Cellular metabolic and immune pathways can be acted upon by diverse molecular factors. Some examples include small molecules, regulatory proteins or RNAs, intermediary metabolites and hormones. These factors can also be introduced or induced by pathogens during infections. Indeed, it is known that complex interplay exists between metabolism and immunity. However, the ways in which these interactions occur, and the nature of the players are active subjects of research. Herein, three different studies are presented which investigate the roles of three distinct modulators of metabolism and/or immunity. Firstly, a natural product produced by a pathogenic fungus is shown to activate the aryl hydrocarbon receptor and induce the expression of xenobiotic metabolizing enzymes. Secondly, the modulation of lipid metabolism by an immunometabolic antiviral microRNA, microRNA-185, is deconvoluted using activity-based protein profiling (ABPP), transcriptomic and lipidomic analysis. This study also identifies a novel enzymatic target of microRNA-185 which can be targeted pharmacologically to reduce hepatitis C virus infectivity. Finally, a third study investigates the link between a poorly characterized enzyme, lysophospholipase-like 1 (LYPLAL1), and hepatic glucose metabolism using a specific activity-based probe. Overall, the work presented in this thesis makes use of various molecular and chemical biology methods to probe pathways which are acted upon by structurally diverse factors to improve our understanding of host-pathogen interactions and metabolism.

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Advanced water treatment (AWT)/reuse has become a necessity for many utilities across the globe as the quantity and quality of water resources has been diminished. In some locations including California, the full-advanced treatment (FAT) train is mandated including membrane filtration, reverse osmosis, and UV advanced oxidation. The application of carbon-based treatment has emerged as a cost-effective alternative to FAT in locations that cannot manage brine disposal. However, considering the relative novelty of this treatment technology for water reuse, the process still requires full-scale validation of treatment goals including pathogen reduction. While there are many constituents of concern in water reuse, exposure to pathogens remains the greatest acute health risk. The studies described herein examine pathogen and microbial surrogate reduction both full-scale and pilot-scale floc/sed-ozone-biofiltration advanced water treatment facility. Both culture and molecular-based methods were used to demonstrate removal in this case and pilot challenge testing was employed to address the shortcomings of full-scale monitoring and to address additional research objectives. The reduction of Cryptosporidum, Giardia, enteric viruses, pathogenic bacteria and their corresponding surrogate microorganisms (e.g. spore forming bacteria, coliphage) was quantified across the upstream wastewater treatment process and the AWT. In general, the removal of surrogate microorganisms was less than or equal that of the pathogens of interest thereby justifying their use in full-scale monitoring. Several limitations of full-scale monitoring were noted including low starting concentrations which resulted in large sample volume required to demonstrate log-reduction. Additionally, while molecular methods were sufficient to demonstrate reduction by physical treatment steps, they are unable to demonstrate inactivation. Therefore, ozone pilot testing was performed to evaluate the use of capsid integrity PCR for showing inactivation by ozonation. Additional testing was also performed to relate the LRV shown with culture methods to the LRV shown with PCR so as to create a relationship that can be used in future monitoring. While pathogen inactivation is a major concern in water reuse, these objectives must also be balanced with the formation of disinfection byproducts (DBPs) through ozonation. Given the elevated concentration of dissolved organic matter, relatively higher ozone doses are required in reuse applications when compared with water treatment applications in order to achieve the desired treatment goals (oxidation, disinfection). Pilot scale ozone testing was performed to evaluate ozone disinfection performance in unfiltered secondary effluent while balancing the formation of bromate and oxidation of trace organic contaminants (TrOCs). Two chemical bromate control methods were compared including preformed monochloramine (NH2Cl), and hydrogen peroxide (H2O2). Neither of these bromate control methods had any demonstrable impact on virus or coliform inactivation, however H2O2 eliminated measurable ozone exposure which is necessary for the inactivation of more resistant spore forming bacteria. Additionally, NH2Cl was shown to suppress *OH exposure and thus negatively impacted the oxidation of ozone resistant TrOCs, while H2O2 marginally improved TrOC oxidation. Finally, the use of H2O2 for bromate control necessitates the validation of an alternative framework for ozone process control. The existing ozone Ct framework has been shown to be prohibitively conservative especially for virus inactivation. In this study, the applied specific ozone dose (O3:TOC) and the change in