Search results

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.
Show less

Title: EPIDEMIOLOGY OF ANTIBIOTIC RESISTANT SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) AND NON-TYPHOIDAL SALMONELLA (NTS) IN MICHIGAN
Creator: Mukherjee, Sanjana
Date: 2018
Collection: Electronic Theses & Dissertations
Description: The enteric pathogens, Shiga toxin-producing E. coli (STEC) and non-typhoidal Salmonella (NTS), are leading causes of foodborne infections in the US, resulting in 265,000 and 1.2 million illnesses every year, respectively. The emergence of antibiotic resistance in these pathogens has been documented and is of great concern due to negative patient health outcomes and the possibility of transfer of resistance genes to other clinically relevant pathogens. However, there is a scarcity in...
Show moreThe enteric pathogens, Shiga toxin-producing E. coli (STEC) and non-typhoidal Salmonella (NTS), are leading causes of foodborne infections in the US, resulting in 265,000 and 1.2 million illnesses every year, respectively. The emergence of antibiotic resistance in these pathogens has been documented and is of great concern due to negative patient health outcomes and the possibility of transfer of resistance genes to other clinically relevant pathogens. However, there is a scarcity in information about frequencies of antibiotic resistant and factors associated with resistant STEC and NTS infections in Michigan. It is necessary to have a complete understanding about the of emerging antibiotic resistance and factors driving the rise of resistance in STEC and NTS to help develop effective control strategies. In this dissertation, 980 STEC isolates collected from patients in Michigan between 2001 and 2014 were examined for resistance to clinically relevant antibiotics. The examination of STEC strains for resistance, revealed high frequencies of resistance to ampicillin and trimethoprim-sulfamethoxazole, with significant increases in antibiotic resistance rates observed over this 14-year period. Multivariate logistic regression analysis identified non-O157 serotypes to be independently associated with antibiotic resistance. The recent increase in incidence of non-O157 serotypes observed in the US, coupled with the high frequencies of antibiotic resistance observed in this study, suggest the emergence of antibiotic resistant non-O157s as important human pathogens. Additionally, antibiotic resistant STEC isolates from patients in recent years (2010-2014) were more likely to cause hospitalizations than pansusceptible STEC isolates, suggesting that resistant STEC infections may result in adverse patient outcomes. Using whole genome sequencing, we also identified chromosomal mutations and 33 horizontally acquired genes present in the genomes of non-O157 STEC, likely conferring resistance. Importantly, by creating a co-occurrence network of these genes, we identified the co-occurrence of certain resistance genes, which are possibly present on the same mobile genetic element, thus resulting in multi-drug resistance. In addition to examining resistance in STEC, a total of 198 clinical NTS isolates collected between 2011 and 2014 were also examined for antibiotic resistance in this dissertation. Resistance to tetracycline, trimethoprim-sulfamethoxazole and ampicillin were commonly observed. Concerningly, high frequencies of multidrug resistant NTS were also observed with significant increases in their prevalence observed between 2011 and 2014. These high multidrug resistant rates have important implications on patient care as the efficacy of multiple antibiotics is reduced. Antibiotic resistant NTS isolates were also found to result in significantly longer mean hospital stays compared to pansusceptible NTS. Serovar specific differences in frequencies of antibiotic resistance were observed; S. Enteritidis were observed to have lower resistance frequencies than other serovars. Lastly, to better understand the role that cattle reservoirs play in harbouring antibiotic resistant STEC strains, we examined 121 STEC isolates collected in 2012 from six cattle farms in Michigan for antibiotic resistance. While high resistance frequencies to tetracycline and trimethoprim-sulfamethoxazole were observed in certain herds, no resistance to ampicillin was observed, unlike what was observed in STEC isolates collected from patients. While different populations of resistant STEC may be circulating in the clinical and agricultural environments, continuous monitoring of resistance in the cattle reservoir is warranted to determine if animal reservoirs can serve as potential sources of resistant infections in humans.
Show less

Title: Ecological and evolutionary consequences of exometabolites 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.
Show less

Title: Effects of dietary fiber sources on the gastro-intestinal microbiota, fermentation metabolites, and listeria monocytogenes in vivo and in vitro
Creator: Walker, Ryan (Ryan Adam)
Date: 2019
Collection: Electronic Theses & Dissertations
Description: Dietary fiber consumption influences the gastro-intestinal microbiota, gastro-intestinal function, and health. This research investigated the effects of dietary fiber sources on bacterial composition, short-chain fatty acid (SCFA) production, gastro-intestinal barrier function, and Listeria monocytogenes challenge in mice and human colon cells. L. monocytogenes causes life-threatening illness in humans and animals. Some dietary fibers protect animals against illness in models of infection,...
Show moreDietary fiber consumption influences the gastro-intestinal microbiota, gastro-intestinal function, and health. This research investigated the effects of dietary fiber sources on bacterial composition, short-chain fatty acid (SCFA) production, gastro-intestinal barrier function, and Listeria monocytogenes challenge in mice and human colon cells. L. monocytogenes causes life-threatening illness in humans and animals. Some dietary fibers protect animals against illness in models of infection, while others enhance infection. Therefore, it is unclear if consuming certain dietary fiber sources confers protection against foodborne illnesses. The objectives of this research were: 1) describe how dietary fiber sources affect bacterial composition in vivo and in vitro; 2) quantify SCFAs produced by bacterial fermentation; 3) determine if fiber sources differentially affect L. monocytogenes infection in vivo; and 4) determine if bacterial metabolites promote or inhibit L. monocytogenes infection by affecting in vitro epithelial barrier function.In this project, dietary fiber sources differentially affected gastro-intestinal bacterial composition in vivo and in vitro, L. monocytogenes infection in mice, and enhanced barrier integrity in vitro. The overall results of this project demonstrate that dietary fiber sources differentially influence certain gastro-intestinal bacterial populations, measures of diversity, and that resulting compositional changes are important in the pathogenicity of L. monocytogenes. Additionally, fermentation end products enhance gastro-intestinal barrier function in this in vitro model, which may be an important factor in the prevention of enteric infection in humans. In summary, both the gastro-intestinal microbiota and its metabolites are important factors for maintaining gastro-intestinal health.
Show less

