Browsing by Author "Hansmeier, Nicole"
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Item Open Access Blocks in Tricarboxylic Acid Cycle of Salmonella enterica Cause Global Perturbation of Carbon Storage, Motility, and Host-Pathogen Interaction(American Society for Microbiology, 2019-12-11) Noster, Janina; Hansmeier, Nicole; Persicke, Marcus; Chao, Tzu-Chiao; Kurre, Rainer; Popp, Jasmin; Liss, Viktoria; Reuter, Tatjana; Hensel, MichaelThe tricarboxylic acid (TCA) cycle is a central metabolic hub in most cells. Virulence functions of bacterial pathogens such as facultative intracellular Salmonella enterica serovar Typhimurium (S. Typhimurium) are closely connected to cellular metabolism. During systematic analyses of mutant strains with defects in the TCA cycle, a strain deficient in all fumarase isoforms (ΔfumABC) elicited a unique metabolic profile. Alongside fumarate, S. Typhimurium ΔfumABC accumulates intermediates of the glycolysis and pentose phosphate pathway. Analyses by metabolomics and proteomics revealed that fumarate accumulation redirects carbon fluxes toward glycogen synthesis due to high (p)ppGpp levels. In addition, we observed reduced abundance of CheY, leading to altered motility and increased phagocytosis of S. Typhimurium by macrophages. Deletion of glycogen synthase restored normal carbon fluxes and phagocytosis and partially restored levels of CheY. We propose that utilization of accumulated fumarate as carbon source induces a status similar to exponential- to stationary-growth-phase transition by switching from preferred carbon sources to fumarate, which increases (p)ppGpp levels and thereby glycogen synthesis. Thus, we observed a new form of interplay between metabolism of S. Typhimurium and cellular functions and virulence.Item Open Access Development and Evaluation of the Sequential Clustering Technique for the Faster Study of the Human Microbiome(Faculty of Graduate Studies and Research, University of Regina, 2020-08) Haidl, Emily Grace; Stavrinides, John; Butz, Cortney J.; Hansmeier, Nicole; Links, MatthewThe human microbiome is the set of microbial communities that live on and in the human body. These microbes can play a large role in human health and disease, and bioinformatic methods to study these relationships are becoming increasingly important as the amount of human microbiome data increases. Most methods are slow, which makes them difficult to use for the analysis of large, multi-project datasets. We propose a Sequential Clustering (SC) technique using the software VSEARCH that is able to cluster all reads across large sample sets in a sequential manner. When applied, the SC method was able to identify the genera in a mock community in abundances similar to those found using a traditional clustering technique, while working much faster, and with the added feature of being easily accelerated with parallelization. The SC method was able to recover clusters similar to those generated using traditional methods and with greater taxonomic consistency depending on the sequence identity threshold pairs chosen. The technique was also able to identify the distribution of unclassified bacteria across 15 different human body sites sampled in three independent microbiome projects, in a reduced-size dataset analysis. The SC technique is a valuable, time-saving approach that can facilitate large-scale analyses across thousands of datasets, and shows promise as an alternative to traditional clustering techniques.Item Open Access Elucidating mechanisms of acid tolerance and antibiotic resistance in Salmonella and Klebsiella using transposon insertion sequencing (INSeq) and whole genome sequencing(Faculty of Graduate Studies and Research, University of Regina, 2024-04) Amin, Mohammad Ruhul; Cameron, Andrew; Yost, Christopher; Hansmeier, Nicole; Dahms, Tanya; Tahlan, KapilSalmonella enterica and Klebsiella pneumoniae are two major public health concerns that are responsible for millions of illnesses every year throughout the world. Much remains to explore the genes and mechanisms that are critical for their pathogenesis and antibiotic resistance. I used transposon Insertion Sequencing (INSeq), a powerful high-throughput screening technique that can link bacterial genes to phenotypes and identify genes that are essential for bacterial survival, to determine