endosymbiont was extracted from the genome sequencing data of its host. In the second study, a highly divergent endosymbiont was characterized from the assembled genome of its host. In the third study, genome sequences from a novel bacterium were extracted from both the raw sequencing data and assembled genome of a eukaryote that contained significant amounts of sequence from multiple competing bacteria. Taken together, these results demonstrate the usefulness of the described approach in singularly disparate situations, and strongly argue for a sophisticated, multifaceted, supervised approach to the characterization of host-associated microbes and their interactions. / Ph. D.
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