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Title: UNDERSTANDING THE ROLES OF INTERKINGDOM MICROBIAL INTERACTIONS, MICROBIAL TRAITS, AND HOST FACTORS IN THE ASSEMBLY OF PLANT MICROBIOMES
Creator: Liber, Julian Aaron
Date: 2021
Collection: Electronic Theses & Dissertations
Description: The community of organisms that associate with plants are vital to both the survival of the host plant but also the diseases which may kill it. The processes by which this community, called the microbiome, assemble and function can contribute to the traits of the host, including plants that humans rely on for food, resources, and ecosystems services. This thesis focuses on understanding the assembly of microbiomes at the scale of microbe-microbe interactions and traits of individual microbes,...
Show moreThe community of organisms that associate with plants are vital to both the survival of the host plant but also the diseases which may kill it. The processes by which this community, called the microbiome, assemble and function can contribute to the traits of the host, including plants that humans rely on for food, resources, and ecosystems services. This thesis focuses on understanding the assembly of microbiomes at the scale of microbe-microbe interactions and traits of individual microbes, as well as how characters of the host may change this process. I first address this by examining the in vitro and in planta interactions within small synthetic communities of root-inhabiting bacteria and fungi and with the plant host and viral disease of the host. While intermicrobial interactions in vitro were not predictive of in planta interactions, adding host disease or additional organisms to the system altered the assembly process. I then show the development and applications of the CONSTAX2 classifier, a taxonomic assignment tool for metabarcoding studies, which offers improved accuracy and ease of use for conducting metabarcoding studies exploring the diversity and structure of microbial communities. Last, I present a study testing which factors affected the composition of forest fungal communities to understand the ecology of litter-inhabiting fungi and improve methodologies for sampling leaf-associated fungal communities. The factors affecting the assembly of plant microbiomes are complex and varied but connecting individual interactions to community composition and ultimately function may improve our abilities to predict and manage microbiome processes.
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Title: DYNAMIC DESERTS : THE SYNERGISTIC EFFECTS OF FUNGI, SUNLIGHT, AND NON-RAINFALL MOISTURE ON PLANT LITTER DECOMPOSITION IN DRYLANDS
Creator: Logan V, James Robert
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Drylands cover well over one third of the Earth’s land and are an important part of the global carbon cycle. Despite this, most models underestimate carbon turnover in arid and semi-arid systems, limiting our ability to predict how they will respond to changing climates. This is partly because many models are driven by rainfall, assuming little to no decay between precipitation events. Unlike in many wetter systems though, plant litter decomposition in drylands is largely controlled by non...
Show moreDrylands cover well over one third of the Earth’s land and are an important part of the global carbon cycle. Despite this, most models underestimate carbon turnover in arid and semi-arid systems, limiting our ability to predict how they will respond to changing climates. This is partly because many models are driven by rainfall, assuming little to no decay between precipitation events. Unlike in many wetter systems though, plant litter decomposition in drylands is largely controlled by non-rainfall processes including photodegradation and biotic decomposition supported by non-rainfall moisture (fog, dew, and water vapor; “NRM”). Despite their importance however, few studies have examined how these drivers interact with one another and with fungal communities to influence carbon turnover. In this dissertation, I demonstrate how photodegradation, NRM, and fungal decomposers interact to accelerate carbon turnover in drylands. To do this, I leveraged a natural gradient of NRM frequency in the Namib Desert that receives intense solar radiation. In one study, I used a reciprocal transplant design to show that moisture regime exerts a strong influence on litter-associated fungal communities and show that the relationship between NRM and litter decay rates depends on the composition of the decomposer community. In another study, I manipulated solar radiation for three years and found that photodegradation of the plant cuticle allows litter to absorb more water during NRM events, accelerating biotic decomposition. By examining litter-associated fungal communities under these same radiation treatments, I also show that fungi are largely insensitive to radiation stress and that photodegradation mainly affects decomposition rates in this system through photochemical changes in litter that increase subsequent biotic decomposition. Finally, to quantify the relationship between NRM and carbon turnover on multi-year timescales, I measured mass loss for 30 months along a moisture gradient spanning an order of magnitude of NRM frequency. By coupling these data with continuous meteorological measurements over the same period, I show that accounting for NRM and temperature sensitivity substantially improves the performance of a simple exponential litter decay model. These findings build on previous work demonstrating the importance of solar radiation and NRM as crucial drivers of litter decomposition and point a way forward for future studies to examine how these two processes may interact under future climate scenarios. Drylands are undergoing significant changes from anthropogenic climate change and understanding the drivers of litter decomposition allows us to better predict how these ecosystems are responding to global change.
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Title: INSIGHTS INTO ORGANOSULFUR ASSIMILATION IN STAPHYLOCOCCUS AUREUS
Creator: Lensmire, Joshua Michael
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Bacterial pathogens deploy sophisticated strategies to acquire vital nutrients from the host during infection. Staphylococcus aureus is a considerable human pathogen due to its capacity to cause numerous life-threatening diseases. As such S. aureus has an intricate metabolism that promotes proliferation in distinct host environments. However, little is known regarding the sulfur sources the pathogen scavenges from host tissues. Sulfur is an essential nutrient due to its extensive redox...
Show moreBacterial pathogens deploy sophisticated strategies to acquire vital nutrients from the host during infection. Staphylococcus aureus is a considerable human pathogen due to its capacity to cause numerous life-threatening diseases. As such S. aureus has an intricate metabolism that promotes proliferation in distinct host environments. However, little is known regarding the sulfur sources the pathogen scavenges from host tissues. Sulfur is an essential nutrient due to its extensive redox capacity and consequently, it is a critical component of many cofactors. Prior studies started to define sulfur sources S. aureus can use including the inorganic sulfur sources, sulfide and thiosulfate, and organosulfur sources, cysteine, cystine, and glutathione. While we understand some of the sulfur sources S. aureus can use, we do not know the genetic determinants facilitating assimilation. The present studies sought to explain how S. aureus imports and catabolizes organic sources of sulfur. First, we wanted to uncover the proteins allowing S. aureus to utilize cystine and cysteine as sulfur sources. The S. aureus homologues of characterized cystine transporters, TcyP and TcyABC, were experimentally validated as cystine and cysteine transporters. We expanded the sulfur sources S. aureus can utilize to include homocystine and N-acetyl cysteine and show that both TcyABC and TcyP support growth on N-acetyl cysteine while only TcyABC supports growth on homocystine. Finally, a tcyP mutant is impaired in murine heart and liver when competing with WT S. aureus suggesting import of TcyP substrates is important for heart and liver colonization. While a tcyP mutant is reduced in competition with WT in murine heart and liver colonization is not fully ablated signifying more sulfur sources must be catabolized. We next examined how S. aureus imports and catabolizes GSSG. To identify S. aureus GSSG utilization strategies, we used a chemically defined medium containing GSSG as the sulfur source and isolated mutants harboring transposon insertions within a putative ABC-transporter and -glutamyl transpeptidase that fail to proliferate. The mutants also do not grow in medium supplemented with GSH. Consistent with these findings, we named the locus the glutathione import system (gisABCD-ggt). Biochemical analysis of recombinant Ggt confirms in silico functional predictions by demonstrating that Ggt cleaves both GSH and GSSG. Though Gis mutants display wildtype virulence, we find that Gis-Ggt promotes competition with Staphylococcus epidermidis when GSH or GSSG is supplied as the sole sulfur source in vitro. S. aureus resides as a nasal commensal in 30% of the population and once inside the body can infect nearly every organ. Throughout the changing host environments, S. aureus must sense and acclimate to nutrient availability. We sought to define how sulfur starvation and growth on different sulfur sources changes the transcriptional profile of S. aureus. We described the transcriptional changes when WT S. aureus or a CymR, the sulfur transcriptional regulator, mutant were grown in sulfur replete and deplete conditions. We show sulfur starvation leads to significant expression changes including upregulation of iron acquisition encoding genes and oxidative stress encoding genes. Furthermore, we provide evidence showing upregulation of CymR dependent sulfur transporters when S. aureus is grown on GSH and thiosulfate both of which are conditions in which CymR repression should be occurring. Finally, this dissertation ends with areas of future exploration of sulfur source utilization in S. aureus. These avenues include examining nutrient sulfur available in distinct infection sites and expansion of the sulfur sources S. aureus can use. Overall, the work presented here substantially contributes to our understanding of what sulfur sources S. aureus imports and catabolizes and how different sulfur sources change the transcriptional states of the cell.
