Browsing by Author "Wasmuth, James D."

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    Characterizing antimicrobial resistance and virulence genes in Calgary wastewater Escherichia coli isolates
    (2020-01) Lam, Linh; Dong, Tao G.; Wasmuth, James D.; DeVinney, Rebekah
    Antibiotic resistance is becoming a global health issue. The number of infection cases caused by multi-drug resistant pathogens is increasing, causing loss of lives and burdening healthcare costs. As antibiotic resistance is a problem that affects the health of humans, animals, and the environment, solutions require multidisciplinary approaches. One area of focus in research is characterizing antibiotic resistance genes in the aquatic environment, and in particular, wastewater treatment plants (WWTPs). Not only do WWTPS serve to reduce the impact of human activity on the environment, but they also play an important role as a hotspot for horizontal gene transfer, which could accelerate the spread of antibiotic resistance genes. The goal of this project was to characterize antibiotic resistance and virulence genes from a library of multi-drug resistant minicipal wastewater Escherichia coli (E. coli) isolates located in Calgary, Alberta, Canada. We performed preliminary screening for antibiotic resistance of wastewater E. coli isolates collected from 2014 to 2017 (n=9242). Multi-drug resistant isolates were selected to build a sub-library of 400 isolates which were subjected to Dr. Neumann’s group at the University of Alberta for further antibiotic resistance profiling and biochemical testing. From the detailed resistance profile, a sub-library of 82 E. coli isolates with multi-drug resistance was selected for this study. Genomic DNA was extracted from the isolates, then their DNA libraries were prepared before being sent for whole-genome sequencing. Bioinformatics tools were used to analyse their genomic contents and detect resistance and virulence genes in silico. The 82 multi-drug resistant isolates were found to have an open pangenome model, with a large reservoir of accessory genes for adaptation. Several isolates, including ones that were collected after the ultraviolet treatment, clustered closely to known pathogenic strains on the phylogenetic tree and carry typical characteristic virulence genes for various E. coli pathotypes. The results from this study indicate the presence of potential pathogenic E. coli with multi-drug resistance in the environment and highlight the potential of using whole-genome sequencing as a robust tool for diagnostic tests.
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    Creation of Bioinformatics Approaches to Understand Host-Pathogen Molecular Mimicry
    (2024-09-10) Rich, Kaylee Dawn; Wasmuth, James D.; Gilleard, John; Lewis, Ian; Peters, Nathan
    Imitation is not the sincerest form of flattery; for some it is the key to survival. Across phyla, pathogens imitating aspects of their host is crucial to their continued survival and proliferation. Also called molecular mimicry, imitation of host biomolecules has been observed to target a wide range of processes, including host signaling pathways and the immune system. Identification of molecular mimicry can provide insights into pathogen virulence and pathogenicity. Additionally, in silico detection methods can predict currently unknown host-pathogen interactions. Here, I describe my development of a bioinformatic method for mimicry detection using protein sequence data, with the ultimate aim to further our collective understanding of molecular mimicry. In Chapter 2, I resurrected and improved upon a bioinformatic k-mer based mimicry detection method, which I assessed on 32 human-infecting pathogens. During my assessment, I exposed potential concerns with the method and recommended avenues for further improvement. Chapter 3 describes my critical evaluation of each step in the pipeline and subsequent development of mimicDetector, a new pipeline for molecular mimicry identification. During my evaluation, I assessed the performance of five protein search tools with two amino acid substitution scoring matrices on varying query lengths. From these results, I determined the combination best suited to mimicry detection and implemented a new filtering method which better assesses similarity between pathogen and host protein sequences. Lastly, in Chapter 4, I applied mimicDetector to a focused investigation of the causative agent of human tuberculosis, Mycobacterium tuberculosis (Mtb). Among my predicted mimics were potentially novel Mtb-human protein interactions as well as a protein frequently implicated in Mtb molecular mimicry. My investigation highlights strategies used by Mtb to subvert host defenses and provides insight into the role mimics play in pathogenicity and virulence.