UV254 absorbance were evaluated as ozone monitoring frameworks across a range of water quality characteristics. Elevated temperature and pH were shown to significantly impact ozone decay kinetics, and only marginally impact virus inactivation. Both frameworks that were evaluated were shown to be valid across all water quality conditions evaluated. Validating pathogen reduction across carbon-based reuse treatment trains is imperative in order to allow for more widespread application and regulatory confidence in the technology. Coagulation, floc/sed, ozone, and biofiltration were shown to be robust barriers for pathogen and surrogate reduction and recommended concentration and quantification methods are presented herein. The ozone challenge testing results also provide guidance to utilities using ozone for disinfection while controlling DBPs and enhancing organics oxidation in water reuse applications. / Doctor of Philosophy / Water reuse has become a necessity for many utilities across the globe as the quantity and quality of water resources has been diminished. In some locations including California, the full-advanced treatment (FAT) train is required including membrane filtration, reverse osmosis, and UV advanced oxidation. The application of carbon-based treatment has emerged as a cost-effective alternative to FAT in locations that cannot manage brine disposal. However, considering the relative novelty of this treatment technology for water reuse, the process still requires full-scale validation of treatment goals including pathogen reduction. While there are many constituents of concern in water reuse, exposure to pathogens remains the greatest acute health risk. The studies described herein examine pathogen and microbial surrogate reduction both full-scale and pilot-scale floc/sed-ozone-biofiltration advanced water treatment facility. Both culture and molecular-based methods were used to demonstrate removal in this case and pilot challenge testing was employed to address the shortcomings of full-scale monitoring and to address additional research objectives. The reduction of protozoa, viruses, bacteria and their corresponding surrogate microorganisms was quantified across the upstream wastewater treatment process and the water reuse treatment train. In general, the removal of surrogate microorganisms was less than or equal that of the pathogens of interest thereby justifying their use in full-scale monitoring. Several limitations of full-scale monitoring were noted including low starting concentrations which resulted in large sample volume required to demonstrate log-reduction. Additionally, while molecular methods were sufficient to demonstrate reduction by physical treatment steps, they are unable to demonstrate inactivation. Therefore, ozone pilot testing was performed to evaluate several methods to adapt these methods to reflect inactivation. While pathogen inactivation is a major concern in water reuse, these objectives must also be balanced with the formation of disinfection byproducts through ozonation. Given the elevated concentration of dissolved organic matter, relatively higher ozone doses are required in reuse applications when compared with water treatment applications in order to achieve the desired treatment goals (oxidation, disinfection). Pilot scale ozone testing was performed to evaluate ozone disinfection performance in wastewater effluent while balancing the formation of byproducts and oxidation of trace organic contaminants. Two chemical byproduct control methods were compared including preformed monochloramine, and hydrogen peroxide. Neither of these bromate control methods had any demonstrable impact on virus or coliform inactivation, however H2O2 eliminated measurable ozone exposure which is necessary for the inactivation of more resistant spore forming bacteria. Additionally, monochloramine was shown to suppress hydroxyl radical exposure and thus negatively impacted the oxidation of ozone resistant organic contaminants, while hydrogen peroxide marginally improved oxidation. Finally, the use of hydrogen peroxide for bromate control necessitates the validation of an alternative framework for ozone process control. The existing framework that relies on ozone exposure has been shown to be conservative especially for virus inactivation. In this study, the applied specific ozone dose and the change in UV254 absorbance were evaluated as ozone monitoring frameworks across a range of water quality characteristics. Elevated temperature and pH were shown to impact ozone decay kinetics and virus inactivation to varying degrees. Both frameworks that were evaluated were shown to be valid across all water quality conditions evaluated. Validating pathogen reduction across carbon-based reuse treatment trains is imperative in order to allow for more widespread application and regulatory confidence in the technology. Coagulation, flocculation/sedimentation, ozone, and biofiltration were shown to be robust barriers for pathogen and surrogate reduction and recommended concentration and quantification methods are presented herein. The ozone challenge testing results also provide guidance to utilities using ozone for disinfection while controlling disinfection byproducts and enhancing organics oxidation in water reuse applications.