genes that are associated with survivability of Salmonella Typhimurium in Luria Bertani (LB), E-minimal medium (EMM) and under acid stress, and of K. pneumoniae under exposure to five classes of antibiotics. Growing a pool of 450,000 mutants on LB identified a total of 362 essential genes, the majority of which (90.6%) are within the S. Typhimurium core genome. The pCol1B9 replication initiation and its regulator, repZ and repY, are among the essential genes found in the strain's accessory genome. Comparing essential genes identified in LB to two earlier studies of S. Typhimurium strains and identifying genes that are conditionally essential in EMM suggest that a single growth environment and strain cannot provide a comprehensive understanding of essential genes at the species level. S. Typhimurium is hypothesized to have a special acid tolerance system in which the cytoplasm becomes more acidic. I applied INSeq to find genes that contribute to acid tolerance at pH 4.0 and pH 5.0. For which I developed a modified INSeq approach capable of identifying genes required for persistence in non-growth conditions. In addition to several known genes, this project identified novel acid tolerance genes including trxB (thioredoxin reductase), pykF (pyruvate kinase), sspA (starvation protein), and revealed that as the stress increases through time and decreasing pH, additional tolerance mechanisms are required to protect cells. Next, I used INSeq to find intrinsic resistance genes in a clinical isolate of multidrug resistant K. pneumoniae. We found pstB (ABC transporter ATP binding protein), gltA (Citrate synthase), tgt (tRNA guanosine transglycosylase), fabF (fatty acid synthase), and glycosyltransferase encoding genes: pgaptmp_000142, pgaptmp_000147, and pgaptmp_000148, each contribute to resistance across multiple classes of antibiotics. Considering all the INSeq data, healthy cell envelopes were found to be crucial for optimum growth and cell protection, regardless of the growth environment, which included laboratory conditions under acid and antibiotic stresses. In addition, numerous known genes were identified for corresponding features, such as phoP-phoQ system for acid tolerance and acrAB-tolC for multidrug resistance, confirming the effectiveness of INSeq. I next used whole genome sequencing to find genetic changes in S. Typhimurium isolates to characterize a sugar metabolism and an antibiotic resistance phenotype. First, I helped characterize how a C→T transition in the dctA promoter allows for growth at lower orotate concentrations by creating an improved binding site for the transcriptional activator CRP. Secondly, by progressively challenging cells with higher concentrations of antibiotics, I discovered an A→T transition in codon 466 of gyrB reduces ciprofloxacin sensitivity in a S. Typhimurium mutant that cannot synthesize the intracellular signaling molecule cAMP. Both Salmonella and Klebsiella are considered top priority pathogens for research and development of new antibiotics; this PhD thesis provides an improved understanding of the biology of both organisms and simultaneously identifies high-quality candidate genes that can be targeted for the development of improved antibiotics and other therapeutics.Item Open Access Establishing wastewater surveillance for SARS-CoV-2 in Southern Saskatchewan(Faculty of Graduate Studies and Research, University of Regina, 2023-06) Reuter, Sonja Tamara; Hansmeier, Nicole; Chao, Tzu-Chiao; Yost, Christopher; Van Hamme, JonathanWastewater surveillance has become a crucial part in the monitoring of the COVID-19 pandemic. Infected people shed SARS-CoV-2 in their feces, therefore, virus levels in wastewater reflect the trends in infection numbers in the population that contributes to the wastewater. Wastewater surveillance offers information about the spread of SARS-CoV-2 independent of testing strategies and individual choices and therefore better reflects population health than individual testing. In this study we compared five RNA extraction kits and reagents and different enrichment methods for wastewater surveillance of SARS-CoV-2 RNA and determined that the solids fraction of wastewater was most suitable for RNA extraction using the AllPrep PowerViral DNA/RNA Kit by Qiagen. We also examined the impact of storage on wastewater samples