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Title
Coevolutionary implications of envelope-mediated resistance to phage
Creator
Burmeister, Alita
Date
2017
Collection
Electronic Theses & Dissertations
Description
This dissertation concerns the coevolution of pathogens and their hosts. For my thesis, I have worked with Escherichia coli and phage using experimental evolution, molecular biology, and theory that describes general host-pathogen interactions. This work centers on three themes of broad interest to evolutionary biology and microbiology: coevolutionary origins of novelty and diversity, the role of tradeoffs in constraining evolvability, and the ecological impacts of host resistance.In Chapter...
Show moreThis dissertation concerns the coevolution of pathogens and their hosts. For my thesis, I have worked with Escherichia coli and phage using experimental evolution, molecular biology, and theory that describes general host-pathogen interactions. This work centers on three themes of broad interest to evolutionary biology and microbiology: coevolutionary origins of novelty and diversity, the role of tradeoffs in constraining evolvability, and the ecological impacts of host resistance.In Chapter 1, I worked with a set of experimentally coevolved E. coli/ communities to investigate the bacterial genes that gained mutations and whether fitness tradeoffs had constrained the evolution of those mutations. To do this, I isolated bacteria from the coevolution experiment and sequenced the genomes of the isolates. I found resistance mutations that modified the expression or sequence of proteins used by during infection: LamB and OmpF at the outer membrane, and ManY and ManZ at the inner membrane. To test for fitness tradeoffs, I estimated the isolates’ fitness in the presence and absence of . Phage selection strongly favored resistance mutations, despite those mutations incurring pleiotropic costs related to resource acquisition and homeostasis.For my second chapter, I was particularly interested in the E. coli manY and manZ mutations that allowed phage to adsorb to the outside of the cell but limited the phage’s ability to eject its genome into the cytoplasm. I thought that these mutations might effectively “trap” phage in non-productive infections, thereby accelerating the rate of phage loss from the extracellular environment. However, I found no evidence for such traps; instead, had evolved independence of manY and manZ. These results indicate that for each of the known resistance-conferring mutations evolved by E. coli, the coevolving populations discovered evolutionary routes to circumvent the resistance. For Chapter 3, I shifted briefly from laboratory experiments to mathematical theory to further investigate the trap idea. Although it turned out that the manY and manZ mutations don’t act as traps, I was more generally interested in host defenses on the inside of the cell, such as CRISPR-Cas defense and restriction enzymes, which exist for many bacterial species. Comparable to other studies on parasite traps in animal hosts, I used theory to predict that in the presence of trap alleles, bacteriophage densities would be lower than they otherwise would be, even if more permissive hosts were available to them.In my final chapter, I returned to the coevolution experiment with E. coli and . Although it was known that laboratory populations of phage evolved to use OmpF, and that this function required multiple mutations in the phage J gene, it was unknown how those mutations accumulated. I studied how both phage J gene mutations and bacterial malT (a positive regulator of lamB) mutations influenced the phage’s adaptive landscape. I found that bacterial evolution strongly affected selection patterns on different phage genotypes: in many cases the evolution of host resistance more strongly favored increased phage adsorption rate. Because of that, the evolutionary intermediates between the ancestral and OmpF-infecting phage were positively selected, revealing that host coevolution can increase the rate at which phage evolve to use novel host structures.
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Title
Identifying the genetic basis of attenuation in Marek's disease virus via experimental evolution
Creator
Hildebrandt, Evin
Date
2014
Collection
Electronic Theses & Dissertations
Description
Marek's disease virus (MDV), an oncogenic alphaherpesvirus of chickens, causes up to $2 billion in loses a year due to Marek's disease (MD). Therefore control of this economically important disease is critical. The primary method to control MD is vaccination. Attenuated, or weakened, strains of MDV have been generated via repeated in vitro serial passage to generate avirulent MDV strains that have been used as successful MD vaccines. Despite introduction of several vaccines since the 1970's,...