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Title: SOCIOECOLOGICAL PREDICTORS OF MICROBIOME VARIATION IN WILD POPULATIONS OF AFRICAN MAMMALS
Creator: Rojas, Connie A.
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Host-associated microbial communities (e.g. microbiomes) influence multiple aspects of their host’s phenotype. Over a decade of research shows that the microbiome can vary with both host factors and environmental factors. However, much of the existing literature has been limited to intestinal microbiomes and to laboratory and domesticated animals. Multi-body site and longitudinal analyses of the microbiomes of wild mammals are lacking. Here, I address these gaps in knowledge and use DNA...
Show moreHost-associated microbial communities (e.g. microbiomes) influence multiple aspects of their host’s phenotype. Over a decade of research shows that the microbiome can vary with both host factors and environmental factors. However, much of the existing literature has been limited to intestinal microbiomes and to laboratory and domesticated animals. Multi-body site and longitudinal analyses of the microbiomes of wild mammals are lacking. Here, I address these gaps in knowledge and use DNA sequencing to survey the microbiomes of a highly gregarious carnivore, the spotted hyena (Crocuta crocuta). Due to their complex societies, spotted hyenas offer an excellent model system for investigating how host physiology and ecology interact with the microbiome, and for elucidating the contributions of the microbiome to host function. In this dissertation, I leverage over three decades of data and samples collected by my adviser from wild hyenas residing in the Masai Mara National Reserve, Kenya (MMNR). Because this dissertation involved many collaborations with other scientists, I use the first person plural throughout this dissertation. In Chapter 1, I evaluate whether the microbiomes at six body-sites vary with host age, sex, and social rank in spotted hyenas, and find that the microbiome is distinct among body sites, and that this differentiation in microbiomes occurs early in life. For Chapter 2, I conduct a longitudinal analysis of the gut microbiome across 3 generations of spotted hyenas from 4 lineages, and elucidate the potential ways gut microbes may be contributing to their host’s digestion of animal carcasses. Findings show that the composition of the gut microbiome is highly variable across time, but its functional repertoire of genes is highly consistent. Furthermore, our analyses reveal that the abundances of bacterial taxa are associated with long-term ecological changes in livestock grazing, anthropogenic disturbance, and herbivore densities that occurred in the Masai Mara reserve. Chapter 3 inquires whether host social interactions and close associations between individuals shape gut microbiota similarity and diversity in a social group of spotted hyenas, which exhibit fission-fusion dynamics. Consistent with our hypothesis, close hyena affiliates share a greater number of bacterial types than hyena dyads that rarely encountered one another, but contrary to our hypothesis, more socially connected individuals do not harbor more diverse gut microbiotas than more isolated individuals. Chapter 4 compares the gut microbiomes of 11 species of sympatric African herbivores from the MMNR and Laikipia region in Kenya, and determines the relative influence of host diet and host phylogenetic relatedness in structuring the microbiome. My findings indicate that across distantly related hosts, herbivore gut microbiotas are strongly shaped by host phylogenetic relatedness and taxonomy, but among closely related hosts, host diet explains the most variation in the gut microbiota. Findings suggest that the gut microbiota is species-specific, but can be further modified by host ecology, including host diet and geography, especially among closely related host species. Overall, my dissertation provides novel insight regarding the factors shaping the gut microbiome in wild carnivores and herbivores, at individual, group-level, and ecosystem-wide scales.
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Title: CHEMICAL BIOLOGY AND GENETIC STUDIES TARGETING THE MYCOBACTERIAL CELL ENVELOPE
Creator: Williams, John Tison
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Mycobacterium tuberculosis is one of the leading causes of death due to a single infectious pathogen. The evolution and spread of drug resistant strains requires new antibiotics to control the TB pandemic. Over the last decade, the lipid flippase MmpL3 has been identified as a potential drug candidate based on its essential nature for cell viability and repeated identification as the lead target of small molecule inhibitors of Mtb growth. Using a combined untargeted and targeted whole cell...