Title: Environmental Microbial Surveillance : From Source Tracking in Watersheds to Pathogen Monitoring in Sewersheds
Creator: Flood, Matthew Thomas
Date: 2022
Collection: Electronic Theses & Dissertations
Description: Understanding of the connections between water and health, through the use of water quality monitoring, surveys and surveillance, can help to address the impacts of anthropomorphic changes on the environment. This study sought to understand these connections through the water quality monitoring within watershed basins as well as pathogen surveillance within sewersheds. Specifically, this dissertation sought to 1) understand the sources of pollution and their connections with land use in the...
Show moreUnderstanding of the connections between water and health, through the use of water quality monitoring, surveys and surveillance, can help to address the impacts of anthropomorphic changes on the environment. This study sought to understand these connections through the water quality monitoring within watershed basins as well as pathogen surveillance within sewersheds. Specifically, this dissertation sought to 1) understand the sources of pollution and their connections with land use in the various subsections of watersheds; 2) to find a cost-effective way to surveil the spread of SARS-CoV-2 using wastewater surveillance; and 3) to understand the differences in wastewater surveillance between communities. Water quality monitoring using microbial source tracking (MST) was performed with a survey of five mixed-use watersheds in Michigan. Through the use of spatial clustering, it was found that temporal contamination was primarily driven by precipitation and its associated variables (e.g., streamflow, turbidity, overland flow), while spatial contamination is driven by land uses (e.g., septic tank density, tile drain proportions, and tillage). Additionally, porcine fecal contamination was more often correlated with nutrients in streams than either bovine or human contamination. The development of a cost-effective workflow for the detection and quantification of SARS-COV-2 in wastewater was undertaken. Wastewater from communities around Michigan were collected and analyzed along with viral surrogates for SARS-CoV-2 to investigate different workflow options. The Pseudomonas phage Phi6 was seeded in different wastewater matrices to test concentration and recovery by ultrafiltration-based method and polyethylene glycol (PEG) precipitation. The PEG method provided better virus recovery than the ultrafiltration-based methods as measured using RT-ddPCR. The comparison of two communities (A and B) wastewater results for SARS-CoV-2 analyzed against case data was undertaken. These results were significantly correlated with cases in both communites, but the level of correlation differed based on spatial (e.g., zipcode vs county level cases) and temporal (e.g., date of symptom(s) onset vs. the referral date for cases) resolution. Wastewater surveillance was more representative of higher spatial resolution (zipcode data) of cases in both communities. When examining the temporal resolution of the communities, community B’s wastewater results were more closely tied to the onset of symptoms and not the case referral date. The ability to monitor indicators of pollution in watersheds and surveil etiological agents of disease in sewersheds provide non-intrusive methods for evaluating the potential risks and current burdens to community health. The first part of the work could be considered “downstream” monitoring identifying sources and potential exposures with the goal of reducing waterborne disease. While “upstream” monitoring was used for identifying the disease trends in the community and was focused on public health measures to prevent transmission. This project contributed novel methods, results and analysis providing valuable knowledge ultimately addressing the role of monitoring strategies to protect public health.
Show less

Title: Environmental regulation of cyclic di-GMP turnover in vibrio cholerae
Creator: Koestler, Benjamin J.
Date: 2014
Collection: Electronic Theses & Dissertations
Description: 3', 5'-cyclic diguanylic acid (c-di-GMP) is an intracellular bacterial second messenger that mediates the transition between a sessile, biofilm forming lifestyle to a motile, virulent lifestyle. Diguanylate cyclase (DGC) enzymes synthesize c-di-GMP, whereas c-di-GMP specific phosphodiesterase (PDE) enzymes hydrolyze the second messenger. Although numerous bacterial behaviors are regulated by c-di-GMP, the regulatory inputs of this system remain mostly undefined. Here, I examine how the marine...
Show more3', 5'-cyclic diguanylic acid (c-di-GMP) is an intracellular bacterial second messenger that mediates the transition between a sessile, biofilm forming lifestyle to a motile, virulent lifestyle. Diguanylate cyclase (DGC) enzymes synthesize c-di-GMP, whereas c-di-GMP specific phosphodiesterase (PDE) enzymes hydrolyze the second messenger. Although numerous bacterial behaviors are regulated by c-di-GMP, the regulatory inputs of this system remain mostly undefined. Here, I examine how the marine bacterium and human pathogen Vibrio cholerae utilizes c-di-GMP signaling to interpret and respond to environmental cues. The central hypothesis that underpins my research is that V. cholerae senses environmental signals with DGCs and PDEs to modulate c-di-GMP concentrations in different environments. As c-di-GMP is a widely conserved second messenger utilized by many different bacteria, the mechanisms by which V. cholerae utilizes c-di-GMP in different environments can be applied to other bacterial systems to further comprehend how they behave in and adapt to various surroundings. To examine the influence of environmental factors on c-di-GMP synthesis and hydrolysis, I have developed a novel method named The Ex vivo Lysate c-di-GMP Assay (TELCA) that systematically measures total DGC and PDE cellular activity. I have shown that V. cholerae grown in different environments exhibits distinct intracellular levels of c-di-GMP, and using TELCA have determined that these differences correspond to changes in both c-di-GMP synthesis and hydrolysis. These findings highlight that modulation of both total DGC and PDE activity alters the intracellular concentration c-di-GMP.While searching for specific environmental cues that regulate c-di-GMP, I have found that bile acids, a prevalent constituent of the human proximal small intestine, increase intracellular c-di-GMP in V. cholerae. This bile-mediated increase in c-di-GMP is quenched by bicarbonate, the intestinal pH buffer secreted by intestinal epithelial cells. These findings lead me to propose that V. cholerae senses distinct microenvironments within the small intestine, using bile and bicarbonate as chemical cues, and responds by modulating intracellular c-di-GMP.In addition to its function as a bacterial second messenger, c-di-GMP has potent immunostimulatory properties in eukaryotes; these properties make c-di-GMP a prime candidate for use as a vaccine adjuvant. Here, I present a novel method of delivering c-di-GMP into eukaryotic cells using adenovirus. I have demonstrated that c-di-GMP can be synthesized in vivo by delivering DGC DNA into the cell, and that this c-di-GMP increases the secretion of numerous cytokines and chemokines. This novel adenovirus c-di-GMP delivery system offers a more efficient and cost-effective method to administer c-di-GMP as an adjuvant to stimulate innate immunity.
Show less