Human pathogenic yeasts of the genus Candida and 𝘊𝘳𝘺𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘯𝘦𝘰𝘧𝘰𝘳𝘮𝘢𝘯𝘴 are responsible for about 10% of hospital-acquired infections. In addition, drug-resistant yeasts are rapidly emerging with the use of anti-fungal drugs. Common drugs such as Fluconazole and Amphotericin B target the ergosterol pathway in yeast. Learning about other metabolic differences in yeasts may also give a new understanding to their role as pathogens. Metabolomics is a field of study about the large spectrum of metabolites necessary for the growth and survival of an organism. Gas chromatography-mass spectrometry (GC-MS) was used to analyze the metabolome of different yeasts in three different studies. The first study was a targeted analysis of the ergosterol pathway in 𝘊𝘢𝘯𝘥𝘪𝘥𝘢 𝘢𝘭𝘣𝘪𝘤𝘢𝘯𝘴 and a double drug-resistant mutant was found to have several changes in its sterol composition while Fluconazole-resistant strains were similar to the wild-type. In the second study, a comprehensive analysis of the polar and lipid metabolite profiles of six pathogenic yeasts revealed that lipid profiles were more conserved than polar profiles, thereby better reflecting their taxonomical relationship according to 265 rRNA sequences. However, there were several potential species-specific metabolites and short regions in the metabolite profiles with enough peak differentiation that could be used to rapidly distinguish between these yeasts by visual inspection. In the third study, the metabolic phenotypes of three strains of 𝘊𝘳𝘺𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘯𝘦𝘰𝘧𝘰𝘳𝘮𝘢𝘯𝘴 were analysed to determine the extent of contribution of the metabolite phenotypes from two parents to their hybrid offspring. While the lipid metabolite phenotypes of all strains resembled each other, the polar metabolite phenotype of the hybrid offspring strongly resembled one parent but not the other. / Thesis / Master of Science (MS)

Plants are surrounded by various threats from the environment such as pathogens, abiotic stresses, and animal attacks. Nutrient content and distribution are essential for plant growth and development as well as plant immunity. Pathogens extract nutrients from host plants to benefit their own growth and reproduction. Sulfate, amino acids, and phosphate are indispensable elements for plant growth, plant nutrition, and plant resistance/susceptibility to disease. However, the role of these nutrients in plant-oomycete interactions is an unexplored area. We developed a hydroponic system to precisely control the nutrients applied to plants. We used Arabidopsis thaliana and Nicotiana benthamiana (N. b) as model plants. Hyaloperonospora arabidopsidis as well as two Phytophthora species, Phytophothora capsici (P. cap) and Phytophothora nicotianae (P. nic) were used as model oomycete pathogens. Hpa is an obligate biotrophic pathogen that obtains nutrients directly from the host plant without causing cell death, while P. cap and P. nic are hemibiotrophic pathogens that display a biotrophic phase followed by a necrotrophic phase where they feed on dead cells. Genomic evidence suggests that these pathogens might obtain nutrients including sulfur in different forms from the host (organic and inorganic respectively). We have optimized the hydroponic system and used Taqman PCR assays and sporangiophore counts to assay the influence of sulfur nutrients on Hpa and P. cap infections. We found that (1) sulfur transporter and metabolism genes play essential roles in plant-oomycete interactions; (2) sulfur is critical components for HR responses against Hpa; (3) low sulfur induces pathogenesis related genes as a systemic acquired response. RNA-seq analysis on Phytophthora-infected Arabidopsis suggested that sulfur transport, assimilation, and metabolism play an important role in plant-oomycete interactions. A second project used RNA-seq analysis on P. nic infected N. b, to identify potential nutrition-related-plant genes that are necessary for full pathogen virulence. RNAi knockdowns of N. b AAP6 (amino acid permease 6) and PHT4 (phosphate transporter 4) genes showed an inhibition of oomycete colonization. These experiments together advance the study on the interplay between nutrient assimilation/metabolism in host plants and oomycete infection which will provide insight into the mechanisms how pathogens intercept nutrients from host. In the long-term, this research could reveal new traits applicable for disease resistance to promote crop and food production. / Doctor of Philosophy / Plants are surrounded by diverse threats from the environment such as pathogens, abiotic stresses, and animal attacks. Oomycetes are the most destructive group of pathogens, triggering