Theses and Dissertations from UMD
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Item FOOD SAFETY IN THE ERA OF NEXT-GENERATION SEQUENCING: GENOMIC CHARACTERIZATION OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI AND METAGENOMIC SURVEILLANCE OF IRRIGATION SURFACE WATER(2023) Huang, Xinyang; Meng, Jianghong; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In this study, we first utilized high-throughput next-generation sequencing (NGS) and bioinformatic analyses to characterize potential public health threats posed by non-top-7 Shiga toxin-producing Escherichia coli (STEC). NGS allowed us to detect virulence (n = 46) and antimicrobial resistance (AMR) (n = 27) factors within the genomes of the STEC strains, to make genome-wide comparisons with published human clinical isolates, and to characterize three novel O-antigen gene clusters. We found that the distribution of 33 virulence genes and 15 AMR determinants exhibited significant differences among serotypes (P < 0.05), and that 47 strains were genetically related to human clinical strains inferred from a pan-genome phylogenetic tree. We secondly developed a web tool, PhyloPlus, that allowed users to generate customized bacterial and archaeal phylogenies, which can be incorporated into their own microbial community studies. We also utilized two public datasets (human microbiome, n = 60; fermented food metagenomes, n = 62) to illustrate how application of phylogeny improved our analyses. We showed that the integration of phylogenies introduced alternative phylogeny-based diversity metrics and allowed more conservative null model constructions, thereby reducing potential inflation of type I errors. Finally, we employed deep metagenomic shotgun sequencing, and our developed web tool, to investigate on a collection of 404 surface water samples collected from four regions in Latin America. We reported the high detection rates of pathogenic and contaminant bacteria in these samples, including Salmonella (29.21%), Listeria (6.19%), and E. coli (35.64%), necessitating the monitoring and proper treatment on these surface waters. We also described the regional differences in terms of sample taxonomic composition and the resistome, and further presented key factors that drove the separation patterns for each sampling region. We utilized recent metagenomic assembly and binning algorithms to report the construction of 1,461 de-replicated metagenome-assembled genomes (MAGs) that were of at least medium quality. The incorporation of the MAGs into the taxonomic classifier Kraken2’s database led to a 12.85% increase in classifiable sequence reads. Additionally, we conducted network analysis on AMR genes and the genus-level taxonomy, based on assembled contigs, to provide information to better understand the dynamics of the transferring of AMR genes.Item Diversity, dynamics, and dissemination of microbial communities in reclaimed and untreated surface waters used for agricultural irrigation(2019) Chopyk, Jessica; Sapkota, Amy R; Public and Community Health; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)High quality freshwater is a vital resource for sustaining agriculture and feeding a growing global population. Yet, due to increasing declines in groundwater, key food production regions across the world face uncertainty with regard to water availability. Nontraditional irrigation water sources, such as reclaimed water (advanced treated municipal wastewater) and untreated surface water (e.g. creeks, ponds, and brackish rivers), may contribute to sustainable solutions to conserve groundwater supplies. However, the microbial community composition and dynamics within these water sources are typically poorly characterized and comparative analysis of their microbial communities are rare. Using high-throughput, cultivation-independent sequencing methodologies, this dissertation research focused on three aims: 1) exploring the functional and taxonomic features of bacteria in nontraditional irrigation water sources; 2) assessing the bacterial and viral communities of agricultural pond water in relation to seasonality; and 3) describing the dynamics, composition, and potential dissemination of irrigation water microbiota from a freshwater creek to an irrigated field. The first aim was addressed through a broad investigation of bacteria within agricultural ponds, freshwater creeks, brackish rivers, and reclamation facilities. Through metagenomic-based analyses, features of the bacterial community, such as antimicrobial resistance genes (ARGs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) arrays, were found to vary by sampling date and specific site. For the second aim, agricultural pond water was sampled over two time periods and found to harbor diverse bacteria and bacteriophage species, the abundance and composition of which were influenced by factors characteristic of the pond’s topography and seasonality. For the final aim, samples from a creek used actively for agricultural irrigation, as well as samples of pre- and