and determined that while short-term storage does not affect the samples significantly, longer storage changes the measured viral levels. We then used our protocol to establish a wastewater surveillance for SARS-CoV-2 viral levels in Regina and nine other locations in Southern Saskatchewan, Canada. We observed that the three waves of COVID-19 during the study time were caused by the variants Alpha, Delta and Omicron and that the SARSCoV- 2 RNA levels in wastewater were a good reflection of the reported active COVID-19 case numbers.Item Open Access Evaluating the effect of spaceflight on the host–pathogen interaction between human intestinal epithelial cells and Salmonella Typhimurium(Nature Research, 2021-03-09) Barrila, Jennifer; Sarker, Shameema F.; Hansmeier, Nicole; Yang, Shanshan; Buss, Kristina; Briones, Natalia; Park, Jin; Davis, Richard R.; Forsyth, Rebecca J.; Ott, C. Mark; Sato, Kevin; Kosnik, Cristine; Yang, Anthony; Shimoda, Cheryl; Rayl, Nicole; Ly, Diana; Landenberger, Aaron; Wilson, Stephanie D.; Yamazaki, Naoko; Steel, Jason; Montano, Camila; Halden, Rolf U.; Cannon, Tom; Castro-Wallace, Sarah L.; Nickerson, Cheryl A.Spaceflight uniquely alters the physiology of both human cells and microbial pathogens, stimulating cellular and molecular changes directly relevant to infectious disease. However, the influence of this environment on host–pathogen interactions remains poorly understood. Here we report our results from the STL-IMMUNE study flown aboard Space Shuttle mission STS-131, which investigated multi-omic responses (transcriptomic, proteomic) of human intestinal epithelial cells to infection with Salmonella Typhimurium when both host and pathogen were simultaneously exposed to spaceflight. To our knowledge, this was the first in-flight infection and dual RNA-seq analysis using human cells.Item Open Access Exploring the Evolution of Pathogenicity in a Mult-host Bacterial Pathogen Using Comparative Genomics(Faculty of Graduate Studies and Research, University of Regina, 2021-03) Soutar, Craig David; Stavrinides, John; Hansmeier, Nicole; Butz, Cortney J.; Yost, Christopher; Tambong, James T.Pantoea is a Gram-negative bacterial genus within the recently created Erwiniaceae. Pantoea members are found in association with a wide range of hosts, and some species have been reported as the causative agent of human infections. Despite this, little research has focused on the human pathogenicity of Pantoea. This thesis aims to explore the evolution of pathogenicity in Pantoea using phylogenetic and comparative genomic approaches, with a focus on human pathogenicity. Chapter 1 provides a broad review of Pantoea, virulence, and phylogenetic and genomic concepts. Chapter 2 describes molecular typing of clinical Pantoea isolates obtained from hospital patients. Of 54 clinical isolates, 41 were confirmed as Pantoea, with the majority of these isolates found to belong to P. septica. Chapter 3 involves verification of the taxonomy of Pantoea, the P. septica lineage, and the Erwiniaceae using phylogenomics. From over 100 strains, a 308 core protein dataset was used to perform a phylogenetic analysis. Genomes of all strains were used to conduct average nucleotide identity and average amino acid identity analyses. The phylogenetic analysis showed that the P. septica lineage, including the proposed genus Kalamiella, nested within Pantoea with strong support. This work indicates no basis for establishment of Kalamiella, or taxonomic revision of the P. septica lineage. Chapter 4 involves characterizing the evolutionary history of siderophore gene clusters in Pantoea. Siderophores are noted virulence factors involved in iron acquisition; however, the siderophore complement of Pantoea has been largely unexplored. Through comparative genomic and phylogenetic analyses, biosynthetic gene clusters for three distinct siderophores were found across Pantoea, with each having a unique distribution and evolutionary history. Notably, the aerobactin gene cluster was acquired by two human pathogenic species, P. septica and P. ananatis, in two independent horizontal transfer events; both from human pathogenic genera of Enterobacteriaceae. This thesis confirmed the identity of dozens of human Pantoea isolates and demonstrated that P. septica specifically appears to be adept at infecting humans. This