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.
Show less

Title: Fate and detection of Bacillus anthracis spores in pasteurized milk, juice and eggs
Creator: Shah, Sandip H.
Date: 2009
Collection: Electronic Theses & Dissertations

Title: Genetic and chemical biology studies of Mycobacterium tuberculosis pH-driven adaptation
Creator: Dechow, Shelby J.
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Mycobacterium tuberculosis (Mtb) endures robust immune responses by sensing and adapting to its host environment. One of the first cues the bacterium encounters during infection is acidic pH, a characteristic of its host niche – the macrophage. Targeting the ability of Mtb to sense and adapt to acidic pH has the potential to reduce survival of Mtb in macrophages. A high throughput screen of a >220,000 compound small molecule library was conducted to discover chemical probes that inhibit Mtb...
Show moreMycobacterium tuberculosis (Mtb) endures robust immune responses by sensing and adapting to its host environment. One of the first cues the bacterium encounters during infection is acidic pH, a characteristic of its host niche – the macrophage. Targeting the ability of Mtb to sense and adapt to acidic pH has the potential to reduce survival of Mtb in macrophages. A high throughput screen of a >220,000 compound small molecule library was conducted to discover chemical probes that inhibit Mtb growth at acidic pH. From this screen, AC2P20 was identified as a chemical probes that kills Mtb at pH 5.7 but is inactive at pH 7.0. Through a combination of transcriptional profiling, mass spectrometry, and free thiol abundance and redox assays, I show that AC2P20 likely functions by depleting intracellular thiol pools and dysregulating redox homeostasis. Findings from this study have helped define new pathways involved in Mtb’s response to acidic pH using a chemical genetic approach.Upon sensing acid stress, Mtb can adapt accordingly by entering a nonreplicating persistent state, resulting in increased tolerance to host immune pressures and antibiotics. During growth in vitro, when given glycerol as a sole carbon source, Mtb responds to acidic pH by arresting its growth and entering a metabolically active state of nonreplicating persistence, a physiology known as acid growth arrest. To answer how Mtb regulates and responds to acidic pH, I performed genetic selections to identify Mtb mutants defective in acid growth arrest. These selections identified enhanced acidic growth (eag) mutants which all mapped to the proline-proline-glutamate ppe51 gene and resulted in distinct amino acid substitutions: S211R, E215K, and A228D. I demonstrated that expression of the PPE51 variants in Mtb promotes significantly enhanced growth at acidic pH showing that the mutant alleles are sufficient to cause the dominant gain-of-function, eag phenotype. Furthermore, I performed single carbon source experiments and radiolabeling experiments showing that PPE51 variants preferentially uptake glycerol at an enhanced rate, suggesting a role in glycerol acquisition. Notably, the eag phenotype is deleterious for growth in macrophages, where the mutants have selectively faster replication but reduced virulence in activated macrophages as compared to resting macrophages. This supports that acid growth arrest is a genetically controlled, adaptive process that could act as a potential targetable physiology in future TB therapeutics. My work with the carbonic anhydrase inhibitor, ethoxzolamide, sought to combine genetic and chemical biology to better understand pH-adaptation in Mtb. Ethoxzolamide is a potent inhibitor of Mtb carbonic anhydrase activity and the PhoPR regulon, suggesting a previously unknown link between carbon dioxide and pH-sensing. We hypothesized that the production of protons from carbonic anhydrase activity could be modulating PhoPR signaling. Mtb has three carbonic anhydrases (CanA, CanB, and CanC), and by using CRISPRi and gene knockout, I show that CanB is required for pathogenesis in macrophages, but I did not observe a function in controlling PhoPR signaling. However, transcriptional profiling at different pH and CO2 concentrations show that PhoPR is induced by high CO2 and also revealed a core subset of CO2 responsive genes independent of PhoPR or acidic pH regulation. Overall, these studies defined new functions for thiol- and redox-homeostasis, glycerol uptake, and CO2-concentration in regulating Mtb adaptation to acidic environments and provide new targets for the development of acidic pH-dependent therapeutics.
Show less