Show moreMycobacterium tuberculosis is one of the leading causes of death due to a single infectious pathogen. The evolution and spread of drug resistant strains requires new antibiotics to control the TB pandemic. Over the last decade, the lipid flippase MmpL3 has been identified as a potential drug candidate based on its essential nature for cell viability and repeated identification as the lead target of small molecule inhibitors of Mtb growth. Using a combined untargeted and targeted whole cell phenotypic screen I identified novel inhibitors of this valued target. A combination of lipid profiling and an innovative competitive binding assay supported MmpL3 as the target of these inhibitors. Cross resistance profiling of MmpL3 inhibitors against twenty-four unique mmpL3 Mtb mutants demonstrated that the level of resistance is associated with the proximity of resistant mutants to essential residues for protein function. Further, these resistance profiles suggested that MmpL3 inhibitors fall into separate clades depending on their chemical scaffolds. The results of this screen led to the development of novel potent analogs for one of the identified MmpL3 inhibitors, HC2099. These analogs were active against clinically relevant drug resistant Mtb strains that cause treatment failure in patients. Active analogs were able to kill Mtb inside of infected macrophages, an infectious niche of Mtb, without inducing cytotoxicity against these important immune cells. One of these analogs, MSU-43085, was orally bioavailable and successfully inhibited Mtb growth in infected mice, supporting further development and highlighting the therapeutic potential of this series. High throughput screens are often used to identify new inhibitors of Mtb growth. However, prioritized hits form these screens often identify similar targets such as MmpL3, lipid synthesis enzymes, redox cyclers, as well as inhibitors of the electron transport chain. Follow up studies of these inhibitors are often time consuming, costly and result in the rediscovery of previously identified targets. While this is not necessarily detrimental to Mtb drug discovery, as these reoccurring targets have therapeutic potential. The continued prioritization of inhibitors for these common metabolic pathways potentially limits the identification of inhibitors for novel targets. Therefore, additional steps that identify inhibitors of these common pathways could reshape how high throughput screen hits are prioritized. By applying the targeted mutant screen used to identify MmpL3 inhibitors to a non-prioritized library of hits from a high throughput screen, we identified more than fifty new potential MmpL3 inhibitors. Using an iterative strategy of applying additional mutants of commonly identified targets, this strategy promises to lead to parallel follow-up studies of inhibitors with known and unknown mechanisms of action. The ability of Mtb to enter into quiescent states in response to host stresses is one of the leading causes for the extended time to cure and evolution of drug resistance. These states can be induced by several environmental stresses including acidic pH, hypoxia, and others. In an effort to study this adaptation in the rapidly growing mycobacterial species M. smegmatis, we identified a lethal sodium citrate phenotype. Transcriptional profiling and genetic screening of mutants tolerant to sodium citrate indicated that this phenotype was due to the combined action of both chelation and osmotic stresses. Cell viability could be reduced from sodium citrate killing by cation and osmoprotectant supplementation. From these experiments we propose a model that can be applied to study carbon source uptake and probe the role of genes identified from the forward genetic screen with unknown function
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Title: WILDFIRE IMPACTS ON SOIL CARBON POOLS AND MICROBIAL COMMUNITIES IN MIXED-CONIFER FORESTS OF CALIFORNIA
Creator: Adkins, Jaron
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Forest ecosystems are important reservoirs for long term carbon (C) storage. Forests of the western United States account for 20-40% of total U.S. carbon C sequestration, and nearly half of the total C in these forests is stored in soil. However, many forests in the western U.S are experiencing wildfire conditions that diverge from historical fire regimes. Prior to Euro-American settlement, California’s mixed-conifer forests typically experienced frequent surface fires of low to moderate burn...
Show moreForest ecosystems are important reservoirs for long term carbon (C) storage. Forests of the western United States account for 20-40% of total U.S. carbon C sequestration, and nearly half of the total C in these forests is stored in soil. However, many forests in the western U.S are experiencing wildfire conditions that diverge from historical fire regimes. Prior to Euro-American settlement, California’s mixed-conifer forests typically experienced frequent surface fires of low to moderate burn severity, but, due to the combined effects of altered forest structure and climate change, now experience fires that are larger and more severe than historical conditions. Fires have numerous direct and indirect effects on the soil biological, chemical, and physical characteristics that influence the soil C cycle. Understanding how altered soil characteristics influence the cycling and persistence of soil C, and how they vary with severity, is important for managing forests for C storage and for predicting fire-climate feedbacks. My dissertation work incorporates observational and manipulative experiments to understand the direct and indirect effects of burn severity on soil C cycling and microbial communities over the short to intermediate term, with a particular focus on the distribution of soil C between active and slow cycling pools. Soil C can be conceptualized as discrete pools of variable persistence in soil. The active C pool is quickly decomposed, contributing to the return of CO2 to the atmosphere, whereas the non-active C pool is more stable and contributes to long term C storage. I leveraged a burn severity gradient resulting from a wildfire in a California mixed-conifer forest to determine the structure and kinetics of these C pools at an intermediate time point in post-fire recovery (i.e. three years). I found that the size of the non-active C pool was smaller in burned areas than unburned areas, and the kinetic rate of the non-active C pool was negatively related to burn severity. I also characterized the soil microbial communities across this severity gradient and identified the environmental characteristics responsible for differences. I found that fungal-to-bacterial ratio and oligotroph-to-copiotroph bacteria ratio decreased with burn severity, and these effects were driven by differences in live and dead tree basal area, soil nutrients, and pH. Leveraging another burn severity gradient, I then determined whether differences in microbial communities and soil C pools were related one-year post-fire in a mixed-conifer forest. I again found lower non-active C pool kinetic rates, and higher abundances of copiotrophic bacteria in burned compared to unburned areas. Differences in soil C pool kinetics were related to tree basal area, soil nutrients, and bacterial communities. I determined the short-term impacts of fire on soil C pools and cycling using lab experiments in which I manipulated soil heating intensity and pyrogenic organic matter (PyOM) additions. I found that high intensity soil heating can decrease microbial biomass C (MBC) accumulation, whereas PyOM had minimal effects on MBC in the short-term. Finally, I found that the size of the active C pool increased with soil heating intensity, while the kinetic rate of the non-active C pool decreased; PyOM primarily increased the size of the non-active C pool. Taken as a whole, my research suggests that fire induces short-term soil C losses by increasing the size of the active C pool, but, over the intermediate-term, residual soil C is more persistent. Fire severity is predicted to increase globally throughout the 21st century, and my research contributes to understanding how forest C storage will be affected by disrupted wildfire regimes.
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Title: SYNTHESIS AND CHARACTERIZATION OF BIOACTIVE GLASS-CERAMIC PARTICLES WITH ADVANCED ANTIBACTERIAL PROPERTIES FOR APPLICATIONS IN BONE REGENERATION
Creator: Pajares Chamorro, Natalia
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Bacterial infections are major surgical complications, which have worsened due to the continued evolution of drug-resistance. In coping with the decay of the antibiotic era, scientists eagerly search for alternative treatments. Multi-functional biomaterials capable of combating infections while triggering tissue regeneration are of great interest. For example, bioactive glasses have been regularly used to deliver drugs and regenerate tissue owed to their unique bone-bonding ability. Doping...
Show moreBacterial infections are major surgical complications, which have worsened due to the continued evolution of drug-resistance. In coping with the decay of the antibiotic era, scientists eagerly search for alternative treatments. Multi-functional biomaterials capable of combating infections while triggering tissue regeneration are of great interest. For example, bioactive glasses have been regularly used to deliver drugs and regenerate tissue owed to their unique bone-bonding ability. Doping the bioactive glass structure with broad-spectrum biocide ions such as Ag+ confers advanced antibacterial properties. The release of Ag+ is controlled by the degradation process of the glass network, maintaining the dose within a therapeutic window that is not cytotoxic to eukaryotic cells. Despite the extensive research performed on Ag-doped bioactive glasses, their regenerative properties in bone tissues have been rarely investigated. This thesis presents promising interactions between Ag-doped bioactive glass (Ag-BG) microparticles and osteoprogenitor cells, providing evidence of the ability to support bone regeneration. Ag-BG’s degradation provoked cell proliferation and cell differentiation in vitro and demonstrated healing of critical calvaria defects in mice after one month of implantation, thanks to the release of Si and Ca ions. Additionally, Ag-BG was antibacterial against Staphylococcus aureus (S. aureus), the most common cause of bone-degenerative diseases like osteomyelitis, and demonstrated low proclivity to induce resistance. The antibacterial potential originated from the degradation by-products of the structure. The mechanism of inhibition was built upon four main sources from higher to lower contribution: Ag+ release, oxidative stress, mechanical damage by nano-sized debris, and osmotic effect. In addition, Ag-BG was capable of restoring ineffective antibiotics with cell-wall-related inhibitory mechanisms by simple combinatorial therapies, rendering them effective in clearing infections. This unprecedented functionality of Ag-BG was expanded with antibiotic depots, where Ag-BG served as a carrier for an ineffective drug. Bioactive glass nanoparticles (BGNs) have been proposed to advance biological and antibacterial properties compared to their micro-sized counterparts. However, the challenges of producing BGNs with multifold metallic ions in a reproducible manner have limited their use. Here, the Stöber method was comprehensively studied to understand the effect of process variables on BGNs’ composition, structure, and morphology. The use of methanol as solvent and the early addition of metallic ion reagents before catalysis helped improved their cation incorporation within the glass network. Extended stirring was key to achieving the targeted composition and controlling the particle size. Monodispersed 10 nm Ag-doped BGNs (Ag-BGNs) were achieved. These Ag-BGNs were stronger antimicrobial weapons, providing bacterial inhibition within hours of treatment. The biological properties were not significantly advanced in the Ag-BGNs compared to Ag-BG; however, cell proliferation, differentiation, and bone re-growth were still provoked. These Ag-BGNs were used as fillers in hydrogel nanocomposites with natural matrices consisting of collagen type I or extracellular matrix. Ag-BGNs distributed homogeneously along the polymer fibrils and allowed polymerization within hours at physiological conditions. These materials hold potential for injectable devices, designing minimally invasive single-step treatment for debilitating bone infections while promoting tissue recovery.