work also validated the taxonomic assignment of the P. septica lineage as part of Pantoea and provided evidence of virulence factor horizontal acquisition by P. septica from established human pathogens.Item Open Access Genetic interaction landscape of the Escherichia coli transcriptional factor machinery(Faculty of Graduate Studies and Research, University of Regina, 2022-07) Hosseinnia, Ali; Babu, Mohan; Raina-Fulton, Renata; Widdifield, Cory; Hansmeier, Nicole; Kumar, AyushIn Bacteria, transcription factors (TFs) consist of sensory ligand-binding and DNA-binding helix-turn-helix (HTH) domains to respond to the environmental and internal stimuli. Although Escherichia coli is a well-studied model bacterium, yet half of its TFs’ characteristics remain unclear, including 72 genes of unknown function. Using E. coli synthetic genetic array, a quantitative approach to scoring the fitness of single and double mutant genes, I was able to measure pairwise genetic interactions (GIs) among all TFs in rich and minimal media (RM and MM) to create a differential (DF) GI network. DF network analysis demonstrated GIs altered in RM or MM growth conditions. Both static and DF GI networks were also effective in detecting TF pathways, highlighting new roles for uncharacterized TFs (YjdC, YneJ, YdiP) as regulators of cell division, putrescine pathway and efflux pump, and cold shock adaptation, respectively. Pan-bacterial conservation suggests that TF genes with similar GI profiles are co-conserved in bacterial evolution. Moreover, the E. coli TF GI network provided deep insights on conserved genetic association across bacterial phyla that can be valuable to build TF machinery organization, even in pathogens.Item Open Access Identification of an antibiotic biosynthetic gene cluster in pantoea agglomerans B025670(Faculty of Graduate Studies and Research, University of Regina, 2019-07) Sorout, Naveen; Stavrinides, John; Hansmeier, Nicole; Dietz, Heather; Kumar, AyushThe rise of multi-drug resistant isolates of several pathogens, including Gram-negative members of the Enterobacteriaceae, requires the identification of new antimicrobials that can target them. Members of the genus Pantoea, a Gram-negative bacterial group, produce natural products with antimicrobial properties. A survey of a Pantoea collection against clinically relevant pathogens, including VRE, Enterobacter, Klebsiella, Citrobacter and Kosakonia, revealed 25 strains that produce at least one antibiotic against one or more of these pathogens. Of these strains, Pantoea agglomerans B025670 produces at least one antibiotic (denoted PNP-4) that has activity against strains of Enterobacter and Kosakonia. The conventional genetic approach was used to identify the antibiotic-producing biosynthetic gene cluster. This involved transposon mutagenesis of wild type B025670 (B025670-WT) followed by genetic screens on the mutants to identify candidates that no longer produced the antibiotic. The antibiotic production in B025670-WT was disrupted by mutations in several amino acid biosynthetic pathways, including mutations in histidine, leucine, arginine, isoleucine, cysteine and tryptophan operons. However, this was reversed upon supplementation with the corresponding amino acids in the growth medium. Furthermore, none of the candidate mutants appeared to have disruptions in the cluster responsible for PNP-4 production. Comparative genomics was then used to identify the mystery cluster. A comparison of B025670-WT draft genome with closely related non-producers of the antibiotic revealed two clusters that are unique to B025670-WT. Mutations in one of these clusters, potentially involved in fatty acid biosynthesis, results in a disruption of antibiotic production. Furthermore,the distribution of this cluster across Pantoea strains is limited.Item Open Access Impact of ROS-Induced Damage of TCA Cycle Enzymes on Metabolism and Virulence of Salmonella enterica serovar Typhimurium(Frontiers Media, 2019-04-24) Noster, Janina; Persicke, Marcus; Chao, Tzu-Chiao; Krone, Lena; Heppner, Bianca; Hensel, Michael; Hansmeier, NicoleSalmonella enterica serovar Typhimurium (STM) is exposed to reactive oxygen species (ROS) originating from aerobic respiration, antibiotic treatment, and the oxidative burst occurring inside the Salmonella-containing vacuole (SCV) within