post-irrigated soil, were analyzed. ARGs and virulence factors were identified in the water and soil samples, with the majority being specific to their respective environment. Moreover, analyses of CRISPR arrays from the creek samples indicated the persistence of certain bacterial lineages, as well as specific interactions between creek bacteriophage and their hosts. Overall, this research improves scientific knowledge of bacterial and viral composition, dynamics, and interactions that can be utilized to assess the suitability and safety of nontraditional irrigation water sources.Item METABOLIC VIRULENCE DETERMINANTS AND RAPID MOLECULAR DIAGNOSTICS OF PATHOGENIC SPIROCHETES(2016) Backstedt, Brian; Pal, Utpal; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Borrelia burgdorferi and Leptospira interrogans are pathogenic spirochetes that elicit serious health threats, termed as Lyme disease and leptospirosis. Key areas of spirochete research involve a better understanding of their intriguing biology and infection, including identification of novel virulence factors and improvements in diagnostic and preventive methods. Notably, certain bacterial metabolic enzymes are surface-exposed, having evolved to acquire additional functions referred to as protein moonlighting that contributes in microbial virulence. Comparative genome analysis revealed that certain components of sugar metabolism pathways are either absent or seemingly inactive in pathogenic spirochetes, which were studied herein for their potential roles as metabolic virulence factors. Of nine borrelial enzymes investigated, only phosphomannose isomerase (PMI) was found to be surface-exposed and remained enzymatically active in the spirochete outer membrane. PMI is critical for mannose metabolism and facilitates the interconversion of fructose 6-phosphate and mannose-6-phosphate, although its occurrence in borrelial surface remains enigmatic. PMI may provide a critical function for B. burgdorferi viability as it is constitutively expressed and all attempts to create genetic mutants remained unsuccessful. Active immunization studies using recombinant PMI did not influence the outcome of infection within tick or murine hosts, although a significant reduction in bacterial levels within the joints of mice was recorded, suggesting its involvement in spirochete persistence in a tissue-specific manner. Despite substantial advancement, the development of more effective diagnostics for leptospirosis and Lyme disease still remains a critical need since human vaccines are unavailable. Antibiotic treatment can resolve these infections but is most effective when administered early during infection, prior to pathogen dissemination to distant organs. As diagnostic methods for spirochete infection still depends on ineffective and antiquated technologies, we sought to develop novel RNA-based assays for better detection of early spirochete infection. Results indicated that targeting specific regions of 16S and 23S ribosomal RNA targets provided the highest possible sensitivity and specificity of detection, which was far superior to current serological, microbiological or molecular methods used to detect presence of invading pathogens.Item Characterization of the TrxSR Two-Component Signal Transduction System of Streptococcus pyogenes and its Role in Virulence Regulation(2011) Gold, Kathryn; McIver, Kevin; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The Gram-positive group A streptococcus (GAS) is a strict human pathogen, which causes a wide variety of infections, ranging in severity from minor to life threatening. In order to cause such a diverse array of diseases, GAS utilizes two-component signal transduction systems (TCS) to coordinately regulate sets of virulence genes in response to changing host conditions. The present study investigates the role of the TrxSR TCS in the regulation of virulence of the GAS. Using an insertional inactivation mutation in TrxR in serotype M1 MGAS5005, transcriptome studies established that TrxR activates transcription of Mga-regulated virulence genes, a separate non-TCS regulatory pathway controlling factors important for immune evasion and colonization. Transcriptional reporter fusions of Pmga to firefly luciferase revealed that the TrxR regulation occurs through the Pmga promoter. Additionally, electrophoretic mobility shift assays using purified His-MBP tagged TrxR established specific binding of TrxR to Pmga, although the interaction appeared to be transient. To determine the importance of signal transduction for TrxR-mediated regulation of the Mga regulon and virulence, an in vitro reconstitution assay was performed with purified TrxR and TrxS. Using both wild type and mutated forms of the TrxSR proteins, we demonstrated that TrxSR is a functional two-component phosphorelay system. Interestingly, phosphorylation of TrxR did not appear to be critical for DNA binding and regulation, since a TrxR D55A mutation did not change the expression of TrxR regulated genes in GAS based on EMSA and