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Title: ENVIRONMENTAL DRIVERS AND EVOLUTIONARY CONSEQUENCES OF HORIZONTAL GENE TRANSFER IN SOIL BACTERIA
Creator: Kittredge, Heather
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Horizontal gene transfer (HGT) is a driving force in bacterial evolution and could drive rapid adaptation in bacterial communities. Natural transformation is one mechanism of HGT that allows bacteria to pick up extracellular DNA (eDNA) from the environment and integrate it into their genome. But the rate of HGT in natural environments, and the role this process plays in facilitating rapid adaptation remains unknown. As climate change threatens the stability of environments worldwide,...
Show moreHorizontal gene transfer (HGT) is a driving force in bacterial evolution and could drive rapid adaptation in bacterial communities. Natural transformation is one mechanism of HGT that allows bacteria to pick up extracellular DNA (eDNA) from the environment and integrate it into their genome. But the rate of HGT in natural environments, and the role this process plays in facilitating rapid adaptation remains unknown. As climate change threatens the stability of environments worldwide, understanding how quickly bacteria can adapt to novel environments is essential. My dissertation research characterizes the environmental drivers and evolutionary consequences of natural transformation in a highly transformable model soil bacterium Pseudomonas stutzeri.Despite decades of research on understanding HGT at the molecular level, less is known about the ecological drivers of HGT. To understand the soil conditions relevant for transformation, I first measured eDNA in the field over a short-term drying rewetting disturbance (Ch. 2). I found that eDNA increased in response to the rewetting disturbance but quickly disappeared from soil, suggesting a small portion of this eDNA could be transformed by bacterial cells recovering from the disturbance. To test the efficiency of transformation under the conditions in which eDNA disappeared, I created a novel microcosm system for quantifying transformation in soil (Ch. 3). Here, I inoculated soil with live antibiotic-susceptible, and dead antibiotic-resistant P. stutzeri. I then tracked the evolution of antibiotic resistance over a range of soil conditions and eDNA concentrations. Transformation drove the evolution of antibiotic resistance across a wide range of soil moistures and increased in response to larger inputs of dead cells (eDNA source), with antibiotic resistance repeatedly appearing in antibiotic free soil. Despite the prevalence of transformation across bacterial species, the evolutionary origins and consequences of transformation are still largely unknown. Transformation presumably provides a fitness benefit in stressful or continuously changing environments, but few studies have quantified changes in transformation in response to adaptive evolution. Here, I evolved P. stutzeri at different salinities and tested how the growth rate and transformation efficiency changed in response to salt adaptation (Ch. 4). Overall, the growth rate increased in response to adaptation, but the transformation efficiency declined, with only ~50% of the evolved populations transforming eDNA at the end of experiment – as opposed to 100% of ancestral populations transforming eDNA. Overall, my dissertation research elucidates the factors driving transformation in soil, setting the stage for future experiments to scale up estimates of transformation to the whole community level. I find that transformation occurs under most soil conditions and allows genetic variants to arise at low frequencies in the absence of selection. I also report novel experimental evidence that transformation efficiency can change dramatically, and in a highly variable manner, over just ~330 generations. Taken together, this body of research highlights a role for transformation in many natural systems of ecological significance, and points to dead cells as an important but often overlooked source of genetic diversity.
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Title: METABOLIC AND RESPIRATORY PATHWAYS CONTROLLING VIBRIO CHOLERAE COLONIZATION
Creator: Van Alst, Andrew John
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Vibrio cholerae is an enteric pathogen of the human small intestine that proliferates to high cell density during human infection. Although not typically classified as a virulence factor, metabolism is a cornerstone for fitness in the host environment. In this work, I explore the essential role of aerobic metabolism, including oxidative respiration, for successful colonization of V. cholerae in the infant mouse model. Oxidative respiration is the most efficient energy generating metabolic...
Show moreVibrio cholerae is an enteric pathogen of the human small intestine that proliferates to high cell density during human infection. Although not typically classified as a virulence factor, metabolism is a cornerstone for fitness in the host environment. In this work, I explore the essential role of aerobic metabolism, including oxidative respiration, for successful colonization of V. cholerae in the infant mouse model. Oxidative respiration is the most efficient energy generating metabolic pathway in living organisms and supports the rapid proliferation of V. cholerae in the small intestinal environment. Despite knowledge that oxygen diffuses from the host epithelium into the gut lumen, the role of oxygen in supporting colonization and proliferation of V. cholerae had not been explored prior to the work presented here in Chapters 2 and 3.In Chapter 2, by targeting the pyruvate dehydrogenase (PDH) complex, an enzyme required to convert pyruvate to acetyl-CoA under aerobic conditions, I show that aerobic metabolism through the PDH complex is required for population expansion in the infant mouse. As the gut was predominantly considered anaerobic and exists in a state of low oxygen tension, I also examined the contribution of anaerobic metabolism to infant mouse colonization. By targeting cognate pyruvate formate-lyase (PFL) that similarly converts pyruvate to acetyl-CoA, but only under anaerobic conditions, I determined that anaerobic respiration is dispensable for colonization. In Chapter 3, I directly test the importance of aerobic and anaerobic respiration by targeting the complete set of terminal oxidases and terminal reductases encoded by V. cholerae. Using a modified Multiplex Genome Editing by Natural Transformation (MuGENT) approach, I generated strains denoted Aero7 and Ana4. Aero7 is a functionally strict anaerobe derivative of V. cholerae, lacking all four terminal oxidases (cbb3, bd-I, bd-II, and bd-III), whereas Ana4 lacked functionality in each of the four terminal reductase complexes (fumarate, trimethylamine-N-oxide, nitrate, and biotin sulfoxide reductases). Disruption in the oxidase complexes in strain Aero7 severely attenuated V. cholerae colonization in the infant mouse, however, no attenuation was observed for Ana4. These data supported our findings in Chapter 2 that aerobic, but not anaerobic metabolism was critical for V. cholerae growth in the infant mouse. Furthermore, I determined that the bd-I oxidase, and to a lesser extent the cbb3 oxidase, support oxidative respiration during infection with bd-II and bd-III oxidases being dispensable for colonization. In summation, aerobic metabolism through the PDH complex and the terminal reduction of oxygen by the bd-I oxidase are essential to V. cholerae colonization of the infant mouse. Through this work, I uncovered a role for oxidative metabolism for V. cholerae colonization. These findings expand our knowledge of V. cholerae biology and pathogenicity in the gastrointestinal tract and implicate oxygen as a critical electron acceptor that shapes the progression of enteric infections.
