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Title: Molecular epidemiology, pangenomic diversity, and comparative genomics of Campylobacter jejuni
Creator: Rodrigues, Jose Alexandre
Date: 2022
Collection: Electronic Theses & Dissertations
Description: Campylobacter jejuni, the leading cause of bacterial gastroenteritis in the United States, is often resistant to commonly used antibiotics and has been classified as a serious threat to public health. Through this work, we sought to evaluate infection trends, quantify resistance frequencies, identify epidemiological factors associated with infection, and use whole-genome sequencing (WGS) as well as comparative phylogenomic and pangenomic approaches to understand circulating C. jejuni...
Show moreCampylobacter jejuni, the leading cause of bacterial gastroenteritis in the United States, is often resistant to commonly used antibiotics and has been classified as a serious threat to public health. Through this work, we sought to evaluate infection trends, quantify resistance frequencies, identify epidemiological factors associated with infection, and use whole-genome sequencing (WGS) as well as comparative phylogenomic and pangenomic approaches to understand circulating C. jejuni populations in Michigan. C. jejuni isolates (n=214) were collected from patients via an active surveillance system at four metropolitan hospitals in Michigan between 2011 and 2014. Among the 214 C. jejuni isolates, 135 (63.1%) were resistant to at least one antibiotic. Resistance was observed for all nine antibiotics tested yielding 11 distinct resistance phenotypes. Tetracycline resistance predominated (n=120; 56.1%) followed by resistance to ciprofloxacin (n= 49; 22.9%), which increased from 15.6% in 2011 to 25.0% in 2014. Notably, patients with ciprofloxacin resistant infections were more likely to report traveling in the past month (Odds Ratio (OR): 3.0; 95% confidence interval (CI): 1.37, 6.68) and international travel (OR: 9.8; 95% CI: 3.69, 26.09). To further characterize these strains, we used WGS to examine the pangenome and investigate the genomic epidemiology of this set of C. jejuni strains recovered from Michigan patients. Among the 214 strains evaluated, 83 unique multilocus sequence types (STs) were identified that were classified as belonging to 19 previously defined clonal complexes (CCs). Core-gene phylogenetic reconstruction based on 615 genes identified three clades, with Clade I comprising six subclades (IA-IF) and predominating (83.2%) among the strains. Because specific cattle-associated STs, such as ST-982, predominated among strains from Michigan patients, we also examined a collection of 72 C. jejuni strains from cattle recovered during an overlapping time period by WGS. Several phylogenetic analyses demonstrated that most cattle strains clustered separately within the phylogeny, but a subset clustered together with human strains. Hence, we used high quality single nucleotide polymorphism (hqSNP) profiling to more comprehensively examine those cattle and human strains that clustered together to evaluate the likelihood of interspecies transmission. Notably, this method distinguished highly related strains and identified clusters comprising strains from both humans and cattle. For instance, 88 SNPs separated a cattle and human strain that were previously classified as ST-8, while the human and cattle derived ST-982 strains differed by >200 SNP differences. These findings demonstrate that highly similar strains were circulating among Michigan patients and cattle during the same time period and highlight the potential for interspecies transmission and diversification within each host. In all, the data presented illustrate that WGS and pangenomic analyses are important tools for enhancing our understanding of the distribution, dissemination, and evolution of specific pathogen populations. Combined with more traditional phenotypic and genotypic approaches, these tools can guide the development of public health prevention and mitigation strategies for C. jejuni and other foodborne pathogens.
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Title: Characterization of Broadly Conserved AvcID Toxin-Antitoxin System and Its Mechanism to Inhibit Phage by Disrupting Nucleotide Metabolism
Creator: Hsueh, Brian Yifei
Date: 2022
Collection: Electronic Theses & Dissertations
Description: ABSTRACTCHARACTERIZATION OF BROADLY CONSERVED AVCID TOXIN-ANTITOXIN SYSTEM AND ITS MECHANISM TO INHIBIT PHAGE BY DISRUPTING NUCLEOTIDE METABOLISMByBrian Yifei HsuehThe prevalence of antiphage defense systems, which have recently been shown to be located on mobile genetic elements in bacteria, have sparked interest to understand the coevolutionary arms race of bacteria and bacteriophage (phage). Bacteria and phages have coexisted for billions of years, and phages are widely distributed in...