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    Development and application of ultra-sensitive tools for the detection of malaria
    (2020-01) Mohon, Md Abu Naser; Pillai, Dylan R.; Wasmuth, James D.; Parkins, Michael D.
    The goal to eliminate malaria has been challenged by the lack of accurate diagnostic tools to identify symptomatic, asymptomatic, and drug-resistant malaria carriers. In this dissertation, we have shown the potential of the Loop-mediated Isothermal Amplification (LAMP)-based diagnostic approaches to be a powerful tool available for malaria elimination. We have validated the combination of the Non-instrumented Nucleic Acid (NINA) platform heater (PATH, Seattle) with a commercial LAMP kit (LoopAmp malaria Pan/Pf detection kit), with a view to deploying it in extremely resource-limited settings in the future. An ultrasensitive (US)-LAMP assay was also developed and validated to identify asymptomatic malaria reservoirs. Moreover, a novel strategy for detecting single nucleotide polymorphisms (SNPs) by the LAMP method was designed and deployed for spotting artemisinin resistance in P. falciparum. We conclude that the NINA-LAMP assay can be a convenient test for detecting symptomatic malaria cases with a sensitivity of 100% and specificity of 98.6% compared to the gold standard nested PCR. Additionally, the US-LAMP assay was able to achieve a limit of detection (LOD) between 25 to 100 parasites/mL from dried blood spots. We have also found that the overall prevalence of asymptomatic malaria was 22.1% in the Gambella region of Ethiopia, detected by the US-LAMP assay. The sensitivity and the specificity of the US-LAMP assay were 92.6% and 97.1%, respectively compared to an ultrasensitive quantitative reverse transcriptase PCR. Additionally, the SNP-LAMP assay was 100% sensitive and 97.3% specific to identify the C580Y mutation in the kelch 13 propeller gene, which is known as the major genetic determinant of artemisinin resistance in Southeast Asia. Furthermore, we conclude that artemisinin resistance-linked kelch 13 propeller mutations are absent in the Bangladeshi P. falciparum isolates. However, two cases of the A578S SNP in the kelch 13 propeller gene were found in those P. falciparum isolates, although this SNP was not associated with artemisinin resistance. In conclusion, as the LAMP-based diagnostic approaches are simple, low-cost, and accurate compared to currently available nucleic acid tests, they can be used at different aspects to diagnose malaria and expedite elimination.
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    The impact of transposable elements on genomes of parasitic nematodes
    (2020-07-06) Dunemann, Sonja Maria; Wasmuth, James D.; Lynch, Tarah; Deardon, Rob; Yeaman, Sam
    Parasitic nematodes infect many animal and plant species. In humans, they cause significant levels of morbidity and death in low and middle income countries. In livestock and crop plants, they have a significant, negative economic impact, globally. To better understand the biology of these organisms, we must understand their genetic makeup. Up to 36% of parasitic nematode genomes consist of transposable elements (TEs). TEs are mobile, repetitive elements, and act as major players of evolution due to their ability to transfer horizontally between different taxa, move within genomes, and impact the phenotype of their host. However, TEs are often sidelined in standard genomic and transcriptomic analyses. Hence, we know little about their impact on and interaction with nematodes. Here I show that TEs can be horizontally transferred between parasitic nematodes and their hosts, and that TEs are actively mobilizing themselves throughout the life-cycle of two parasitic species. I compare different phylogenetic methods to test for horizontal transfer of TEs between taxa using AviRTE, a LINE element that has been shown previously to have horizontally transferred between birds and parasitic nematodes. I find that phylogenetic trees of TEs based on coding regions differ from trees based on full-length sequences. I identify another TE, RTE1\_Sar, that was horizontally transferred between parasitic nematodes and the common shrew. To rule out contamination of the shrew genome, I develop a pipeline called ConTest that tests for contamination by comparison of TE flanking sequences to two sequence databases. To better understand potential consequences of TE insertions, I investigate underlying mechanism of TE expression. I find that TE expression is specific to developmental stage, and that genes and TEs have potential transcript chimera. This thesis shows that transposons might have transferred more often between parasites and hosts than previously thought, and provides a pipeline to test if a genomic sequence is based on contamination. Furthermore, this work lays an early foundation to study TE impact on parasite genomes by showing that the majority of TE expression arises from read-through transcription, but that younger LINE elements are active and continue to shape genome evolution.
