Theses and Dissertations

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Open Access
A circuitous journey of virus characterization and surveillance in North and Central America
(Colorado State University. Libraries, 2023) McMinn, Rebekah J., author; Ebel, Gregory D., advisor; Quackenbush, Sandra, committee member; Brault, Aaron, committee member; Neuwald, Jennifer, committee member
The burden of ticks and the pathogens they carry is increasing worldwide. Powassan virus (POWV, Flaviviridae: Flavivirus), the only known North American tick-borne flavivirus, is of particular concern due to rising cases and the severe morbidity of human disease. In this dissertation we evaluated the recent emergence of POWV from a culmination of field (chapter 2), in vitro (chapter 3), and in-vivo (chapter 4) studies. In addition, we determined the applicability of a vector-enabled surveillance method (xenosurveillance) in Central America (chapter 5). We first used a genetic approach to evaluate the emergence of lineage II POWV, known as deer tick virus (DTV), in parts of North America where human cases occur. We detected DTV-positive ticks from eight of twenty locations in the northeastern United States with an average infection rate of 1.4%. High-depth whole genome sequencing of eighty-four new and archival POWV and DTV samples allowed us to assess geographic and temporal phylodynamics. We observed both stable infection in the northeastern United States and patterns of geographic dispersal within and between regions. Bayesian skyline analysis demonstrated DTV population expansion over the last fifty years. This is concordant with the documented expansion of Ixodes scapularis tick populations and suggests increasing risk of human exposure as the vector spreads. Finally, we isolated sixteen novel viruses in cell culture and demonstrated limited genetic change after passage, a valuable resource for future studies investigating this emerging virus. We then assessed in vitro phenotypes of POWV on human neuronal cells using 16 genetically diverse isolates obtained from a broad geographic and temporal range. We determined over a 10,000-fold range in peak viral titer and significantly decreased cell mortality for two Midwest DTV isolates, though no clear correlation between in vitro phenotype and geo-temporal characteristics could be made. We then performed whole genome sequencing of virus post neuronal cell passage to identify potential residues of interest. Again, no residues could be linked to phenotype, though several interesting residues with increased frequency post-neuronal cell culture were identified. Based on the significant in vitro diversity observed, we sought to assess pathogenesis and tick transmission phenotypes between isolates. We noted neurological disease in mice in both lineages of POWV, with potential low-virulence strains derived from coastal New York. Additionally, we observed an early neuroinvasion phenotype for a Midwest DTV isolate. The ability to infect I. scapularis ticks was determined by feeding on infected host mice (viremic) and through an artificial infection method. Surprisingly, infection rates in ticks via viremic or artificial infection remained consistent between all five isolates tested, resulting in 12-20% infection rate. Taken together, these data demonstrate potential genotype-independent ability to infect ticks and conversely, strain-dependent differences in pathogenesis. In chapter 5, we evaluated a vector-enabled surveillance method ('xenosurveillance') in rural Guatemala. Surveillance methods that permit rapid detection of circulating pathogens are desperately needed. Xenosurveillance is a novel surveillance approach that takes advantage of mosquito feeding behavior to identify blood-borne pathogens that may be circulating in human and animal hosts. This approach circumvents invasive blood sampling of individuals and results in an abundant sample source derived from both humans and animals. In this study, twenty households from two villages (Los Encuentros and Chiquirines) in rural, southwest Guatemala were enrolled and underwent weekly prospective surveillance for 16 weeks. When febrile illness was reported in a household, recently blood-fed mosquitoes were collected from within dwellings and blood samples taken from each member of the household. Mosquitoes were identified to species and blood sources identified by sequencing. Shotgun metagenomic sequencing was used to identify circulating viruses. Culex pipiens (60.9%) and Aedes aegypti (18.6%) were the most abundant mosquitoes collected. Bloodmeal sources were most commonly human (32.6%) and chicken (31.6%), with various other mammal and avian hosts detected. Several mosquito-specific viruses were detected, including Culex orthophasma virus. Human pathogens were not detected. While more intensive sampling may be needed to detect human pathogens, sampling mosquitoes that feed on humans and domestic animals may prove valuable for monitoring pathogens with zoonotic potential.