severe food security issues. Phytophthora is an oomycete genus causing serious economic loss. Traditional disease control managements including pesticides, crop rotation and culture practices, are not time- or financially- efficient due to the difficulty in managing oomycete spread and oomycete resistance to chemicals. Thus, new plant genes for resistance to oomycete diseases would have a major impact. Plant nutrients are critically important for plant fitness in every aspect of plant growth and plant immunity. Cellular regulatory networks for sulfur, amino acids, and phosphate assimilation and metabolism networks connect to every aspect of plant activity such as functioning enzymes, formation of chlorophyll, synthesis of proteins, and plant immunity. These nutrients are part of the plant defense system but also can be beneficial nutrients fed to the invading pathogens. Studying how nutrients are involved in the responses to oomycete invasions will provide information to introduce resistance strategies into crops. We utilized oomycete pathogens with different lifestyles to study the interactions and found that some sulfate transporter genes, an amino acid transporter and a phosphate transporter might be manipulated by oomycete to obtain nutrients. Sufficient nutrition is a critical factor for successfully triggering plant immunity but also could be reprogrammed by pathogens for successful infection and development. Our studies gave useful information to understand which plant nutrient genes are important during plant–oomycete interactions. These findings could be useful in identifying or engineering new plant genes to control plant diseases.

Fusobacterium are Gram-negative anaerobic bacteria that colonize a variety of eukaryotes including cattle and humans. In humans, Fusobacterium coordinates the central architecture of the oral biofilm by expressing an abundance of outer membrane adhesins that mediate bridging between early and late colonizing bacteria. While Fusobacterium are mostly considered commensal microorganisms, they can also become an opportunistic pathogen that spreads throughout the human body and promote the development of oral and extra-oral infections and diseases including colorectal cancer. Importantly for this work, many Fusobacterium species and strains are recalcitrant to genetic manipulation, the majority of which has led to hindrance in the study of their biology, molecular mechanisms, and pathogenesis. The genetic intractability of Fusobacterium is an obstacle for the development of future treatments for diseases associated with these anaerobic bacteria. Therefore, the creation of tools to enhance genome editing in target species is crucial to understand the molecular mechanisms driving Fusobacterium infections. This dissertation exploits innate and adaptive defense systems present in Fusobacterium for their use as molecular tools for genome editing. Accordingly, we first investigated restriction-modification systems with a focus on the role of DNA methyltransferases and endonucleases in host defense and genetic recalcitrance in several strains of Fusobacterium through bioinformatic and biochemical approaches. Altogether, over 15 DNA methyltransferases were characterized. Most notably, we identified and characterized two type II DNA methyltransferases that are capable of methylating plasmid DNA by treating with purified enzymes in-vitro and coexpression approaches in Escherichia coli strains, enabling an statiscally improved transformation efficiency via electroporation in F. nucleatum. Also contained in this dissertation is the first detailed description of CRISPR-Cas adaptive immunity systems present in Fusobacterium strains. Most of the discovered CRISPR-Cas systems in Fusobacterium belong to Class 1 systems. Nonetheless we identified Type II-A and Type VI-C Class 2 systems. The discovery of Cas9 and Cas13c effectors respectively from these systems will be crucial in the development of a new generation of genome-editing tools in Fusobacterium. The studies included in this dissertation provide the framework for overcoming Fusobacterium genetic recalcitrance by the implementation of host mimicking techniques. By utilizing restriction-modification system enzymes and the adaptive immunity CRISPR-Cas systems, we will gain a better understanding of how Fusobacterium modulates infections and diseases, and ultimately explore the potential of novel therapeutic treatments. / Doctor of Philosophy / The oral cavity has one of the most diverse and largest microbial populations, where microorganisms are capable of colonizing hard surfaces of the teeth and the soft tissues of the oral mucosa. A fundamental member of the oral microbiome is Fusobacterium, a