Title: Genetic diversity of clinical and bovine non-o157 shiga toxin-producing escherichia coli (stec
Creator: Blankenship, Heather Marie
Date: 2019
Collection: Electronic Theses & Dissertations
Description: Shiga toxin-producing Escherichia coli (STEC) is a leading cause of foodborne infection resulting in 265,000 illnesses and more than 3,600 hospitalizations annually. Since its identification in 1982 associated with an outbreak of haemorrhagic colitis, serotype O157:H7 has been the primary focus of research and surveillance. However, the increasing incidence of other serogroups, or non-O157 STEC, that are associated with clinical illness has since surpassed the incidence of O157 and has raised...
Show moreShiga toxin-producing Escherichia coli (STEC) is a leading cause of foodborne infection resulting in 265,000 illnesses and more than 3,600 hospitalizations annually. Since its identification in 1982 associated with an outbreak of haemorrhagic colitis, serotype O157:H7 has been the primary focus of research and surveillance. However, the increasing incidence of other serogroups, or non-O157 STEC, that are associated with clinical illness has since surpassed the incidence of O157 and has raised questions about the genetic diversity of this pathogen population. Six serogroups, O26, O45, O103, O111, O121, and O145, have been denoted as "big six" non-O157 STEC serogroups since they are frequently associated with clinical outcomes.In this dissertation, 895 non-O157 STEC isolates recovered from patients in Michigan between 2001-2018 were analyzed using whole genome sequencing (WGS) to identify virulence gene profiles and apply new typing methods to better discriminate closely related strains. The recovery of a wide range of serogroups from cases presenting with symptoms ranging from mild diarrhea to hemorrhagic colitis, indicates that genetic diversity and variation may have an impact on disease outcomes. The number and richness of serogroups identified over the past 18 years has been steadily increasing and serogroup alone lacks the discriminatory capabilities to classify related isolates. Indeed, strains representing the same sequence types (ST) were often found to be unrelated by serogroup. Notably, some serogroups, STs, virulence gene profiles and alleles were associated with clinical outcomes and patient demographics. Contrast to national surveillance, cases between 11 and 29 years of age had the highest frequency of STEC infections in Michigan.Additionally, a subset of 44 non-O157 STEC recovered from Michigan patients between 2000 and 2006 were examined more comprehensively while making comparisons to 114 clinical STEC isolates from Connecticut to examine the impact of geographic location on risk factors for non-O157 STEC infections. Lastly, a subset of STEC isolates associated with outbreaks in Michigan were examined to identify the impact of WGS on identification of strain relatedness for surveillance compared to pulsed-field gel electrophoresis.While most of the work outlined in this dissertation focused on characterizing clinical non-O157 STEC isolates, a comparative analysis of cattle isolates was also performed since cattle are an important reservoir of STEC. Indeed, numerous outbreaks and illnesses have been traced back to contaminated cattle-based food products or fecal contamination of water and crops. The ability of STEC to persist in the cattle reservoir and farm environment may give rise to more pathogenic strains due to the accumulation of horizontally acquired genes. 66 STEC isolates recovered from a beef herd over four samplings were examined to identify the genetic diversity within the cattle population and longitudinal persistence. The ability of a strain to form a strong biofilm was associated with the ability to persist and be recovered at multiple sampling phases from the same animal. Further, to better understand the genetic diversity of STEC recovered from the cattle reservoir, an additional 12 STEC isolates from three bovine herds (n = 78) and 241 clinical O157 STEC isolates (n = 1,135) were included to identify shared profiles. The similarity in serogroups and virulence gene profiles warrant a continued surveillance of the cattle environment to better understand crossover events and the ability of strains to evolve into new virulent STEC lineages. The work described in this dissertation helped to elucidate the genetic characteristics important for clinical outcomes and identified targets for future surveillance to better understand lineages that may be important for disease.
Show less

Title: Harnessing peat-based gnotobiotic plant growth to characterize microbiota-mediated immunocompetence in Arabidopsis
Creator: Paasch, Bradley Carlton
Date: 2021
Collection: Electronic Theses & Dissertations
Description: The increasingly evident involvement of microbes in basic host function has led to a more holistic perspective of plants to be considered. Here, plants and their associated microbiotas interact with each other as holobionts in performing various biological functions in natural ecosystems and crop fields as a single ecological unit. The extent to which microbial components of the holobiont contribute to host health, however, is not fully understood, especially in natural environments. Here, I...
Show moreThe increasingly evident involvement of microbes in basic host function has led to a more holistic perspective of plants to be considered. Here, plants and their associated microbiotas interact with each other as holobionts in performing various biological functions in natural ecosystems and crop fields as a single ecological unit. The extent to which microbial components of the holobiont contribute to host health, however, is not fully understood, especially in natural environments. Here, I applied the use of peat-based gnotobiotic growth systems to generate plants grown with and without exposure to microbiota to characterize the role of microbiota on the development of plant immunocompetence.In my first chapter, I review the current understanding on the interplay between microbiota and plant health and immunity. I begin with background on microbial detection and response in plants. I then discuss plant-associated microbiota and provide examples of how perturbation to microbiota homeostasis, such as during dysbiosis for example, can be associated with positive and negative impacts on host health. Host factors regulating microbiota homeostasis in plants are discussed. Lastly, I describe tools and approaches that can be used to the study of plant-microbiota interactions and highlight recent findings involving the modulation of plant immune responses by plant microbiotas. In my second chapter, I highlight the contributions I made to the improvement of two peat-based gnotobiotic plant growth systems for plant microbiome research recently developed in Dr. Sheng Yang He’s laboratory: the FlowPot and GnotoPot systems. I adapted the FlowPot system to use a field soil, highlighting its versatility, and optimized several abiotic conditions associated with plant growth in GnotoPots. Additionally, I used 16S rRNA gene amplicon sequencing to characterize the colonization of a natural, soil-derived microbial community and several preparations of a synthetic bacterial community, demonstrating the use of GnotoPots for colonization studies. We expect both systems to be useful tools for the research community to address a wide variety of questions related to plant-microbiota interactions. In my third chapter, I implement the optimizations made to peat-based gnotobiotic growth systems described in the previous chapter to characterize the role of basal microbiota colonization on plant immunocompetence. I found that compared to plants colonized by a soil-derived microbiota, axenic plants grown without exposure to a microbiota lacked robust age-dependent immunity. Axenic plants were defective in several aspects of pattern-triggered immunity including flg22-induced production of reactive oxygen species, signaling through MAPK pathways, and induction of defense-related genes and hypersusceptible to disease a bacterial foliar pathogen. Additionally, I found that a synthetic microbiota composed of culturable leaf endosphere bacteria was able to restore immunocompetence similar to plants inoculated with a soil-derived community in a growth substrate-dependent manner. These results demonstrate a role of microbiota in immunocompetence and age-dependent immunity, which was previously thought to be an intrinsic trait of plants.
Show less