qPCR. In order to investigate whether there is a functional conservation of TrxR's involvement in GAS virulence regulation, mutations were made in serotype M4 and M49 strains representing either throat only or generalist strains. We have determined that TrxR regulates mga and Mga-regulated genes (emm, arp) in the M4 and M49 backgrounds, suggesting conservation of TrxR's role in virulence regulation. Overall, TrxSR represents a functional TCS that appears to directly regulate the Mga virulence regulon independent of phosphorelay. Furthermore, the functional conservation of TrxR regulation of Mga in other serotypes suggests a conserved role for its involvement in virulence regulation in GAS.Item CHARACTERIZATION OF TWO HIGHLY CONSERVED POXVIRUS TRANSMEMBRANE PROTEINS OF UNKNOWN FUNCTION(2009) Sood, Cindy Leigh; Moss, Bernard; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The vaccinia virus I5L open reading frame encodes a 79-amino-acid protein, with two predicted transmembrane domains, conserved among all sequenced members of the chordopoxvirus subfamily. No nonpoxvirus homologs or functional motifs have been recognized, and the role of the I5 protein remains unknown. I5 synthesis was dependent on viral DNA replication and occurred exclusively at late times, consistent with a consensus late promoter motif adjacent to the start of the open reading frame. I5 was present in preparations of purified virions and could be extracted with nonionic detergent, suggesting membrane insertion. Transmission electron microscopy of immunogold-labeled thawed cryosections of infected cells revealed the association of an epitope-tagged I5 with the membranes of immature and mature virions. Viable I5L deletion and frameshift mutants were constructed and found to replicate like wild-type virus in a variety of cell lines, indicating that the protein was dispensable for in vitro cultivation. However, mouse intranasal challenge experiments indicated that a mutant virus with a frameshift resulting in a stop codon near the N terminus of I5 was attenuated compared to control virus. The attenuation correlated with clearance of mutant viruses from the respiratory tract and with less progression and earlier resolution of pathological changes. We suggest that I5 is involved in an aspect of host defense that is evolutionarily conserved although a role in cell tropism should also be considered. The vaccinia virus A43R open reading frame encodes a 168-amino acid protein with a predicted N-terminal signal sequence and a C-terminal transmembrane domain. Although A43R is conserved in all sequenced members of the orthopoxvirus genus, no non-orthopoxvirus homolog or functional motif was recognized. Biochemical and confocal microscopic studies indicated that A43 is expressed at late times following viral DNA synthesis and is a type-1 membrane protein with two N-linked oligosaccharide chains. Neither mature nor enveloped virions contained appreciable amounts of A43, which was detected in Golgi and plasma membranes. Loss of A43R expression had no discernible effect on plaque size or virus replication in cell culture and little effect on virulence in a mouse intranasal infection model. Although the A43 mutant produced significantly smaller lesions in the skin of mice than the control, the amounts of virus recovered from the lesions were similar.Item Regulation of Virulence by BarA-UvrY Two-Component system and LuxS in Extraintestinal Pathogenic Escherichia coli.(2007-12-05) Palaniyandi, Senthilkumar; Mukhopadhyay, Suman; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Pathogenic E. coli cause intestinal or extraintestinal infections in many host species. E. coli strains enter the intestinal tract through food and colonize the intestinal epithelium to cause infections. In animals and humans, E. coli causes gastroenteritis, neonatal meningitis and urinary tract infections. In birds, E. coli causes a complex syndrome called avian colibacillosis. The orthologs of BarA-UvrY two-component (TCS) system is known to regulate a number of phenotypic traits in gamma proteobacteria, although their role in Extraintestinal pathogenic Escherichia coli (ExPEC) virulence is yet to be determined. The barA gene is membrane bound sensor kinase protein and the uvrY gene encodes the cognate response regulator in E. coli. Work in this study has focused how the BarA-UvrY and LuxS system regulates in vivo virulence in uropathogenic E. coli (UPEC) and avian pathogenic E. coli (APEC) during infection. The main goal of this study is to look at how BarA-UvrY TCS and LuxS regulate virulence in APEC 7122 and UPEC CFT073. In this study, we studied the role of BarA-UvrY TCS system in regulation of virulence in the aforementioned ExPEC strains using animal model and tissue culture system and the role of LuxS in regulation of virulence determination in ExPEC. Our results indicate that BarA-UvrY regulates multiple virulence properties in APEC 7122 and UPEC CFT073 and that LuxS regulates partial virulence properties in APEC 7122 and UPEC CFT073.