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Title: COMPUTATIONAL APPROACHES TO COMPLEX BIOLOGICAL PHENOMENA
Creator: Franklin, Joshua Luke
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Biological systems can be difficult to understand due to a vast array of interacting phenom-ena. The result is that some seemingly "easy" questions go unanswered. For example, we have long known that bacteria utilize many distinct flagellar configurations, but in most cases it remains unclear why they do so. We know that cell differentiation is critical to many biological processes, yet we still do not fully understand how such spatiotemporal patterning occurs. Despite mutation being one of...
Show moreBiological systems can be difficult to understand due to a vast array of interacting phenom-ena. The result is that some seemingly "easy" questions go unanswered. For example, we have long known that bacteria utilize many distinct flagellar configurations, but in most cases it remains unclear why they do so. We know that cell differentiation is critical to many biological processes, yet we still do not fully understand how such spatiotemporal patterning occurs. Despite mutation being one of the driving forces of evolution, we still have a hazy understanding of how organisms respond and adapt to high mutation rates. However, ad- vances in technology, modeling, and experimental techniques have enabled us to investigate the small and nuanced effects that can answer these questions. In this dissertation, I have used modern computational tools and statistical techniques to investigate evolutionary and behavioral processes. Agent-based models of evolution and flagellar inheritance have allowed me to investigate the evolution of mutational robustness and trade-offs associated with flagellar motility, respectively. By writing Bayesian mixed- effect models, I have been able to precisely quantify the metabolic cost of producing flagella and describe spatiotemporal patterns of cell differentiation within fruiting bodies of Myxo- coccus xanthus. Careful quantitative modeling of biological phenomena can help cut through the complexity of these systems. As computational power continues to grow and software continues to become more sophisticated, these computational approaches will both become more powerful and easier to use. Computational approaches will not replace experiments in most cases; instead, computational models can direct experiments, which can themselves direct new modeling efforts, in an iterative loop of progressing knowledge.
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Title: The Control of Phenotypic Diversity in Vibrio cholerae during the Transition between Motility and Attachment
Creator: Lee, John Seungwu
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Vibrio cholerae is a causative agent of human intestinal disease, cholera. It requires both flagellar-based motility and biofilm formation to colonize the small intestine. The secondary messenger molecule, c-di-GMP, plays a central role in controlling transition between motility and biofilm formation. However, the switch between these two lifestyles has been studied on an average population scale, overlooking the heterogeneous phenotypic response that can occur at a single-cell level. V....
Show moreVibrio cholerae is a causative agent of human intestinal disease, cholera. It requires both flagellar-based motility and biofilm formation to colonize the small intestine. The secondary messenger molecule, c-di-GMP, plays a central role in controlling transition between motility and biofilm formation. However, the switch between these two lifestyles has been studied on an average population scale, overlooking the heterogeneous phenotypic response that can occur at a single-cell level. V. cholerae infections are characterized by the co-occurrence of cells with motile and sessile behaviors, but the determinant of this phenotypic diversity remains poorly understood. We used single-cell tracking to examine the motile behaviors of two V. cholerae strains (El Tor C6706 and Classical O395) in response to direct manipulations of c-di-GMP concentration. Both motile and non-motile cells are present in a well-mixed batch culture with distributions of these phenotypes that change depending on the growth phase. We determined that the proportion of motile cells differs between El Tor and Classical strains because they maintain different levels of c-di-GMP. However, even in conditions that promote biofilm formation, V. cholerae still generates a sub-population of motile cells. C-di-GMP is known to inversely regulate assembly of mannose-sensitive haemagglutinin (MSHA) pili and flagella. We found that the most cells in clonal populations are flagellated, but MSHA piliation affords the variable opportunity to attach at single-cell level, driving behavioral switching between motile and sessile behaviors. Our results support the hypothesis that c-di-GMP regulates phenotypic diversity in V. cholerae, and it does so by simultaneous elaboration of MSHA pili and flagellum.
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Title: THE EFFECT OF FREEZE-THAW CYCLES ON MICROBIAL RESILIENCE ALONG A CROP BIODIVERSITY GRADIENT
Creator: Liang, Brian Wan
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Freeze-thaw cycles (FTCs) are cyclical periods of soil disturbance that are increasing in number and intensity due to climate change effects on winter precipitation and temperature patterns and are not well characterized within an agroecosystem environment. First, I review the literature and discuss the effects of FTCs on soil properties, explore the nuances of characterizing FTCs in experiments, and assess the knowledge gaps of FTC studies in agroecosystems. I conducted a laboratory...
Show moreFreeze-thaw cycles (FTCs) are cyclical periods of soil disturbance that are increasing in number and intensity due to climate change effects on winter precipitation and temperature patterns and are not well characterized within an agroecosystem environment. First, I review the literature and discuss the effects of FTCs on soil properties, explore the nuances of characterizing FTCs in experiments, and assess the knowledge gaps of FTC studies in agroecosystems. I conducted a laboratory experiment using soils from a crop rotational diversity gradient and froze them at three distinct FTC frequencies. My results indicate that increased crop rotational diversity did not moderate FTC disturbance effects at any frequency level. Increased FTC frequencies generally increased soil organic C losses as CO2, decreased ammonium (NH4+), increased nitrate (NO3-) pools, and increased extracellular enzyme activities (EEA). The respiratory burst after each freezing period was the predominant contributor to differences by FTC in cumulative CO2 respiration by the end of the incubation. Interestingly, the medium FTC frequencies facilitated the highest EEA for select enzymes with minimal reductions in microbial biomass. This suggests that microbes and their EEA are impacted too severely with high frequency FTCs to maintain function. My study revealed that the novel microbial communities and soil processes found along a crop rotational diversity gradient are not resilient against climate change effects of FTCs in soils. Accelerated soil organic C loss and nutrient turnover are expected to occur throughout agroecosystems that experience increased FTCs.