host cells. ROS damage cellular compounds, thereby impairing bacterial viability and inducing cell death. Proteins containing iron–sulfur (Fe–S) clusters are particularly sensitive and become non-functional upon oxidation. Comprising five enzymes with Fe–S clusters, the TCA cycle is a pathway most sensitive toward ROS. To test the impact of ROS-mediated metabolic perturbations on bacterial physiology, we analyzed the proteomic and metabolic profile of STM deficient in both cytosolic superoxide dismutases (ΔsodAB). Incapable of detoxifying superoxide anions (SOA), endogenously generated SOA accumulate during growth. ΔsodAB showed reduced abundance of aconitases, leading to a metabolic profile similar to that of an aconitase-deficient strain (ΔacnAB). Furthermore, we determined a decreased expression of acnA in STM ΔsodAB. While intracellular proliferation in RAW264.7 macrophages and survival of methyl viologen treatment were not reduced for STM ΔacnAB, proteomic profiling revealed enhanced stress response. We conclude that ROS-mediated reduced expression and damage of aconitase does not impair bacterial viability or virulence, but might increase ROS amounts in STM, which reinforces the bactericidal effects of antibiotic treatment and immune responses of the host.Item Open Access Manipulation of microvillar proteins during Salmonella enterica invasion results in brush border effacement and actin remodeling(Frontiers Media, 2023-03-02) Felipe-Lopez, Alfonso; Hansmeier, Nicole; Danzer, Claudia; Hensel, MichaelEnterocyte invasion by the gastrointestinal pathogen Salmonella enterica is accompanied by loss of brush border and massive remodeling of the actin cytoskeleton, leading to microvilli effacement and formation of membrane ruffles. These manipulations are mediated by effector proteins translocated by the Salmonella Pathogenicity Island 1-encoded type III secretion system (SPI1- T3SS). To unravel the mechanisms of microvilli effacement and contribution of SPI1-T3SS effector proteins, the dynamics of host-pathogen interactions was analyzed using live cell imaging (LCI) of polarized epithelial cells (PEC) expressing LifeAct-GFP. PEC were infected with S. enterica wild-type and mutant strains with defined defects in SPI1-T3SS effector proteins, and pharmacological inhibition of actin assembly were applied. We identified that microvilli effacement involves two distinct mechanisms: i) F-actin depolymerization mediated by villin and ii), the consumption of cytoplasmic G-actin by formation of membrane ruffles. By analyzing the contribution of individual SPI1-T3SS effector proteins, we demonstrate that SopE dominantly triggers microvilli effacement and formation of membrane ruffles. Furthermore, SopE via Rac1 indirectly manipulates villin, which culminates in F-actin depolymerization. Collectively, these results indicate that SopE has dual functions during F-actin remodeling in PEC. While SopE-Rac1 triggers F-actin polymerization and ruffle formation, activation of PLCg and villin by SopE depolymerizes F-actin in PEC. These results demonstrate the key role of SopE in destruction of the intestinal barrier during intestinal infection by Salmonella.Item Open Access Prioritization of biomarker targets in human umbilical cord blood: identification of proteins in infant blood serving as validated biomarkers in adults(National Institute of Environmental Health Sciences (NIEHS), 2012-05-01) Hansmeier, Nicole; Chao, Tzu-Chiao; Goldman, Lynn R; Witter, Frank R; Halden, Rolf UEarly diagnosis represents one of the best lines of defense in the fight against a wide array of human diseases. Umbilical cord blood (UCB) is one of the first easily available diagnostic biofluids and can inform about the health status of newborns. However, compared with adult blood, its diagnostic potential remains largely untapped.Item Open Access Proteomes of Host Cell Membranes Modified by Intracellular Activities of Salmonella enterica*(Elsevier, 2015-01) Vorwerk, Stephanie; Krieger, Viktoria; Deiwick, Jörg; Hensel, Michael; Hansmeier, NicoleItem Open Access Proteomic Analysis of Salmonella-modified Membranes Reveals Adaptations to Macrophage Hosts*(American Society for Biochemistry and Molecular Biology (ASBMB), 2020-05) Reuter, Tatjana; Vorwerk, Stephanie; Liss, Viktoria; Chao, Tzu-Chiao; Hensel, Michael; Hansmeier, NicoleSystemic