Gram-negative bacterium which coordinates the oral biofilm formation by interacting with other microorganisms. In recent studies, Fusobacterium has been associated with oral and extra-oral infections and diseases including periodontitis, preterm birth, Lemiere syndrome, inflammatory bowel disease and colorectal cancer. Importantly, many Fusobacterium species and strains are challenging to study due to their inability to uptake exogenous DNA and lack of genetic tools, which has hindered the study of their biology, molecular mechanisms and pathogenesis. The challenges in the genetic manipulation of Fusobacterium present a significant obstacle for the development of future treatments for diseases associated with these bacteria. Therefore, the creation of tools to expand bacterial transformation of exogenous DNA and genome editing to more than just one Fusobacterium species is crucial to understand how Fusobacterium is causing these infections. This dissertation explores the presence and utilization of defense systems, which defend bacteria from phage attack, as an alternative to improve Fusobacterium genetics. Accordingly, we first studied a set of over 15 enzymes that recognize a specific DNA pattern and add a methyl group (DNA methyltransferases) to specific nucleotides in several strains of Fusobacterium. We discovered that two of these enzymes improve Fusobacterium's ability of importing and genomically incorporating exogenous DNA after an electric discharge permeabilizes the bacterial membrane. Furthermore, for the first time we have described the composition of CRISPR-Cas bacterial defense systems, that detect invading DNA from viruses and provide protection to Fusobacterium strains. These systems have previously been successfully used as genetic tools to achieve genome editing. Thus, their further characterization is warranted to create novel molecular tools in Fusobacterium. Altogether, these discoveries will lead to a better comprehension of Fusobacterium biology in infections and diseases, while exploring novel therapeutic strategies.

Various control methods used in the meat and poultry processing environment to mitigate listeriosis were evaluated using a dynamic in-plant Monte Carlo model. These control methods included food contact surface testing, sanitation, post-processing lethality treatment, and product formulation with microbial growth inhibitors. The dynamic in-plant model served as an input into the risk assessment model developed by the FDA and FSIS in 2003 which predicts the number of deaths and illnesses resulting from the use of each control method. The use of growth inhibitors combined with a post-processing lethality step was estimated to save over 200 more lives than the FSIS proposed minimum sampling standard. An analysis of data collected by the National Alliance for Food Safety and Security (NAFSS) found that retail-sliced deli meats have a greater prevalence and concentration of L. monocytogenes than prepackaged deli meats. Cross contamination at the retail level is suspected due to clustering of sample positives by store and the influence of sampling time of day on the prevalence of L. monocytogenes. The comparative risk of Listeria monocytogenes in retail sliced versus prepackaged deli meats was evaluated using a modified version of the 2003 FDA-FSIS risk assessment model which considered slicing location and the use of growth inhibitors. The comparative risk ratio for the number of deaths from retail-sliced versus prepackaged deli meats was found to be 9.1 and retail-sliced product with a growth inhibitor was found to be at greater risk for listeriosis than prepackaged product without growth inhibitor. / Master of Science

Comparative analysis of multiple genomes presents many challenges ranging from management of information about thousands of local similarities to definition of features by combination of evidence from multiple analyses and experiments. This research represents the development stage of a database-backed pipeline for comparative analysis of multiple genomes. The genomes of three recently sequenced species of Brucella were compared and a superset of known and hypothetical coding sequences was identified to be used in design of a discriminatory genomic cDNA array for comparative functional genomics experiments. Comparisons were made of coding regions from the public, annotated sequence of B. melitensis (GenBank) to the annotated sequence of B. suis (TIGR) and to the newly-sequenced B. abortus (personal communication, S. Halling, National Animal Disease Center, USDA). A systematic approach to analysis of multiple genome sequences is described including a data model for storage of defined features is presented along with