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Title: INFLUENCE OF AQUATIC MICROBES ON MOSQUITO OVIPOSITION BEHAVIOR AND LIFE HISTORY
Creator: Receveur, Joseph
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Mosquito-microbe interactions have major roles in mediating mosquito life histories and the capacity of mosquitoes to transmit pathogens to humans. While essential to better understanding mosquito life histories, our knowledge of how microbes influence oviposition behavior and development remains limited, especially in complex polymicrobial communities. This dissertation tested the relationship between aquatic microbial communities and mosquito oviposition behavior. Field studies were...
Show moreMosquito-microbe interactions have major roles in mediating mosquito life histories and the capacity of mosquitoes to transmit pathogens to humans. While essential to better understanding mosquito life histories, our knowledge of how microbes influence oviposition behavior and development remains limited, especially in complex polymicrobial communities. This dissertation tested the relationship between aquatic microbial communities and mosquito oviposition behavior. Field studies were conducted in Michigan to investigate how different leaf-litter derived microbial communities (and modifications of the microbes present in those communities) effected mosquito oviposition and survivorship. Additionally, field (Ouidah, Benin) and laboratory assays were used to determine the effect of a bacterial toxin (mycolactone, produced by Mycobacterium ulcerans) on other aquatic microbes and the downstream impacts of those interactions on mosquito oviposition. Ae. japonicus japonicus (Theobald) mosquitoes preferred red oak (Quercus rubra) leaf-derived leachates over other leaf species tested (sycamore Platanus occidentalis, and honeysuckle Lonicera maackii). Leachates displayed distinct bacterial and fungal communities with sterilization of these communities causing a reduction in oviposition. Larval mosquito growth and survivorship were influenced by both leaf type and leachate concentration. The addition of mycolactone, hypothesized to inhibit microbial quorum sensing, to environmental water samples altered bacterial community composition and reduced Aedes egg laying. In addition to reducing oviposition of Aedes aegypti (L.) in a dose-dependent manner, mycolactone up- and down-regulated expression levels of multiple taxa (N = 13) and functional groups (N = 13), suggesting the toxin plays an important role in interactions between M. ulcerans, other environmental microbes, and mosquito behavior. These studies highlight the essential role mosquito-microbe interactions play in oviposition behavior and life history and expand our knowledge of the microbial underpinnings of mosquito oviposition behavior. Additionally, experiments testing the relationship between the microbiota of aquatic macroinvertebrates and environmental conditions were conducted in Ostana, Italy using high-throughput amplicon sequencing. While both environmental conditions and macroinvertebrate species influenced the internal microbiome, species had a larger effect than environmental conditions, suggesting that physiological conditions in macroinvertebrate guts plays a role in what microbes are able to colonize and survive.
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Title: ECOLOGICAL AND EVOLUTIONARY CONSEQUENCES OF EXOMETABOLTIES FOR MICROBIAL INTERACTIONS
Creator: Chodkowski, John Luke
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Interspecies interactions have fundamental roles in shaping microbial communities. Microbial community members can produce and release a diverse set of extracellular small molecules, collectively referred to as exometabolites. Interspecies interactions that result from exometabolites can alter the response and behaviors of microbial community members. This dissertation work demonstrates the establishment of a synthetic community system that facilitates the study of exometabolite-mediated...
Show moreInterspecies interactions have fundamental roles in shaping microbial communities. Microbial community members can produce and release a diverse set of extracellular small molecules, collectively referred to as exometabolites. Interspecies interactions that result from exometabolites can alter the response and behaviors of microbial community members. This dissertation work demonstrates the establishment of a synthetic community system that facilitates the study of exometabolite-mediated interspecies interactions. This system was then used to understand the consequences of exometabolite-mediated interactions in a 3-member synthetic microbial community over stationary phase using a multi-omics approach. Lastly, an experimental evolution showed the consequences of long-term exometabolite interspecies interactions on the evolution of antibiotic resistance. This work advances knowledge on the dynamic response and behaviors of microbial community members’ during non-growth states. This work also demonstrates how bacterial-bacterial interactions in a simple environment can lead to the emergence of antibiotic resistance.
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Title: Microbial Community Dynamics within a Serpentinization-Influenced Aquifer : Characterization of Community Assembly Processes and Responses to Drilling-Induced Perturbations
Creator: Putman, Lindsay Irene
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Serpentinization is the hydrothermal alteration of ultramafic rock, which results in high pH, reducing fluids that are low in dissolved inorganic carbon, and have high levels of methane and hydrogen. While endemic microbial communities have been well-characterized from a variety of marine and terrestrial serpentinizing ecosystems, to date the ecological processes that contribute to microbial community assembly, community dynamics, and the impact of disturbances in serpentinizing environments...
Show moreSerpentinization is the hydrothermal alteration of ultramafic rock, which results in high pH, reducing fluids that are low in dissolved inorganic carbon, and have high levels of methane and hydrogen. While endemic microbial communities have been well-characterized from a variety of marine and terrestrial serpentinizing ecosystems, to date the ecological processes that contribute to microbial community assembly, community dynamics, and the impact of disturbances in serpentinizing environments have not yet been assessed. The work in this dissertation was performed at the Coast Range Ophiolite Microbial Observatory, CA, USA, where a series of wells were drilled in 2011 to access serpentinization-influenced fluids directly from the subsurface. Geochemical and 16S rRNA gene amplicon datasets were collected directly from these wells and a series of microcosm experiments were performed on fluids from the site. Samples collected over the course of six years were used to assess community assembly processes and the biogeochemical impacts of drilling fluid injection into the subsurface. A series of microcosm experiments were also performed to better understand the response of microbial populations to geochemical changes observed in situ following drilling. Results from this work will inform studies of biogeochemical dynamics relevant to modern and ancient Earth and extraterrestrial sites such as Mars. These data further our understanding of microbial community responses to environmental perturbations and provide information that will aid in the development of future drilling and monitoring projects focused on learning about life in the deep subsurface.
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Title: Postmortem microbiome computational methods and applications
Creator: Kaszubinski, Sierra Frances
Date: 2020
Collection: Electronic Theses & Dissertations
Description: Microbial communities have potential evidential utility for forensic applications. However, bioinformatic analysis of high-throughput sequencing data varies widely among laboratories and can potentially affect downstream forensic analyses and data interpretations. To illustrate the importance of standardizing methodology, we compared analyses of postmortem microbiome samples using several bioinformatic pipelines, while varying minimum library size or the minimum number of sequences per sample...
Show moreMicrobial communities have potential evidential utility for forensic applications. However, bioinformatic analysis of high-throughput sequencing data varies widely among laboratories and can potentially affect downstream forensic analyses and data interpretations. To illustrate the importance of standardizing methodology, we compared analyses of postmortem microbiome samples using several bioinformatic pipelines, while varying minimum library size or the minimum number of sequences per sample, and sample size. Using the same input sequence data, we found that pipeline significantly affected the microbial communities. Increasing minimum library size and sample size increased the number of low abundant and infrequent taxa detected. Our results show that bioinformatic pipeline and parameter choice significantly affect the resulting microbial communities, which is important for forensic applications. One such forensic application is the potential postmortem reflection of manner of death (MOD) and cause of death (COD). Microbial community metrics have linked the postmortem microbiome with antemortem health status. To further explore this association, we demonstrated that postmortem microbiomes could differentiate beta-dispersion among M/COD, especially for cardiovascular disease and drug-related deaths. Beta-dispersion associated with M/COD has potential forensic utility to aid certifiers of death by providing additional evidence for death determination. Additional supplemental files including tables of raw data and additional statistical tests are available in supplemental files online, denoted in the text as table 'S'.