Title: IMPACT OF AGRICULTURAL MANAGEMENT AND MICROBIAL INOCULATION ON SOYBEAN (GLYCINE MAX) AND ITS ASSOCIATED MICROBIOME
Creator: Longley, Reid
Date: 2022
Collection: Electronic Theses & Dissertations
Description: Soybean (Glycine max) is a globally important crop with uses as food, cooking oil livestock feed, and biodiesel. Soybean can be considered holobionts because they host diverse microbiomes which extend plant genotypes and phenotypes through various microbial functions such as nitrogen fixation and increased disease resistance. My research focused on assessing the impact of three agricultural management strategies on the soybean holobiont. Soybean cropping systems can be managed using various...
Show moreSoybean (Glycine max) is a globally important crop with uses as food, cooking oil livestock feed, and biodiesel. Soybean can be considered holobionts because they host diverse microbiomes which extend plant genotypes and phenotypes through various microbial functions such as nitrogen fixation and increased disease resistance. My research focused on assessing the impact of three agricultural management strategies on the soybean holobiont. Soybean cropping systems can be managed using various strategies, including conventional tillage, no-till, and organic management regimes. These management systems have been shown to impact the microbiomes of soybean-associated soils, however, their impacts on plant-associated microbiomes are still not well understood. In this study, I assessed the impact of conventional, no-till, and organic management treatments on soybean microbiomes at Michigan State’s Kellogg Biological Station Long-Term Ecological Research site (KBS LTER). I found that management impacted microbiome composition and diversity in soil, roots, stems, and leaves and that this impact persisted throughout the season. Additionally, when comparing the same soybean genotype grown in conventional and no-till management systems, tillage regime impacted the microbiome throughout the plant and the growing season. This effect impacted microbial taxa which are likely to be plant beneficial, including nitrogen fixing Bradyrhizobium. Another important management tool that is expected to impact plant-associated microbial communities is the application of foliar fungicides. While fungicides are known to protect plants from particular fungal pathogens, non-target impacts of fungicides on crop microbiomes, and the impact of management on microbiome recovery are not well understood. To address this knowledge gap, I assessed the impact of foliar fungicide application on the maize (Headline® fungicide, 2017) and soybean (Delaro® fungicide, 2018) microbiomes in conventional and no-till plots at the KBS LTER. I found that fungicide applications have a non-target impact on Tremellomycete yeasts in the phyllosphere and this impact was greater in soybean than maize. Co-occurrence network analysis and random forest modelling indicated that changes in fungal communities may lead to indirect impacts on prokaryotic communities in the phyllosphere. Importantly, this work demonstrated that phyllosphere communities of soybeans under no-till management had greater recovery from fungicide disturbance. This novel finding exemplifies how tillage regime can impact phyllosphere microbiomes and their responses to disturbance. Microbial inoculants in agriculture have long been used for biocontrol of pathogens, but there is also interest in their use to dampen the impacts of abiotic stress including drought. In this study, I tested whether inoculating soybeans with hub taxa identified through network analysis from no-till soybean root microbiome data from the KBS LTER could provide protection against water limitation. Soybean seedlings were enriched in consortia of hub bacteria and fungi and were grown in no-till field soil. Seedlings were then exposed to low-moisture stress, and plant phenotypes, plant gene expression, and amplicon sequencing of microbial DNA and cDNA were assessed throughout the stress period. Inoculation increased plant growth, nodule numbers, and led to increased expression of nodulation-associated genes. 16S sequencing of cDNA revealed higher levels Bradyrhizobium in inoculated samples. These results indicate that inoculation with hub microbes can benefit soybean plants, possibly through interaction with other microbes, interaction with the plant, or both. In summary, fungicide, tillage, and inoculation all impact the soybean microbiome, indicating that management choices impact the entire holobiont.
Show less