Show moreABSTRACTCHARACTERIZATION OF BROADLY CONSERVED AVCID TOXIN-ANTITOXIN SYSTEM AND ITS MECHANISM TO INHIBIT PHAGE BY DISRUPTING NUCLEOTIDE METABOLISMByBrian Yifei HsuehThe prevalence of antiphage defense systems, which have recently been shown to be located on mobile genetic elements in bacteria, have sparked interest to understand the coevolutionary arms race of bacteria and bacteriophage (phage). Bacteria and phages have coexisted for billions of years, and phages are widely distributed in different environmental niches populated by their bacterial hosts, including the human intestine and marine environment. The evolutionary pressure imposed by phages have led bacteria to evolve diverse strategic systems to protect themselves from phage predation, including CRISPR-Cas, restriction-modification, and abortive infection. Recent studies have begun to reveal that toxin-antitoxin (TA) system are associated with antiphage defense systems. Vibrio cholerae El Tor, the causative agent of current cholera pandemics, has acquired two unique genomic islands of unknown origins, known as Vibrio Seventh Pandemic Islands 1 & 2 (VSP-1 & 2). It is hypothesized that the acquisition of VSP islands increase environmental fitness of El Tor. While both islands encode approximately 36 open reading frames, yet many remain largely uncharacterized. In this work, I characterize a novel TA antiphage system encoded on VSP-1 of V. cholerae, here named AvcID. Chapter 2 describes the biological function of AvcD toxin by which it possesses deoxycytidylate deaminase (DCD) activity and produces dUMP as the final product. Further experiments identify the AvcI antitoxin as a small RNA and determine that it post-translationally inhibits the activity of AvcD. Moreover, AvcD consists of two domains—a N-terminal P-loop NTPase and a C-terminal DCD—and mutations in conserved features of each domain abrogate its activity. AvcD is widely conserved across kingdoms, and virtually all bacteria that encode AvcD also have AvcI homologs. Notably, chromosomal AvcID can solely be activated by transcriptional shutoff in V. cholerae, demonstrating that AvcID is a type III TA system. Unlike canonical type III TA systems, in which the toxin is an endoribonuclease, the AvcD toxin is a deaminase. Importantly, the AvcID system provides antiphage defense in Escherichia coli that lacks this system by corrupting nucleotides for phages to utilize to reduce coliphage replication efficiency. In Chapter 3, I explore the activation mechanism of the AvcID system as well as the consequences to phages after encountering AvcID. During infection, virtually all lytic phages induce transcription shutoff of the host by hijacking host transcription machinery to make virion progeny. I uncover that phage-induced transcriptional shutoff leads to turnover of labile AvcI antitoxin and concomitantly activates the deaminase activity of AvcD, leading to a disruption of nucleotide levels. This disruption of nucleotide levels is shown in both susceptible phages (ex. T5) and resistant phages (ex. T7). Through an unknown mechanism, AvcID also increases the abundance of defective phages that are susceptible to AvcID. In summary, this work has made contributions in the field of TA systems and its association with the antiphage defense paradigm by uncovering the biological function and mechanism in response to phage infection.
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Title: DEVELOPMENT OF 3D BIOACTIVE AND ANTIBACTERIAL SILICATE-BASED SCAFFOLDS FOR BONE TISSUE REGENERATION IN LOAD-BEARING APPLICATIONS
Creator: Marsh, Adam Christoph
Date: 2022
Collection: Electronic Theses & Dissertations
Description: Current gold-standard approaches to addressing the needs of bone defects in load-bearing applications entail the use of either autographs or allographs. Both solutions, however, are imperfect as both autographs and allographs carry the risk of additional trauma, threat of disease transmission, and potential donor rejection respectively. Porous 3D scaffolds are attractive alternatives, illuminating a potential path towards achieving the ideal scaffold for targeting bone tissue regeneration in...