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    Impacts of Lake Physical Characteristics and the Presence of a Non-native Species on Diet Specialization in Threespine Stickleback (Gasterosteus aculeatus)
    (2018-09-21) Curley, Douglas Taylor; Vamosi, Steven M.; Vamosi, Jana C.; Galpern, Paul; Wasmuth, James D.
    The diet and dietary morphological adaptations of populations have been shown to be influenced by both the physical characteristics of their habitat and the presence of non-native species. In this study, I assessed how diet varies within and among populations of threespine stickleback (Gasterosteus aculeatus) in lakes with varying bathymetry in southwestern British Columbia. These lakes also differed in whether they contained the invasive signal crayfish (Pacifastacus leniusculus). Diet was evaluated through gut content analysis. I also investigated how morphology differs between these populations, as morphology has been shown to be linked to diet in stickleback. Diet data did not reveal any significant differences in diet among populations. However, morphology data did reveal significant differences among lakes and between crayfish conditions. The data suggest that diets higher in benthic invertebrates are associated with lakes with more littoral area and with the presence of signal crayfish.
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    Investigating the genetic basis of ivermectin resistance in Haemonchus contortus
    (2018-04-30) Rezansoff, Andrew Mischa; Gilleard, John Stuart; Hansen, David; Rogers, Sean M.; Mains, Paul E.; Wasmuth, James D.; Geary, Tim C.
    Parasitic nematodes have a major impact on human and animal health and their control is threatened by the emergence of resistance to the anthelmintic drugs on which control depends. Although resistance to ivermectin is widespread in many livestock parasites, the genetic mechanisms remain elusive. In this thesis, a number of approaches are undertaken to investigate the genetics of ivermectin resistance in Haemonchus contortus, an important parasitic nematode research model. A transcriptomic comparison of the susceptible MHco3(ISE) and two ivermectin resistant strains, MHco4(WRS) and MHco10(CAVR), was undertaken to investigate if differences in gene expression revealed insights into ivermectin resistance mechanisms. Although this approach did not reveal clear ivermectin resistance gene candidates, the results have some important implications for RNAseq analysis in genetically diverse organisms. First, extremely high levels of genetic diversity in H. contortus had a major impact on RNAseq analysis and needed to be accounted for reliable identification of differentially expressed genes. Second, there were a remarkably large number of genes differentially expressed between the three H. contortus strains. In a novel genetic crossing approach, two independent serially backcrossed H. contortus populations were utilized in which ivermectin resistance loci had been introgressed into the MHco3(ISE) genome. Markers for six leading candidate ivermectin resistance genes from the literature were examined and none were found to show evidence of introgression in the backcross populations. A seventh marker, Hcms8a20 showed clear evidence of introgression and was later confirmed by collaborators using a whole genome sequencing approach to show its location in a 11.2 Mb introgressed region. A set of 25 in vivo or in vitro phenotyped ivermectin resistant H. contortus field populations were then used to investigate evidence of selection on the Hcms8a20 marker and four other loci distributed across the 11.2 Mb introgressed region. A deep amplicon sequencing approach revealed that, of the loci tested, only Hcms8a20 showed consistent evidence of purifying selection in the ivermectin resistant field populations. Overall, the results provide strong evidence that a major ivermectin resistance locus is located within a few megabases of the Hcms8a20 locus in many different ivermectin resistant H. contortus field populations.