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.
Show less

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.
Show less

Title: Identifying the underlying mechanisms of Marek's disease vaccine synergy
Creator: Umthong, Supawadee
Date: 2019
Collection: Electronic Theses & Dissertations
Description: Marek’s disease virus (MDV; Gallid herpesvirus 2, aka, serotype 1) is a ubiquitous and highly oncogenic α-herpesvirus that causes Marek’s disease (MD), a lymphoproliferative disorder affecting chickens with estimated annual costs to the poultry industry of ~$2 billion worldwide. Since 1970, MD has been largely controlled through widespread vaccination. While MD vaccines are very successful in preventing tumors, they do not prevent viral replication and spread. As a consequence, new and more...
Show moreMarek’s disease virus (MDV; Gallid herpesvirus 2, aka, serotype 1) is a ubiquitous and highly oncogenic α-herpesvirus that causes Marek’s disease (MD), a lymphoproliferative disorder affecting chickens with estimated annual costs to the poultry industry of ~$2 billion worldwide. Since 1970, MD has been largely controlled through widespread vaccination. While MD vaccines are very successful in preventing tumors, they do not prevent viral replication and spread. As a consequence, new and more virulent MDV strains have repeatedly emerged in vaccinated flocks. Thus, there is a need to understand how MD vaccines work in order to design future vaccines that are more protective, especially against more virulent MDVs. One promising insight for vaccine development is based upon protective synergism, a phenomenon where two vaccines when combined provide greater protection compared to either original vaccine when administered alone as a monovalent vaccine. The mechanism that underlines the synergistic effect between SB-1 (a Gallid herpesvirus 3, aka, serotype 2 strain) and HVT (herpesvirus of turkey, aka, Meleagrid herpesvirus 1 or serotype 3), two of the most widely used MD vaccines, has never been investigated, and thus, provides a highly relevant and useful model to explore. To investigate the mechanisms of protective synergy of SB-1 and HVT, we used three approaches. First, we investigated how monovalent SB-1 or HVT replicates when they were alone in the host or together as a bivalent vaccine. We observed that the replication patterns of SB-1 and HVT were different with respect to time after administration into the bird and the organs that they were found to replicate in regardless if the other vaccine were present. Based on the observation that HVT replicated primarily early in the bursa, we found that this organ was necessary for protection using both HVT and bivalent HVT + SB-1 vaccines. Second, we measured the effects of CD8 T cells in monovalent SB-1, HVT, and bivalent SB-1+HVT vaccine treatment. Specifically, we reduced CD8 T cells to see their effect of CD8 T cells on MD incidence and vaccinal protection by injecting the chickens with a monoclonal antibody directed against chicken CD8 T cells. In this study, we found that CD8 T cells were necessary for protection induced by vaccines. Third, we identified the cytokine profiles induced by SB-1, HVT, and the bivalent vaccine to see if cytokine synergy could be one of the mechanisms to explain protective synergy. We found that SB-1 induced an innate anti-viral response typified by IFN-α, IFN-β, IL-1β, T-cell proliferation cytokine IL-21, and Th2 cytokine IL-5, while HVT suppressed TGF-β3 and TGF-β4. The early stimulation of IL-1β and IL-21 (IFN-γ-promoting cytokines) at 4 days post vaccination (DPV) by SB-1 combined with the suppression of TGF-β (IFN-γ- suppressing cytokine) at 1 day post challenge (DPC) by HVT could result in the strong induction of IFN-γ found in the bivalent vaccine at 10 DPC. The induction of IFN-γ supports the synergistic effect of cytokines by a cooperative action mechanism where multiple cytokines work together to enhance the signal. Based on these findings, we propose a model to explain bivalent SB-1 and HVT vaccine synergy, which combines the replication of vaccines, T cell response to vaccinations, and cytokine synergy between SB-1 and HVT vaccine. Our proposed mechanism provides insights on how to generate rationally designed MD vaccines.
Show less

Title: Interactions Between Plasma and Material Surfaces for Sterilization and Impurity Adsorption
Creator: Mackinder, Madeline Ann
Date: 2022
Collection: Electronic Theses & Dissertations
Description: As the worldwide population increases, maintaining a standard of public health becomes more critical. Two major concerns in this area are nosocomial infections (NI) and water contamination. Current processes in sterilization and water treatment have limitations that could be overcome using plasma techniques. The unique characteristics of plasma make it a promising alternative for energy-intensive processes. This work investigated the characteristics of plasma that have the greatest impact on...
Show moreAs the worldwide population increases, maintaining a standard of public health becomes more critical. Two major concerns in this area are nosocomial infections (NI) and water contamination. Current processes in sterilization and water treatment have limitations that could be overcome using plasma techniques. The unique characteristics of plasma make it a promising alternative for energy-intensive processes. This work investigated the characteristics of plasma that have the greatest impact on sterilization and the reactivation of activated carbon. Previous studies have researched the physical and chemical surface properties of biochar but have not been able to establish an efficient process to activate biochar with desired characteristics. Plasma treatment would offer a way to etch the surface of biochar and specifically functionalize the surface. Successfully activating biochar would increase its adsorption ability and enable its use for water treatment. This project aims to harness these plasma properties and use plasmas to address three important topics related to public health: sterilization of surfaces, modulating commercial activated carbon (AC), and activations of biochar.Cold plasma sterilization offers an efficient way to sterilize medical components and instruments without the risk of deformation to heat-sensitive materials. This paper reports the use of magnetized plasma to realize low-temperature sterilization. A radio frequency dielectric barrier discharge was created in a quartz tube using a mixture of argon and oxygen gas. Glass slides inoculated with a uniform amount of Escherichia coli were exposed to the plasma afterglow at different pressures with and without a magnetic field. A global model was developed to evaluate the magnetically enhanced dielectric barrier discharges and predict species densities. Optical emission spectroscopy identified the plasma species present and validated the model. The magnetic field significantly promoted the intensity of the plasma and the sterilization efficiency. A process gas pressure of 100 mTorr presented the most effective treatment with a sterilization time less than one minute and sample temperature below 32 °C. The effects of O2 plasma on the adsorption capacity of activated carbon (AC) was investigated by varying treatment times. Transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR), and Zeta potential were used to characterize the surface properties of the AC. The carbon was then applied to remove methylene blue (MB) from an aqueous solution. The adsorption kinetics and isotherm were also studied. Results showed that pseudo-second-order kinetics was the most suitable model for describing the adsorption of MB onto AC. Equilibrium data were well fitted to the Freundlich and Langmuir isotherm models. The highest adsorption capacity resulted from 4 minutes of O2 plasma treatment. This work shows that activation of AC by plasma can open the micropore and increase the effectiveness of chemical removal.Biochar was activated using a combination of O2 plasma and KOH. The adsorption capacity was investigated for different O2 plasma treatment times, KOH concentrations, and treatment temperatures. The adsorption capacity of methylene blue (MB) by the plasma activated biochar was evaluated. The adsorption kinetics and isotherm were also investigated. Results showed that pseudo-second-order kinetics was the most suitable model for describing the adsorption of MB onto biochar. Both the Freundlich and Langmuir isotherm models fit the equilibrium data. The highest adsorption capacity resulted from 10\% KOH + 300 °C for 5 minutes. This work shows that activation of biochar by plasma can improve adsorption capacity. The plasma treated AC and the plasma activated biochar were applied to the removal of PFOA. It was demonstrated that plasma treatment can improve PFOA adsorption. The negative surface charge was shown to negatively impact PFOA adsorption which aligns with the hypothesis that PFOA would preferentially adsorb onto more positive surfaces due to its anionic state in water.
Show less