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Title: Characterization of map3773c, ferric uptake regulator protein, in iron metabolism of mycobacterium avium subsp. paratuberculosis
Creator: Miyagaki Shoyama, Fernanda
Date: 2020
Collection: Electronic Theses & Dissertations
Description: Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD) in ruminants, a chronic inflammation of the intestines characterized by persistent diarrhea that leads to malnutrition and muscular wasting (Rathnaiah et al., 2017). Unfortunately to date, reliable JD diagnostics are lacking. Culture of MAP from feces has been the most reliable method for diagnosis of JD, however MAP requires eight to sixteen weeks to produce colonies in culture, presenting a...
Show moreMycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD) in ruminants, a chronic inflammation of the intestines characterized by persistent diarrhea that leads to malnutrition and muscular wasting (Rathnaiah et al., 2017). Unfortunately to date, reliable JD diagnostics are lacking. Culture of MAP from feces has been the most reliable method for diagnosis of JD, however MAP requires eight to sixteen weeks to produce colonies in culture, presenting a major hurdle to diagnosis (Bannantine et al., 2002). Currently, no current vaccine can protect animals against JD or prevent shedding of MAP (Garcia and Shalloo, 2015). These challenges establish a need for a better understanding of the MAP physiology during infection. Unlike other mycobacteria, MAP has a unique requirement for supplementation of an iron-binding siderophore (mycobactin J) for optimal growth in laboratory media. A whole genome sequence of MAP K10 revealed a truncation of the mbtA gene that was speculated to have led to its mycobactin dependency (Li et al., 2005). Zhu et al (2008) showed that MAP is able to transcribe all mycobactin synthesis genes in an intra-macrophage environment. Furthermore, several genes responsible for iron acquisition in infected tissues, including genes responsible for mycobactin biosynthesis have been shown to be upregulated in naturally infected tissues (Janagama et al., 2010). Its known that iron plays an important role in vital biological processes; however high intracellular concentration of free iron can lead to toxicity to the bacteria. As such, bacteria activate expression of specific group of genes that are controlled by a metal-sensing regulatory transcription factor. In 2009, Janagama and others identified and characterized the iron dependent regulator (IdeR) in MAP. In addition, in a MAP-specific genomic island, MAP carries three putative ferric uptake regulator (Fur) boxes, an iron regulated transcriptional control motif, (Stratmann et al., 2004). To date, nothing is known about the role of Fur in MAP. To elucidate the mechanisms of iron homeostasis in MAP, we investigated LSP15 using a transposon mutant MAP3776c. We demonstrated a phenotype for LSP15 genes in a culture model of cell entry and survival and suggest a function in epithelial cell pathogenesis, however further functional analysis with complementation by a MAP3776c::Tn strain would be necessary to confirm these findings. Additionally, full characterization of a Fur-like protein (MAP3773c) was performed. Using PRODORIC for computational analysis, 23 different pathways that were likely regulated by MAP3773c were identified. These findings were confirmed using a chromatin immunoprecipitation assay followed by high-throughput sequencing (ChIP-seq), that revealed 58 regions where Fur binds under iron replete and deplete conditions. From those, three were directed related to iron regulation MAP3638c (hemophore-like protein), MAP3736c (Fur box) and MAP3776c (ABC transporter). Using the Fur box consensus sequence, we confirmed binding specificity and Mn2+ availability by a chemiluminescent electrophoresis mobility shift assay (EMSA). A transcriptional profile of the parent MAP K10, deletion mutant of MAP3773c and the complemented strains was developed under iron replete and depleted conditions. However, under the current experimental conditions, we are unable to conclude if the lack of transcriptional responses in our study was indicative of a lack of FUR activity.
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Title: Characterizing single-cell behavior of flagellated pathogenic bacteria in mucus and visco-elastic environments
Creator: Nguyen, Nhu Thi Quynh
Date: 2020
Collection: Electronic Theses & Dissertations
Description: Flagellar motility is required for some enteric pathogens to colonize the intestine. Understanding how these pathogens overcome the mucus layer protecting the epithelial tissue is necessary for disease prevention. In this thesis project I examined bacterial motility in mucus to understand factors that facilitate mucus penetration and contrasted this with the motility in different visco-elastic materials.In this thesis, I used single cell tracking to characterize the flagellar motility of...
Show moreFlagellar motility is required for some enteric pathogens to colonize the intestine. Understanding how these pathogens overcome the mucus layer protecting the epithelial tissue is necessary for disease prevention. In this thesis project I examined bacterial motility in mucus to understand factors that facilitate mucus penetration and contrasted this with the motility in different visco-elastic materials.In this thesis, I used single cell tracking to characterize the flagellar motility of Vibrio cholerae and Salmonella enterica in different visco-elastic environments. First, I tested if V. cholerae and S. enterica were still motile in unprocessed pig intestinal mucus. Second, I studied factors that supported their motility in mucus. The first factor I investigated was the effect of pH on V. cholerae motility. I also studied the role of curvature in V. cholerae motility within mucus and some other visco-elastic environments, including liquid and agar. Last, I began investigation of the role of flagellar number in S. enterica motility. In these studies, I focused on analyzing the effective diffusion of bacteria and factors promoting the diffusion in each environment. My findings demonstrate that V. cholerae and S. enterica are able to swim in mucus, and that the torque, and the curvature of V. cholerae, and the flagellar number of S. enterica, play a significant role in bacteria motility in mucus and liquid. Moreover, my findings help elucidate the significance of motility in pathogenesis.
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Title: THE EFFECT OF GROWTH-RESTRICTION ON THE MURINE GUT MICROBIOME
Creator: Quinn, Melissa
Date: 2020
Collection: Electronic Theses & Dissertations
Description: ABSTRACTTHE EFFECT OF GROWTH-RESTRICTION ON THE MURINE GUT MICROBIOME By Melissa QuinnINTRODUCTION. Growth restriction induced by undernutrition in early life increases the risk of developing chronic diseases in adulthood. We hypothesized growth restriction would alter the gut microbiome and metabolome across the lifespan, impairing vital growth signaling processes necessary for proper development, with a primary focus on muscular and hepatic Insulin-like Growth Factor (IGF-1) expression....