Show moreMarek's disease virus (MDV), an oncogenic alphaherpesvirus of chickens, causes up to $2 billion in loses a year due to Marek's disease (MD). Therefore control of this economically important disease is critical. The primary method to control MD is vaccination. Attenuated, or weakened, strains of MDV have been generated via repeated in vitro serial passage to generate avirulent MDV strains that have been used as successful MD vaccines. Despite introduction of several vaccines since the 1970's, more virulent strains of MDV have evolved to break vaccinal protection. Therefore, development of new MD vaccines is necessary. To address this concern, we sought to better understand the molecular basis of attenuation in MDV to provide information that may assist in the rationale design of MD vaccines. Three attenuated replicates of a virulent MDV were serially passed in vitro for over 100 passages. DNA and RNA from attenuated viruses were deep sequenced using Illumina next-generation sequencers to identify changes in DNA sequence or expression following attenuation. Top candidate mutations identified via sequencing were used to generate seven recombinant viruses using red-mediated recombineering for mutations within UL42, UL46, UL5, two involving LORF2 and two mutations within ICP4. These recombinant viruses were tested in vivo to determine the impact of these mutations on MD incidence, in vivo replication and horizontal transmission. Point mutations within UL42, UL46, LORF2-Promoter and ICP4 did not cause observable phenotypic changes compared to the parental virus. A single point mutation within LORF2-Intron and a double mutant involving ICP4 both resulting in 100% MD in challenged birds but failed to transmit horizontally to uninfected contact birds. Finally, a point mutation within UL5 reduced MD incidence by over 90%, significantly reduced in vivo replication, and eliminated horizontal transmission. Further characterization of this UL5 point mutation determined that it increased in vitro replication in growth curves, yet head-to-head competition of the Mut UL5 virus versus parental virus showed the parental virus outcompeted the mutant virus. Furthermore, serial passage of Mut UL5 in vivo did not result in increased in MD incidence, in vivo replication or result in reversion or compensatory mutations to UL5 after passage through birds. Trials testing vaccinal protection of the Mut UL5 virus showed the virus provided partial protection against challenge with virulent MDV, yet did not exceed protection achieved through use of traditional vaccines. Therefore, use of this point mutation in combination with other candidate mutations was tested. Addition of the UL5 mutation with Delta Meq, a candidate vaccine with high protection and replication but also induces bursal-thymic atrophy (BTA), resulted in a recombinant virus that replicated at low levels and did not cause BTA, yet reduced levels of vaccinal protection, indicating an intricate relationship between replication levels, BTA and vaccinal protection. This study shows that a variety of genes are mutated during attenuation, and particularly mutations within DNA replication genes, such as UL5, appear to play an important role in attenuation. We also determined that experimental evolution is a process that not only can identify mutations involved in attenuation, but also offer protection as a vaccine to provide information for further development of MD vaccines.
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Title
Using experimental evolution in Drosophila melanogaster to test predictions about the adaptation of prey to a novel predator
Creator
DeNieu, Michael
Date
2014
Collection
Electronic Theses & Dissertations
Description
One of the primary questions in organismal biology is how evolution has acted to shape the species that we see in nature. Beginning to address this incredibly complex question requires a diverse set of approaches that can be difficult to accomplish in the wild, in part because it requires a relatively robust knowledge of the evolutionary history of given populations. Though it cannot tell us how existing species have evolved, experimental evolution is a powerful tool because it allows one to...