Show moreCurrent gold-standard approaches to addressing the needs of bone defects in load-bearing applications entail the use of either autographs or allographs. Both solutions, however, are imperfect as both autographs and allographs carry the risk of additional trauma, threat of disease transmission, and potential donor rejection respectively. Porous 3D scaffolds are attractive alternatives, illuminating a potential path towards achieving the ideal scaffold for targeting bone tissue regeneration in load-bearing applications, usurping autographs to become the new gold-standard. To unlock the full healing potential of 3D scaffolds, such scaffolds must be multifunctional such that (1) their mechanical performance meets the requisite requirements as dictated by the mechanical performance characteristics of interest for native bone tissue, (2) they stimulate the necessary biological responses for bone tissue regeneration, and (3) they exhibit antibacterial characteristics to combat the threat of infection. To date, no reports document 3D scaffolds exhibiting all three performance characteristics. The aim of this dissertation, therefore, is to deliver 3D scaffolds that are mechanically competent, possess and exhibit inherent and advanced antibacterial characteristics, and are successful at providing the needed biological characteristics for bone tissue regeneration. To achieve this, this dissertation implements a multidisciplinary approach, utilizing comprehensive structural characterization across a wide range of scales to elucidate process – performance relationships to execute scientifically driven modifications to engineer and deliver a 3D scaffold to successfully target bone tissue regeneration in load-bearing applications. A silver-doped bioactive glass-ceramic (Ag-BG) composition was selected as the material for scaffold synthesis due to its inherent and attractive antibacterial and biological performance characteristics. Two fundamentally different processing approaches were utilized for synthesizing Ag-BG scaffolds: the polymer foam replication technique and fused filament fabrication (FFF). The Ag-BG scaffolds studied herein were found to exhibit advanced antibacterial performance characteristics against methicillin-resistant Staphylococcus aureus (MRSA), a common pathogen implicated in osteomyelitis development, able to combat MRSA both in planktonic and biofilm forms. Ag-BG scaffolds demonstrated the ability to form an apatite-like layer when immersed in simulated body fluid (SBF), an indicator that Ag-BG scaffolds will induce the necessary mineralization for bone tissue regeneration, in addition to exhibiting attractive cell viability, proliferation, and differentiation characteristics when studied in vitro. The mechanical performance of Ag-BG scaffolds reported herein saw progressive improvements in each iteration of Ag-BG scaffold synthesis, achieving desirable mechanical competency and reliability as a result of the multidisciplinary approach formulated. In addition to the exploration of developing 3D antibacterial and biological silicate-based scaffolds capable of targeting bone tissue regeneration in load-bearing applications, foundational work towards the development of class II hybrid scaffolds comprised of gelatin methacryloyl (GelMA) and Ag-BG for targeting softer tissue regeneration. The novel syntheses applied to the successful molecular coupling of GelMA and Ag-BG presents an attractive class II hydrogel showing great promise as a compatible ink for 3D bioprinting cell-laden scaffolds capable of targeting tissue regeneration of more sophisticated systems.
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Title: CARBON-MEDIATED ECOLOGICAL AND PHYSIOLOGICAL CONTROLS ON NITROGEN CYCLING ACROSS AGRICULTURAL LANDSCAPES
Creator: Curtright, Andrew James
Date: 2022
Collection: Electronic Theses & Dissertations
Description: The sustainable intensification of agriculture relies on the efficient use of ecosystem services, particularly those provided by the microbial community. Managing for these ecosystem services can improve plant yields and reduce off-site impacts. For instance, increasing plant diversity is linked to positive effects on yield, and these beneficial effects are often mediated by the microbial community and the nutrient transformations it carries out. My dissertation has aimed to elucidate the...
Show moreThe sustainable intensification of agriculture relies on the efficient use of ecosystem services, particularly those provided by the microbial community. Managing for these ecosystem services can improve plant yields and reduce off-site impacts. For instance, increasing plant diversity is linked to positive effects on yield, and these beneficial effects are often mediated by the microbial community and the nutrient transformations it carries out. My dissertation has aimed to elucidate the mechanisms by which plant diversity improves agricultural production. In particular, I have focused on how changes to the amount and diversity of carbon (C) inputs affects soil microorganisms involved in the nitrogen (N) cycle. My work spans multiple scales of observation: from a global meta-analysis to mechanistic studies utilizing denitrification as a model system.In a global meta-analysis, I found that increasing plant diversity through intercropping yields a net increase in extracellular enzyme activity. This effect varied by plant species and soil type suggesting that increases in the quality of nutrient inputs mediates these positive effects on microbial activity. Then, I looked at how intercropping cover crops into corn affects soil nutrient pools and microbial activities in a field experiment. No effect of interseeding cover crops into corn was found on soil nutrient pools or microbial activities. However, by analyzing differences in relationships between nutrient pools and microbial activities at two locations throughout Michigan, I was able to describe how the availability of dissolved organic C (DOC) drives differences in microbial N-cycling processes. I then investigated how C availability drives activity in microbial hotspots within the soil by comparing differences in denitrification potential in bulk soil versus the rhizospheres of corn and interseeded cover crops. Here, I found that denitrification rates were increased in the rhizospheres of all plant types, and this effect varied depending on the species of plant. I was able to further differentiate the impact of DOC and microbial biomass C on the rhizosphere effect and found that C availability was the primary driver of differences in denitrification rates between rhizospheres. Since plants provide many different forms of C to soil microbes, it is important to understand how the chemistry of C inputs affects microbial activity. I used a series of C-substrate additions to determine how C chemistry affects denitrifiers. I found that amino acids and organic acids tended to stimulate the most nitrous oxide (N2O) production and reduction. Although management and site affected overall rates of denitrification, C-utilization patterns of microbes were mostly similar between locations. To identify the mechanisms responsible for these effects, I performed a final experiment to track how denitrifiers utilized different C compounds. The C substrates that stimulated the most complete reduce of N2O also were utilized with the lowest C-use efficiency (CUE). This suggests possible trade-offs between N2O reduction and CUE, with important implications for how to manage microbial communities. Overall, my work demonstrates that land management can impact microbial community activity by influencing the identity of soil C inputs. While the importance of increasing soil C inputs has been known, this dissertation supports the notion that the chemical identity of C inputs can exert significant controls on microbial activity. Moreover, by comparing microbial traits I highlight the importance of trade-offs in how microbially mediated C- and N cycling are coupled.