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    Predicting the Evolutionary and Medical Significance of Human Genetic Variations with Machine Learning
    (2019-04-30) Saha Mandal, Arnab; De Koning, A. P. Jason; Bernier, François P.; Wasmuth, James D.; Rodrigue, Nicolas
    The advent of inexpensive and high-throughput genome sequencing technologies has facilitated the acquisition of patient exome and genome sequences at a vast scale. One of the primary challenges of such data is its functional interpretation, and specifically, the ability to distinguish functionally important, deleterious, and pathogenic variants from neutral or benign variants (“variant impact prediction” or VIP). Over the last two decades, many approaches have been proposed for VIP, which utilize data from patterns of evolutionary conservation, population genomics, protein structures and other sources to inform machine learning classification algorithms. However, existing approaches are fraught with limitations, especially when they are trained on databases of putatively pathogenic variants that may have been identified with reference to existing prediction methods (a type of ‘circularity’). This dissertation identifies shortcomings of existing variant impact prediction methods and discusses how they can be better understood (Chapter 1). Approaches to overcome these shortcomings are presented (Chapter 2), and a new method, TAIGA (Transformation and Integration of Genomic Annotations), is developed. The utility of this method and its accompanying refinements are evaluated (Chapter 3) and later scrutinized (Chapter 4). As part of this work, I have produced TAIGA scores for all protein coding positions of the human genome, and I show these have substantially superior performance in distinguishing known pathogenic variations from neutral variations in a number of high-quality datasets. Variant prediction scores from TAIGA are later integrated with clinical information from human phenotypes (Chapter 5) and this extension demonstrated the highest sensitivity and smallest candidate gene search space over a large set of rare genetic disorders. It is my hope that TAIGA will aide clinicians and researchers alike in the new era of personalized genomic medicine in which we find ourselves.
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    Transcriptional patterns of sexual dimorphism and in host developmental programs in the model parasitic nematode Heligmosomoides bakeri
    (2023-05-28) Pollo, Stephen M. J.; Leon-Coria, Aralia; Liu, Hongrui; Cruces-Gonzalez, David; Finney, Constance A. M.; Wasmuth, James D.
    Abstract Background Heligmosomoides bakeri (often mistaken for Heligmosomoides polygyrus) is a promising model for parasitic nematodes with the key advantage of being amenable to study and manipulation within a controlled laboratory environment. While draft genome sequences are available for this worm, which allow for comparative genomic analyses between nematodes, there is a notable lack of information on its gene expression. Methods We generated biologically replicated RNA-seq datasets from samples taken throughout the parasitic life of H. bakeri. RNA from tissue-dwelling and lumen-dwelling worms, collected under a dissection microscope, was sequenced on an Illumina platform. Results We find extensive transcriptional sexual dimorphism throughout the fourth larval and adult stages of this parasite and identify alternative splicing, glycosylation, and ubiquitination as particularly important processes for establishing and/or maintaining sex-specific gene expression in this species. We find sex-linked differences in transcription related to aging and oxidative and osmotic stress responses. We observe a starvation-like signature among transcripts whose expression is consistently upregulated in males, which may reflect a higher energy expenditure by male worms. We detect evidence of increased importance for anaerobic respiration among the adult worms, which coincides with the parasite’s migration into the physiologically hypoxic environment of the intestinal lumen. Furthermore, we hypothesize that oxygen concentration may be an important driver of the worms encysting in the intestinal mucosa as larvae, which not only fully exposes the worms to their host’s immune system but also shapes many of the interactions between the host and parasite. We find stage- and sex-specific variation in the expression of immunomodulatory genes and in anthelmintic targets. Conclusions We examine how different the male and female worms are at the molecular level and describe major developmental events that occur in the worm, which extend our understanding of the interactions between this parasite and its host. In addition to generating new hypotheses for follow-up experiments into the worm’s behavior, physiology, and metabolism, our datasets enable future more in-depth comparisons between nematodes to better define the utility of H. bakeri as a model for parasitic nematodes in general. Graphical Abstract