Show moreOne of the primary questions in organismal biology is how evolution has acted to shape the species that we see in nature. Beginning to address this incredibly complex question requires a diverse set of approaches that can be difficult to accomplish in the wild, in part because it requires a relatively robust knowledge of the evolutionary history of given populations. Though it cannot tell us how existing species have evolved, experimental evolution is a powerful tool because it allows one to track phenotypic and genotypic changes in populations over time in response to a controlled selection pressure. By imposing a particular selection pressure on populations with a known origin, I can test hypotheses about organismal evolution generated by studying patterns in nature. Here I will discuss a series of experiments conducted on populations of Drosophila melanogaster that have been evolved under predatory selection by nymphs of the Chinese mantis (Tenodera aridifolia sinensis). I first investigated the ability to use phenotypic selection analysis to determine long term evolutionary outcomes. To do this, I measured selection acting on wing size and shape in the base population and then again in the evolved populations after 30 generations of selection, and used this to determine other important morphological and behavioral traits that have likely been targets of selection. I show that evolutionary trajectories are largely predictable, but that unmeasured traits can have profound effects on evolutionary outcomes. I also test the predictions of the risk allocation hypothesis as it pertains to courtship, aggression, and anti-predator behavior. Unlike many previous studies that have focused on learned responses to predation risk, I tested whether populations evolved under differences in variation in predation risk would evolve behavioral patterns consistent with the risk allocation hypothesis. I found that while the hypothesis captured several important aspects of the evolutionary response, the specific predictions failed to accurately describe the actual outcome. my results suggested that the riskiness of different behavioral types played a large role in determining whether they conformed to the predictions of risk allocation. In my final chapter, I investigate a unique behavior in which flies evolved in the presence of predators reduce their propensity to initiate flight. Though my results cannot conclusively determine the cause of the evolution of this new escape strategy, they do suggest that associations with allometric scaling relationships are important in determining the fitness of the divergent strategies observed in the predator-evolved populations.
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Title
An analysis of fitness in long-term asexual evolution experiments
Creator
Wiser, Michael J.
Date
2015
Collection
Electronic Theses & Dissertations
Description
Evolution is the central unifying concept of modern biology. Yet it can be hard to study in natural system, as it unfolds across generations. Experimental evolution allows us to ask questions about the process of evolution itself: How repeatable is the evolutionary process? How predictable is it? How general are the results? To address these questions, my collaborators and I carried out experiments both within the Long-Term Evolution Experiment (LTEE) in the bacteria Escherichia coli, and the...
Show moreEvolution is the central unifying concept of modern biology. Yet it can be hard to study in natural system, as it unfolds across generations. Experimental evolution allows us to ask questions about the process of evolution itself: How repeatable is the evolutionary process? How predictable is it? How general are the results? To address these questions, my collaborators and I carried out experiments both within the Long-Term Evolution Experiment (LTEE) in the bacteria Escherichia coli, and the digital evolution software platform Avida. In Chapter 1, I focused on methods. Previous research in the LTEE has relied on one particular way of measuring fitness, which we know becomes less precise as fitness differentials increase. I therefore decided to test whether two alternate ways of measuring fitness would improve precision, using one focal population. I found that all three methods yielded similar results in both fitness and coefficient of variation, and thus we should retain the traditional method.In Chapter 2, I turned to measuring fitness in each of the populations. Previous work had considered fitness to change as a hyperbola. A hyperbolic function is bounded, and predicts that fitness will asymptotically approach a defined upper bound; however, we knew that fitness in these populations routinely exceeded the asymptotic limit calculated from a hyperbola fit to the earlier data. I instead used to a power law, a mathematical function that does not have an upper bound. I found that this function substantially better describes fitness in this system, both among the whole set of populations, and in most of the individual populations. I also found that the power law models fit on just early subsets of the data accurately predict fitness far into the future. This implies that populations, even after 50,000 generations of evolution in consistent environment, are so far from the tops of fitness peaks that we cannot detect evidence of those peaks.In Chapter 3, I examined to how variance in fitness changes over long time scales. The among-population variance over time provides us information about the adaptive landscape on which the populations have been evolving. I found that among-population variance remains significant. Further, competitions between evolved pairs of populations reveal additional details about fitness trajectories than can be seen from competitions against the ancestor. These results demonstrate that our populations have been evolving on a complex adaptive landscape.In Chapter 4, I examined whether the patterns found in Chapter 2 apply to a very different evolutionary system, Avida. This system incorporates many similar evolutionary pressures as the LTEE, but without the details of cellular biology that underlie nearly all organic life. I find that in both the most complex and simplest environments in Avida, fitness also follows the same power law dynamics as seen in the LTEE. This implies that power law dynamics may be a general feature of evolving systems, and not dependent on the specific details of the system being studied.
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