Title: LISTERIA MONOCYTOGENES INFECTION ALTERS TROPHOBLAST EXTRACELLULAR VESICLES
Creator: Kaletka, Jonathan Matthew
Date: 2022
Collection: Electronic Theses & Dissertations
Description: Listeria monocytogenes (Lm) is a bacterial pathogen that utilizes an intracellular lifecycle to spread throughout the body, including the placenta in pregnant individuals. Placental infection and disease can lead to negative fetal outcomes including spontaneous abortion, birth defects, and stillbirths. Extracellular vesicles (EVs) are tiny particles secreted by nearly every cell type in the body and serve as a cellular signaling mechanism. EVs have been implicated in many cellular functions...
Show moreListeria monocytogenes (Lm) is a bacterial pathogen that utilizes an intracellular lifecycle to spread throughout the body, including the placenta in pregnant individuals. Placental infection and disease can lead to negative fetal outcomes including spontaneous abortion, birth defects, and stillbirths. Extracellular vesicles (EVs) are tiny particles secreted by nearly every cell type in the body and serve as a cellular signaling mechanism. EVs have been implicated in many cellular functions and diseases throughout the body, including those involving the placenta. Placental EVs can have immunomodulatory effects, but during placental disease they can also act in a pro-inflammatory manner, leading to disease progression. EVs can also be proinflammatory during intracellular bacterial infection, where they can communicate the infection and coordinate an immune response. In this dissertation, I investigated how Lm infection of trophoblasts alters the EVs produced by the infected cells, and how they can activate an immune response. Chapter 1 of the dissertation details the current literature on the role that EVs play during bacterial infections and placental development and disease. Chapter 2 focuses on establishing a trophoblast stem cell model (TSC) to study placental infections. TSCs are the source of trophoblasts in the placenta, and cultivation of these cells allow for the continual study of placental disease. Here, I found that TSCs are susceptible to Lm infection, although it requires a higher bacterial load and longer time course compared to other cell types. This chapter details ways to model placenta-pathogen interactions in vitro, allowing for the study of these interactions in a laboratory setting. Chapter 3 investigated how Lm infection of TSCs altered the cargo of the tEVs produced. Previous studies into EVs from infected cells found components from the bacterial cells loaded into the EVs, including bacterial DNA, RNA, and proteins. We found many more unique proteins in the tEVs from infected cells. The infection tEVs had a substantial increase in the number of peptides identified of ribosomal, histone, and tubulin proteins, among others. Gene ontology (GO) analysis showed that the proteins seen in the tEVs from infected TSCs primarily belonged to RNA-binding pathways. This result piqued our curiosity as to if Lm infection also changed the RNA loaded into the tEVs. We performed RNA sequencing to determine the host RNA profiles found in the tEVs. We found different RNA profiles in the tEVs from uninfected and Lm-infected cells. GO analysis on the mRNAs overrepresented in the infection tEVs found that they represent genes from vasculogenesis and placental development pathways. Our results in this chapter show that Lm infection can alter the production and contents of tEVs from TSCs. Chapter 4 of this dissertation aimed to determine how tEVs from Lm-infected TSCs affect immune cells. We found that macrophages treated with infection tEVs produced TNF-α, a pro-inflammatory cytokine. Surprisingly, when we subsequently infected tEV treated cells with Lm, some of the cells became more susceptible to Lm infection. Similar results were seen with treatment with macrophage EVs, where infection EVs made the macrophages susceptible to Lm infection. The work in this chapter suggests that tEVs from Lm-infected TSCs can indeed induce a pro-inflammatory response in macrophages, although this makes the cells more susceptible to infection. Overall, the work presented here explores potential mechanisms as to how the placenta communicates bacterial infections.
Show less