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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.
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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.
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Title: EFFECTS OF PLACENTAL LISTERIA MONOCYTOGENES INFECTION ON FETAL NEURODEVELOPMENT
Creator: Lee, Kun Ho
Date: 2022
Collection: Electronic Theses & Dissertations
Description: Maternal infection can lead to adverse pregnancy outcomes. Numerous epidemiological studies have demonstrated an association between prenatal infection and neuropsychiatric disorders, including autism spectrum disorder (ASD). Different prenatal infections are associated with distinct neurological pathologies, necessitating studies of the diversity of prenatal pathogens and their consequences. Listeria monocytogenes (Lm) is a foodborne pathogen that causes listeriosis, which typically affects...
Show moreMaternal infection can lead to adverse pregnancy outcomes. Numerous epidemiological studies have demonstrated an association between prenatal infection and neuropsychiatric disorders, including autism spectrum disorder (ASD). Different prenatal infections are associated with distinct neurological pathologies, necessitating studies of the diversity of prenatal pathogens and their consequences. Listeria monocytogenes (Lm) is a foodborne pathogen that causes listeriosis, which typically affects immunocompromised individuals, including pregnant mothers. Prenatal infection with Lm can cause detrimental pregnancy outcomes, such as miscarriages, stillbirths, preterm labor, and death in newborns. However, neurological outcomes of maternal listeriosis have not been characterized. Here, I sought to investigate whether placental infection with Lm is associated with altered neurodevelopment by using a bioluminescence strain of Lm and a murine model of pregnancy-associated listeriosis. I show that placental infection affects neurodevelopment during pregnancy and behavior in the offspring.To investigate how placental infection with Lm dysregulates fetal brain development, I performed RNA-seq on fetal brains to quantify the enrichment of genes that were associated with the infection during gestation. The findings of RNA-seq analysis illustrated that placental infection with Lm altered fetal brain transcriptome and showed sexually dichotomous gene expression profiles. I further assessed the effects of different traits, including Lm exposure, the intensity of placental infection, and sex on the fetal transcriptome using systems biology. The genes were grouped into co-expression modules. Notably, maternal infection and its intensity measured by bioluminescence imaging signal were significantly associated with specific modules, suggesting these traits are the main factors driving these transcriptional changes. Lastly, I showed that placental Listeria infection enriched ASD-associated genes. These results demonstrate that maternal listeriosis dysregulates fetal brain transcriptome during gestation. Neurodevelopment is a complex process influenced by various environmental factors during pregnancy. To examine whether prenatal infection with Lm affects cortical lamination and neural activity, I performed hematoxylin and eosin staining and immunohistochemistry. Gross anatomy of the brain structure analysis showed that placental infection with Lm affected cortical lamination in a localized manner. Furthermore, increased neural activity was observed in Lm- exposed male offspring. These results illustrate that placental infection with Lm induces morphological changes in brain tissue during neurodevelopment. Behavioral symptoms of neuropsychiatric disorders are an important component of the diagnosis. Animal behavioral assays and tools have been developed to examine animal behavior such as social interactions, anxiety, and repetitive behaviors. I examined behavior tests that resembled ASD to determine if mouse offspring born following placental infection displayed abnormal behavior. Lm-exposed offspring exhibited altered behaviors and showed sex-dependent behavioral changes. Overall, my work highlights the impact of maternal listeriosis on brain development during pregnancy and its effects on offspring’s behavior and contributes to the understanding of the spectrum of fetal neurodevelopment.
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