Show moreABSTRACTTHE EFFECT OF GROWTH-RESTRICTION ON THE MURINE GUT MICROBIOME By Melissa QuinnINTRODUCTION. Growth restriction induced by undernutrition in early life increases the risk of developing chronic diseases in adulthood. We hypothesized growth restriction would alter the gut microbiome and metabolome across the lifespan, impairing vital growth signaling processes necessary for proper development, with a primary focus on muscular and hepatic Insulin-like Growth Factor (IGF-1) expression. METHODS. A cross-fostering, protein-restricted nutritive model (8% protein) was used to induce undernutrition during gestation (GUN) or lactation (PUN). At 21 days of age (PN21), all mice were weaned to a control diet (CON; 20% protein), isolating undernutrition to specific windows of early life. Fecal samples were collected weekly PN18-PN80 to determine longitudinal programming effects of growth restriction on the gut microbiome (CON N=5, GUN N=6, PUN N=6) and metabolome. Fecal sample DNA was extracted for amplification of the bacterial 16S rRNA genes using PCR, and then the amplicons were sequenced with the Illumina pipeline and analyzed using the Qiita bioinformatics software. Cecum samples were also collected at PN21 (CON N=4, GUN N=6, PUN N=5) and PN80 (CON N=5, GUN N=6, PUN N=6) for microbiome analysis. Liver samples were collected at PN21(CON N=12, GUN N=6, PUN N=7) and PN80 (CON N=13, GUN N=9, PUN N=11) and analyzed along with the cecum for metabolomics via tandem mass spectrometry (LC-MS/MS) and analyzed with the Global Natural Products Social Molecular Networking (GNPS) bioinformatics software. IGF-1 expression in the liver and gastrocnemius (CON N=15, GUN N=12, PUN N=13) was analyzed via a Total Protein NIR western blot to establish a connection between the gut microbiome, tissue metabolome and organ growth. RESULTS. The Beta-Diversity of the fecal microbiome was significantly separated by treatment group using Weighted UniFrac measures (PERMANOVA p=0.0001). Differences in the microbiome were not evident through analysis at the Phylum level (Firmicutes/Bacteroidetes ratio) but were instead driven by longitudinal alterations in the abundance of specific genera and species in PUN. Linear mixed model (LMM) analysis revealed PUN having significantly higher abundance of specific bacteria compared to GUN and CON across the lifespan including: Bacteroides uniformis, B. acidifaciens, B. ovatus, Bifidobacterium sp. and Clostridium sacchrogumia. Rikenellaceae was the only microbe that was significantly lower in abundance in the PUN group over time compared to GUN and CON. Additionally, the PUN metabolome was significantly altered compared to GUN and CON, primarily characterized by reduced: essential amino acids (EAAs: methionine, phenylalanine and tyrosine), riboflavin (B2), primary bile acids, and decreased Dehydroepiandrosterone (DHEA); and increased acylcarnitines and fecal peptides. NIR Western blot analysis revealed significantly lower IGF-1 expression in the liver at PN21 in GUN (p=0.0012) and PUN (p<0.001) as well as overall lower expression in the muscle in PUN (p=0.037) and GUN (p=0.007) compared to CON. CONCLUSION: The gut microbiome and metabolome are altered by early life growth restriction at PN21 and through adulthood. Elevated sugar-fermenting bacteria in the PUN group represent gut microbiome immaturity and delayed development. Temporary metabolic alterations of early life growth restriction are seen in decreased primary bile acids and increased synthesis of liver acylcarnitines, both of which are indicative as adaptations of the pups being calorie-restricted as a result of the low-protein fed dam. More permanent outcomes of growth restriction were evident by increased peptide excretion over the lifespan, significantly decreased methionine and riboflavin – which prevented protein synthesis to occur during early life development, and overall decreased muscle IGF-1 expression and DHEA levels in the PUN mice. Many of the metabolic pathways permanently altered by growth restriction are seen in the liver, making this organ an important site for future research on the development of treatment modalities that can limit growth restriction induced chronic disease.
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Title: Establishment of novel Wolbachia symbiosis and characterization of sex determination genes in dengue mosquito vectors
Creator: Liang, Xiao
Date: 2020
Collection: Electronic Theses & Dissertations
Description: As one of the deadliest animals on earth, mosquitos transmit numerous diseases to humans, including dengue, Zika and malaria, which account for over one million human deaths every year. Due to insufficiency of traditional vector control strategies, significant efforts have recently been made to develop novel genetic approaches to either directly suppress mosquito populations or reduce mosquito’s ability to transmit pathogens to humans. One of them is based on the maternally transmitted...
Show moreAs one of the deadliest animals on earth, mosquitos transmit numerous diseases to humans, including dengue, Zika and malaria, which account for over one million human deaths every year. Due to insufficiency of traditional vector control strategies, significant efforts have recently been made to develop novel genetic approaches to either directly suppress mosquito populations or reduce mosquito’s ability to transmit pathogens to humans. One of them is based on the maternally transmitted intracellular symbiotic bacterium Wolbachia. Estimated to infect more than 60% of arthropods in nature, Wolbachia can spread through host populations by means of a reproduction-interfering referred to as cytoplasmic incompatibility (CI). By altering the host’s physiological environment, including immune priming or metabolic perturbation, Wolbachia can also confer antiviral resistance in mosquito vectors. Successful field trials have been conducted to release Wolbachia-infected mosquito males to induce incompatible matings for population suppression or spread Wolbachia into mosquito populations to reduce or block dengue transmission by population replacement. Both population suppression and replacement require for establishment of an artificial Wolbachia symbiosis in mosquito to make it incompatible with target populations. In order to develop a Wolbachia-based strategy for dengue/Zika control in Singapore and Mexico, I have established the transinfected line WB2. By comparing with another transinfected line WB1 which developed 15 years ago, I have demonstrated that wAlbB maintains a stable symbiosis with Ae. aegypti. Further assays show that Wolbachia induces strong resistance to dengue, Zika and Chikungunya viruses in WB2. WB2 line has now been released for field trials in both Mexico and Singapore. In order to improve Wolbachia-based mosquito control, transinfected mosquitoes must be optimized to display maximum pathogen blocking, the desired CI pattern, and the lowest possible fitness cost. Achieving such optimization, however, requires a better understanding of the interactions between the host and various Wolbabachia strains. Thus, we transferred the Wolbachia wMel strain into Ae. albopictus, resulting in a transinfected line, HM (wAlbAwAlbBwMel), no CI was induced when the triply infected males were crossed with the wild-type GUA females or with another triply infected HC females carrying wPip, wAlbA, and wAlbB, but removal of wAlbA from the HM line resulted in the expression of CI after crosses with lines infected by either one, two, or three strains of Wolbachia. These results show that introducing a novel strain of Wolbachia into a Wolbachia-infected host may result in complicated interactions between Wolbachia and the host and between the various Wolbachia strains, with competition likely to occur between strains in the same supergroup. In order to manage the potential risk of failure in population suppression in Singapore, I developed another Ae. aegypti carrying wMal. The transinfected line showed 100% maternal transmission. To facilitate developing a perfect sex separation approach for Wolbachia-based population suppression, I established the CRISPR/Cas9 approach to characterize the function of sex determination pathway genes in Ae. aegypti. By individually knocking out doublesex (dxl) and transformer-2 (tra-2), two essential genes in mosquito sex determination pathway, we show that dxl is not essential gene for female development while knockout of tra-2 results in male-biased sex ratio and absence of female mosquito with homozygous tra-2. These results indicate that the tra-2 is a potential sex determination target that can be explored to develop the female-specific lethality for mosquito sex separation.
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