Open Access
A comprehensive approach to modeling musculoskeletal aging and injury: an emphasis on Nrf2-related pathogenesis
(Colorado State University. Libraries, 2021) Andrie, Kendra M., author; Santangelo, Kelly S., advisor; Hamilton, Karyn, committee member; Goodrich, Laurie, committee member; Podell, Brendan, committee member; Muñoz Gutiérrez, Juan, committee member; Miller, Benjamin, committee member
Osteoarthritis (OA) is a degenerative joint disease that affects over 730 million people globally, over 30 million Americans, and is the leading cause of disability in adults. The underlying pathogenesis is multifactorial and largely undetermined, with a variety of cellular pathways and risk factors contributing to disease onset and progression. The crux of this work is that downregulation in nuclear factor erythroid-2 related factor-2 (Nrf2)-signaling in musculoskeletal tissue serves as a central driver for persistent low-grade inflammation, dysregulation of redox homeostasis, mitochondrial dysfunction, and protein dyshomeostasis, all of which contribute to OA progression. To explore the role of this pathway in OA, we utilized the Hartley OA-prone guinea pig model, which develops naturally occurring idiopathic disease with pathology that mimics human disease. My global hypothesis is supported by preliminary data that demonstrates that aging Hartley guinea pig knee joint tissues have decreased expression of Nrf2 mRNA and protein, which coincides with disease onset and remains decreased throughout OA progression. We investigated the utility of a novel nutraceutical and Nrf2-activator in delaying both the onset and progression of idiopathic OA in this model. The ultimate goal of this work is to (1) identify key molecular pathways involved in the etiopathogenesis of OA, with a particular focus on the contribution of the Nrf2 pathway; (2) investigate the utility of a novel nutraceutical and Nrf2-activator in delaying the onset and/or progression of OA in the Hartley guinea pig, and (3) examine the effects of Nrf2-activation on long bone strength. The inclusion of a musculoskeletal condition beyond OA was also pursued; as such, the clinical and histologic manifestations of a novel rectus femoris myotendinous junction injury model was characterized in rats. Ultimately, this work seeks to advance the understanding of musculoskeletal aging and injury through the analysis of key structural and functional outcome measures to further develop appropriate therapeutic targets for disease prevention and treatment.
Open Access
A derivatization protocol for mycolic acids detection using liquid chromatography/mass spectrometry
(Colorado State University. Libraries, 2012) Zurita Urrea, Paulina, author; Belisle, John T., advisor; Jones, Robert, committee member; Salman, Mo, committee member
New tools for the diagnosis and control of Tuberculosis are major challenges. In this context the use of biomarkers can be applied for detecting characteristic signatures from the tuberculosis-infected host and the pathogen. Mycolic acids are considered as a hallmark of the Mycobacterium genus being abundant in the mycobacterial cell wall. In this study a derivatization protocol was tested to enhance the detection of mycolic acid after the attachment of a quaternary amine and analysis of the derivatized products in the positive ionization mode with liquid chromatography/mass spectrometry. Three groups were considered i) mycolic acid standard ii) human urine spiked with mycolic acid standard, and iii) human serum spiked with mycolic acid standard. Each group included the analysis of a set of non-derivatized mycolic acids in positive and negative ionization mode, and derivatized mycolic acids in positive mode. The derivatization process applied to the mycolic acid standard and to the urine samples spiked with mycolic did not improve the ion volume value compared to the respective non-derivatized samples. Serum samples, however, showed a significant enhancement in the ion volume of the different mycolic acids analyzed compared to the non-derivatized serum samples (α=0.05). The method detection limit for the three groups was also achieved. Urine and serum samples spiked with mycolic acids showed higher detection limits compared to the mycolic acid standard; this was expected due the lipid extraction protocol and the complex nature of these fluids. The derivatization protocol did not improve the method detection limit compared to the non-derivatized samples. The overall results make the derivatization protocol questionable to be applied routinely in biological samples. However, the results obtained after the derivatization of serum samples could point to the advantages of using a derivatization protocol to study possible interactions between mycolic acids and other molecules present in serum that could be impeding their detection.