necessary descriptive information such as input parameters and scores from the methods used to define features. A collection of adjacency relationships between features is also stored, creating a unified database that can be mined for patterns of features which repeat among or within genomes. The biological utility of the data model was demonstrated by a detailed analysis of the multiple genome comparison used to create the sample data set. This examination of genetic differences between three Brucella species with different virulence patterns and host preferences enabled investigation of the genomic basis of virulence. In the B. suis genome, seventy-one differentiating genes were found, including a contiguous 17.6 kb region unique to the species. Although only one unique species-specific gene was identified in the B. melitensis genome and none in the B. abortus genome, seventy-nine differentiating genes were found to be present in only two of the three Brucella species. These differentiating features may be significant in explaining differences in virulence or host specificity. RT-PCR analysis was performed to determine whether these genes are transcribed in vitro. Detailed comparisons were performed on a putative B. suis pathogenicity island (PAI). An overview of these genomic differences and discussion of their significance in the context of host preference and virulence is presented. / Master of Science

The timely identification of pathogens responsible for disease outbreaks is crucial for implementing effective control measures and minimizing the spread of infectious diseases. Conventional methods of identification are limited to specific pathogen species because they require prior knowledge and pure cultures of the pathogen. Therefore, these methods cannot detect new pathogens responsible for newly emerging diseases. Computational methods that rely on sequencing data have the potential to overcome these limitations. However, the diverse phenotypes among microbial species and strains within the same species pose a challenge in accurately identifying the specific pathogen responsible for the disease. This dissertation highlights the importance of strain-level detection for the identification and characterization of pathogens by employing computational methods that rely on genomic and metagenomic sequencing data. To realize that computational goal, a comparison of different tools, currently used for metagenome classification, was done to illustrate effective detection of bacterial pathogens. To develop computational methods for characterization, genomes of the plant pathogen Ralstonia solanacearum were studied to understand the basis of virulence at cool temperatures. Finally, a new tool was developed that combines accurate detection and characterization at the strain level, through the use of taxonomic databases constructed using genome similarity thresholds. This dissertation work is a contribution to the development of improved approaches to detect and contain disease outbreaks in plants with possible applications in human and animal diseases as well. / Doctor of Philosophy / Detecting and identifying pathogens is crucial for controlling disease outbreaks in humans, animals, and plants. However, currently used methods are limited to identifying only those pathogens that can be grown in a lab. An ideal method for pathogen detection should be broadly applicable to many pathogens. A newer technique called metagenome sequencing allows us to identify known as well as unknown pathogens, including the ones that cannot be grown in a lab. This makes it possible to detect new pathogens from newly emerging diseases. Computational tools that accurately analyze the sequencing data are needed. This dissertation highlights the importance of accurately identifying specific strains of pathogens using computational techniques based on genomic and metagenomic sequencing data. As a result, different tools were evaluated for classifying metagenomes for the successful detection of bacterial pathogens. For the characterization of specific traits responsible for causing disease, genomes of Ralstonia solanacearum, a plant pathogen, were studied to understand how some strains remain harmful at lower temperatures. The dissertation also introduces a novel metagenomic classification tool that combines accurate detection and characterization of pathogen strains by using genome similarity thresholds to create taxonomic databases. This approach improves our ability to identify and understand pathogens at a more specific level. Overall, this research aims to enhance our ability to identify and understand pathogens, allowing for more effective measures to control and prevent disease outbreaks.