infection and proliferation of intracellular pathogens require the biogenesis of a growth-stimulating compartment. The gastrointestinal pathogen Salmonella enterica commonly forms highly dynamic and extensive tubular membrane compartments built from Salmonella-modified membranes (SMMs) in diverse host cells. Although the general mechanism involved in the formation of replication-permissive compartments of S. enterica is well researched, much less is known regarding specific adaptations to different host cell types. Using an affinity-based proteome approach, we explored the composition of SMMs in murine macrophages. The systematic characterization provides a broader landscape of host players to the maturation of Salmonella-containing compartments and reveals core host elements targeted by Salmonella in macrophages as well as epithelial cells. However, we also identified subtle host specific adaptations. Some of these observations, such as the differential involvement of the COPII system, Rab GTPases 2A, 8B, 11 and ER transport proteins Sec61 and Sec22B may explain cell line-dependent variations in the pathophysiology of Salmonella infections. In summary, our system-wide approach demonstrates a hitherto underappreciated impact of the host cell type in the formation of intracellular compartments by Salmonella.Item Open Access Proteomics of intracellular Salmonella enterica reveals roles of Salmonella pathogenicity island 2 in metabolism and antioxidant defense(Public Library of Science, 2019-04-22) Noster, Janina; Chao, Tzu-Chiao; Sander, Nathalie; Schulte, Marc; Reuter, Tatjana; Hansmeier, Nicole; Hensel, MichaelIntracellular Salmonella enterica serovar Typhimurium (STM) deploy the Salmonella Pathogenicity Island 2-encoded type III secretion system (SPI2-T3SS) for the massive remodeling of the endosomal system for host cells. This activity results in formation of an extensive interconnected tubular network of Salmonella-induced filaments (SIFs) connected to the Salmonella-containing vacuole (SCV). Such network is absent in cells infected with SPI2-T3SS-deficient mutant strains such as ΔssaV. A tubular network with reduced dimensions is formed if SPI2-T3SS effector protein SseF is absent. Previous single cell live microscopy-based analyses revealed that intracellular proliferation of STM is directly correlated to the ability to transform the host cell endosomal system into a complex tubular network. This network may also abrogate host defense mechanisms such as delivery of antimicrobial effectors to the SCV. To test the role of SIFs in STM patho-metabolism, we performed quantitative comparative proteomics of STM recovered from infected murine macrophages. We infected RAW264.7 cells with STM wild type (WT), ΔsseF or ΔssaV strains, recovered bacteria 12 h after infection and determined proteome compositions. Increased numbers of proteins characteristic for nutritional starvation were detected in STM ΔsseF and ΔssaV compared to WT. In addition, STM ΔssaV, but not ΔsseF showed signatures of increased exposure to stress by antimicrobial defenses, in particular reactive oxygen species, of the host cells. The proteomics analyses presented here support and extend the role of SIFs for the intracellular lifestyle of STM. We conclude that efficient manipulation of the host cell endosomal system by effector proteins of the SPI2-T3SS contributes to nutrition, as well as to resistance against antimicrobial host defense mechanisms.Item Open Access Purification and proteomics of pathogen-modified vacuoles and membranes(Frontiers Media, 2015-06-02) Herweg, Jo-Ana; Hansmeier, Nicole; Otto, Andreas; Geffken, Anna C; Subbarayal, Prema; Prusty, Bhupesh K; Becher, Dörte; Hensel, Michael; Schaible, Ulrich E; Rudel, Thomas; Hilbi, HubertCertain pathogenic bacteria adopt an intracellular lifestyle and proliferate in eukaryotic host cells. The intracellular niche protects the bacteria from cellular and humoral components of the mammalian immune system, and at the same time, allows the bacteria to gain access to otherwise restricted nutrient sources. Yet, intracellular protection and access to nutrients comes with a price, i.e., the bacteria need to overcome cell-autonomous defense mechanisms, such as the bactericidal endocytic pathway. While a few bacteria rupture the early phagosome and escape