Title: LISTERIA MONOCYTOGENES PLACENTAL COLONZATION AND CONSEQUENCES FOR PREGNANCY OUTCOME
Creator: Conner, Kayla Nicole
Date: 2022
Collection: Electronic Theses & Dissertations
Description: Listeria monocytogenes (Lm) is a Gram-positive bacterium that causes the severe food-borne disease listeriosis. Listeriosis is particularly problematic in pregnant women as Lm colonizes the placenta, resulting in adverse pregnancy outcomes including stillbirth, miscarriage, and preterm labor. Despite numerous studies of placental listeriosis (PL) in various animal models, the mechanisms driving adverse outcomes following PL are largely uncharacterized. This dissertation addresses some of the...
Show moreListeria monocytogenes (Lm) is a Gram-positive bacterium that causes the severe food-borne disease listeriosis. Listeriosis is particularly problematic in pregnant women as Lm colonizes the placenta, resulting in adverse pregnancy outcomes including stillbirth, miscarriage, and preterm labor. Despite numerous studies of placental listeriosis (PL) in various animal models, the mechanisms driving adverse outcomes following PL are largely uncharacterized. This dissertation addresses some of the field’s knowledge gaps by analyzing the changes in placental gene expression and metabolism following infection with Lm and by characterizing a key Lm virulence factor, Internalin P (InlP), which plays a significant role in Lm placental colonization. Chapter 1 gives pertinent background information on the placenta, Lm, and PL and broadly addresses the knowledge gaps to be addressed by the rest of the dissertation. Chapter 2 describes an in vivo study of PL in mice. Infected and control placentas were analyzed for differences in gene expression profiles between the two groups. We identified an enrichment of genes associated with eicosanoid biosynthesis, suggesting perturbations in eicosanoid metabolism in infected tissues. By quantifying placental eicosanoid concentrations through mass spectrometry, we found a significant increase in the concentrations of several eicosanoids with known roles in inflammation and/or labor. This study provides a likely explanation for temporal disruptions of labor following placental infection. Chapters 3 and 4 discuss two studies of the Lm virulence factor InlP, a key player in placental colonization. InlP contributes to Lm’s placental pathogenesis likely by conferring the ability of Lm to transcytose through placental layers. Prior studies reported that no homologs of InlP exist in Listeria species other than Lm. Chapter 3 describes our discovery that at least two other Listeria species, L. ivanovii and L. seeligeri, encode InlP homologs. We characterized the domain architectures and genomic neighborhoods of these homologs and speculated on their implications for Listeria evolution. In chapter 4, I continue discussion of InlP and describe our identification and preliminary characterization of naturally occurring InlP variants. In this study, we used a bioinformatics approach to analyze Lm whole genome sequences (WGS) and identify InlP variants. We uncovered two InlP variants of interest in the Lm population. The first results from a start codon point mutation in the inlP gene, likely resulting in a truncated and potentially nonfunctional InlP protein product. The second is an InlP variant with a PRO to SER substitution in the InlP calcium binding loop, which is hypothesized to play a role in InlP activation or stabilization. These results provide two avenues for further investigation of InlP regulation and function and suggest the potential for InlP-dependent variation in placental colonization potential across Lm isolates. In chapter 5, I summarize this dissertation. This chapter reflects on the results, implications, and challenges of each study outlined in the prior chapters. I discuss the unique challenges faced due to the ongoing COVID-19 pandemic and its effects on my graduate training. Finally, I share concluding remarks and propose future directions for this project and the field of PL. Together, the chapters of this dissertation describe novel findings that contribute to the field by assessing genetic and metabolic changes to the placenta due to listeriosis and further characterizing a known key placental virulence factor.
Show less

Title: MEMBRANE-LOCALIZED TRANSCRIPTION REGULATORS : UNDERSTANDING POST-TRANSLATIONAL REGULATION AND SINGLE-MOLECULE DYNAMICS OF TCPP IN VIBRIO CHOLERAE
Creator: Demey, Lucas Maurice
Date: 2022
Collection: Electronic Theses & Dissertations
Description: Vibrio cholerae is a Gram-negative gastrointestinal pathogen that has evolved an elegant regulatory system to precisely time production of essential virulence factors. A key step in this regulatory system is the transcription of a soluble AraC-like transcription factor, ToxT. ToxR and TcpP, two membrane-localized transcription regulators (MLTRs), positively regulate toxT. Much work has contributed to our understanding of TcpP and ToxR regulation, yet major gaps remain in our knowledge of...
Show moreVibrio cholerae is a Gram-negative gastrointestinal pathogen that has evolved an elegant regulatory system to precisely time production of essential virulence factors. A key step in this regulatory system is the transcription of a soluble AraC-like transcription factor, ToxT. ToxR and TcpP, two membrane-localized transcription regulators (MLTRs), positively regulate toxT. Much work has contributed to our understanding of TcpP and ToxR regulation, yet major gaps remain in our knowledge of these MLTRs. MLTRs are unique one-component signal transduction systems because they respond to extracellular stimuli by influencing gene transcription from their location in the cytoplasmic membrane. In Chapter 2, I explore the prevalence and diversity of MLTRs within prokaryotes to enhance our understanding of TcpP and ToxR. I show that MLTRs are far more common among prokaryotes than previously anticipated and that MLTRs are an understudied class of transcription regulators. In Chapter 3, I describe the use of super-resolution single-molecule tracking to investigate how TcpP, a model MLTR, identifies the toxT promoter. I provide evidence that TcpP binds to the toxT promoter independent of ToxR, and TcpP transitions to a specific diffusion state. The data support the first biophysical model for how TcpP-like MLTRs locate their target promoters. TcpP is subject to a form of post-translational regulation known as regulated intramembrane proteolysis (RIP). RIP of TcpP results in its complete inactivation, resulting in loss of virulence factor production. TcpH inhibits RIP of TcpP under certain pH and temperature conditions. In Chapter 4, I describe the mechanism TcpH employs to inhibit TcpP RIP while V. cholerae is present in the mouse gastrointestinal tract. I demonstrate that the dietary fatty acid α-linolenic acid enhances inhibition. I also show that α-linolenic acid promotes TcpH-mediated inhibition of TcpP RIP by increasing association of both proteins with detergent-resistant membrane (DRM) domains. My work provides the first evidence that DRMs influence virulence factor transcription in V. cholerae and that a dietary fatty acid promotes V. cholerae pathogenesis.
Show less

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.
Show less

MSU Libraries

Digital Repository

Enabled Filters

Pages

Pages

MSU Libraries

Digital Repository

You are here

Search results

Pages

Pages