into the host cytoplasm, most intracellular pathogens form a distinct, degradation-resistant and replication-permissive membranous compartment. Intracellular bacteria that form unique pathogen vacuoles include Legionella, Mycobacterium, Chlamydia, Simkania, and Salmonella species. In order to understand the formation of these pathogen niches on a global scale and in a comprehensive and quantitative manner, an inventory of compartment-associated host factors is required. To this end, the intact pathogen compartments need to be isolated, purified and biochemically characterized. Here, we review recent progress on the isolation and purification of pathogen-modified vacuoles and membranes, as well as their proteomic characterization by mass spectrometry and different validation approaches. These studies provide the basis for further investigations on the specific mechanisms of pathogen-driven compartment formation.Item Open Access A Study of Antibiotic Resistance Plasmids in Fresh Produce and In Wastewater Environments Using Functional Approaches, Comparative Genomics and Abundance Analysis(Faculty of Graduate Studies and Research, University of Regina, 2019-05) Ajayi, Adeyinka Olusegun; Yost, Christopher; Hansmeier, Nicole; Cameron, Andrew; Young, Stephanie; Rubin, JosephStudies have shown that a major reservoir of clinically relevant antibiotic resistance genes (ARGs) is found in non-pathogenic environmental bacteria. Ciprofloxacin, meropenem and erythromycin are important clinical antibiotics due to their broad applications in the treatment of Gram-positive and Gram-negative infections. The capacity to capture, mobilise and control the expression of resistance genes is an important factor in the development of multidrug resistance and the mobilome (the community of mobile genetic elements) is responsible for its genetic plasticity. Plasmids are important constituents of the environmental mobilome. The capacity to replicate independently of the bacterial chromosome coupled with the ability to carry clinically relevant ARGs and other antibiotic resistance-encoding mobile genetic elements means a deep understanding of plasmids is necessary to fully appreciate their roles in the spread and evolution of antibiotic resistance determinants in human-impacted environments. The specific objectives of this study were to: 1) Characterize ciprofloxacin resistance plasmids isolated from fresh spinach using functional approaches and comparative genomics. 2) Investigate the diversity in ciprofloxacin and meropenem resistance plasmids isolated from three geographically-separated wastewater treatment plants (WWTPs) using functional approaches and comparative genomics. 3) Determine how conventional wastewater treatment processes impact the abundance of plasmids encoding ciprofloxacin, meropenem and erythromycin resistance genes, and also incF family incompatibility genes using culture-dependent and culture-independent approaches. During this research, I have isolated and characterized two ciprofloxacin resistanceencoding, multidrug resistance plasmids (pLGP4 and pHTP1) from fresh spinach. I also iii isolated six plasmids encoding ciprofloxacin and meropenem resistance genes from three WWTPs across Canada. The ciprofloxacin resistance plasmids included plasmids pFECG, pFECR, pPICR and pTIC. The meropenem resistance plasmids included plasmids pPIMR and pFEMG. Using enrichment culture and real-time quantitative PCR approaches respectively, it was determined that conventional wastewater treatment processes markedly reduced the abundance of plasmids encoding ciprofloxacin resistance genes and incF family incompatibility genes. Conventional wastewater treatment processes, however, seemed to select for and enrich the population of bacteria carrying a specific erythromycin resistance plasmid. The presence of plasmids encoding clinically relevant ARGs in non-clinical anthropogenic environments suggests there may be a link between the community of antibiotic resistance determinants in clinical and non-clinical environments. Keywords: antibiotic resistance plasmids, spinach, wastewater, ciprofloxacin, meropenem, erythromycin.Item Open Access Systems Biology of Host-Pathogen Protein-Protein Interactions(Faculty of Graduate Studies and Research, University of Regina, 2023-06) Rahmatbakhsh, Matineh; Babu, Mohan; Dahms, Tanya; Hansmeier, Nicole; Hu, PingzhaoDespite undeniable therapeutic developments in infectiology, emerging infectious diseases continue to be a growing threat to public health, as seen by the current COVID- 19 pandemic caused by the novel virus severe acute respiratory syndrome coronavirus (SARS-CoV-2). This virus is classified as an obligate intracellular parasite that co-opts host cellular proteins, often through protein-protein interactions (PPIs), to ensure its replication. Therefore, this thesis aims to integrate high-throughput proteomic approaches with computational modelling to systematically characterize SARS-CoV-2-human networks for a detailed understanding of SARS-CoV-2 pathogenesis. The angiotensin-converting enzyme (ACE2) receptor of SARS-CoV-2 is displayed on many human cells, including the lungs and other organs. However, despite considerable knowledge explaining the SARS-CoV-2 infection mechanism, organ-specific SARS-CoV- 2-host protein interactions remain understudied. In Chapter 2, we carried out an organ/tissue-unbiased proteomic profiling approach of mapping SARS-CoV-2-human protein interactions using high-throughput mass spectrometry (MS)-based proteomic approaches. First, automated machine learning (ML)-based computational workflows with different algorithmic strategies were devised to generate high-quality tissue-specific and tissue-common SARS-CoV-2-human PPIs. Subsequent clustering of highly conserved networks using an optimized complex-based analysis framework uncovered several virally targeted protein complexes (VTCs), reflecting conserved mechanisms of replication. Finally, organ/tissue-specific interaction revealed that NSP3 protein evades host antiviral innate immune signaling by targeting IFIT5 for de-isgylation. Although host interactome is indirectly affected during viral infection, earlier studies have only focused on characterizing the properties of the viral proteins within the host-viral interactions. However, systematically exploring the host-viral interactions from the perspective of the host interactome is essential and should be included in PPI network for a better understanding of viral pathogenesis. In Chapter 3, we combined cofractionation mass spectrometry (CF-MS) with a novel deep learning-based framework, DeepiCE, to map physiologically relevant viral-host and host interactome. First, through comprehensive statistical validations, we demonstrated the remarkable performance of DeepiCE over the state-of-the-art method for network construction. DeepiCE was then applied to co-elution data from salivary samples of individuals infected with SARS-CoV- 2, which led to the generation of high-quality viral-host and host interactome maps highly relevant to SARS-CoV-2 infection. Subsequent clustering of resulting networks using a sophisticated two-stage clustering framework generated high-quality SARS-CoV-2 affected protein complexes, many of which were enriched for diverse cellular processes related to viral pathogenesis and provided new insights into SARS-CoV-2 infection from both the host and pathogen perspective. Despite arduous and time-consuming experimental efforts, PPIs for many pathogenic microbes with their human host are still unknown, limiting our understanding of the intricate interactions during infection and the identification of therapeutic targets. Since computational tools offer a promising alternative, in Chapter 4, we developed a R/Bioconductor package, HPiP software with a series of amino acid sequence property descriptors and an ensemble machine learning classifiers to predict the yet unmapped interactions between pathogen and host proteins. Using SARS-CoV-1 or the novel SARSCoV- 2 coronavirus-human PPI training sets as a case study, we show that HPiP achieves good performance with PPI predictions between SARS-CoV-2 and human proteins, which we confirmed experimentally using several quality control metrics. HPiP also exhibited strong performance in accurately predicting the previously reported PPIs when tested against the sequences of pathogenic bacteria, Mycobacterium tuberculosis and human proteins. Collectively, our fully documented HPiP software will hasten the exploration of PPIs for a systems-level understanding of many understudied pathogens and uncover molecular targets for repurposing existing drugs.