Open Access
A QuPath workflow utilizing machine learning to analyze homing protein specificity and penetration into lung granulomas of Mycobacterium tuberculosis infected mice
(Colorado State University. Libraries, 2024) Patterson, John, author; Gonzalez-Juarrero, Mercedes, advisor; Lyons, Mike, committee member; Ghosh, Soham, committee member
Targeted delivery of drugs to the lungs can improve TB chemotherapy and thus our goal is to develop TB-drug loaded nanoparticles tagged to pulmonary homing peptides. In a previous study, homing peptides to the lungs of TB diseased animals were identified using preclinical TB models (Balb/c and C3HeB/FeJ mice). The selection of homing peptides was carried out using a phage library containing peptides with known homing affinity in other diseases (e.g. cancer). Having identified and selected the homing peptide PL1 (PPRRGLIKLKTS) to granulomas present in the lungs of murine TB models, the PL1 peptide and a negative control (scrambled LinnTT peptide) were tagged to Fluorescein Amidites (FAM). To facilitate tracking in vivo of the nanoparticles to be loaded with TB drugs, silver nanoparticles (SNP) were conjugated to Cy3 fluorochrome, a fluorescent marker used in in vivo tracking studies, followed by functionalization with the PL1 homing peptide (PL1-SNP) or biotin as negative control (Ctrl-SNP). Tracking and homing of the PL1 peptide to granulomas was possible after in vivo administration via intraperitoneal (IP) or intravenous (IV) route of either the FAM tagged synthetic peptides or Cy3-SNPs to Mycobacterium tuberculosis (Mtb) infected C3HeB/FeJ mice. Visualization of the fluorescence-tagged carriers within the lungs was performed using microscopic slides affixed with lung sections from each mouse followed by whole slide imaging. The semi-quantitative analysis of the fluorescence whole slide images performed using the QuPath workflow confirmed that PL1-FAM, or PL1-SNP homed to the granulomas. Thereafter, a QuPath workflow was developed that uses machine learning approaches (MLP) for unbiased identification of tissue types. Other tools were used for characterization and quantification of FAM (synthetic peptides) and Cy3 (SNP) positive cells within granulomatous lesions of the C3HeB/FeJ TB mouse model. Moreover, it was important to quantify the penetration capacity of the FAM tagged peptide as well as the peptide coated SNP into granulomas. QuPath also includes a built in MLP pixel classifier for unbiased segmentation of the whole slide. In addition, a modified QuPath script was developed to segment the granulomas into concentric regions (outer, inner and center) followed by detection and quantification of positive cells for either fluorochrome within each region. Specific colocalization of PL1 with its known receptor (FN-EDB), either as a synthetic peptide or coupled to the SNP, was also studied using lung sections from mice treated with PL1-FAM or PL1-SNP and counter stained with Alexa 647 conjugated anti-FN-EDB monoclonal antibodies. The modified QuPath script was trained to quantify fluorescence from Alexa 647 in cells within granulomas and the Pearson coefficient and QuPath script was used to assess PL1 and FN-EDB colocalization within each region of the granuloma. The results demonstrated that when compared to their respective control samples, the IP route of administration provides equal or better homing of PL1 peptide to the granulomas than the IV route. Both the PL1-FAM and PL1-SNP home to the granulomas and specifically colocalize with its receptor FN-EDB. The FAM tagged peptide and SNP penetrate to the inner and center regions of the granuloma whereas the control SNP were unable to penetrate the barrier in the outer region of the granulomas. The QuPath workflow developed here can be used for tracking and quantification of other homing peptides and nanoparticles for development of new TB therapeutics.
Open Access
A tale of two viruses: the ability of New World mammals to host Old World viruses
(Colorado State University. Libraries, 2022) Lewis, Juliette, author; Schountz, Tony, advisor; Kading, Rebekah, committee member; Vilander, Allison, committee member; Stoner, Kathryn, committee member
To view the abstract, please see the full text of the document.
Open Access
Aedes aegypti and dengue virus investigation of anatomic, genomic, and molecular determinants of vector competence
(Colorado State University. Libraries, 2009) Bernhardt, Scott Arthur, author; Blair, Carol D., advisor; Black, William C., IV, advisor
Dengue (DENV) causes one of the most rapidly expanding diseases in the tropics. Vector competence (VC) in Aedes aegypti for DENV-2 is a quantitative trait and has been shown to be highly variable. Questions remain as to whether variation in VC continues to exist after the primary field observation. What genetic factors contribute to VC and do these factors evolve from arbovirus exposure remain unclear.
Open Access
An exploration of viral RNA-mediated strategies to stall and repress the cellular exoribonuclease XRN1
(Colorado State University. Libraries, 2018) Charley, Phillida A., author; Wilusz, Jeffrey, advisor; Zabel, Mark, committee member; Perera, Rushika, committee member; Reddy, Anireddy, committee member
The regulation of mRNA decay plays a vital role in determining both the level and quality control of cellular gene expression in eukaryotes. Since they are likely recognized as foreign/unwanted transcripts, viral RNAs must also successfully navigate around the cellular host RNA decay machinery to establish a productive infection. This bypass of the cellular RNA decay machinery can be accomplished in many ways, including the sequestering of regulatory proteins or inactivating enzymatic components. One attractive way for RNA viruses to undermine the cellular RNA decay machinery is to target the cellular exoribonuclease XRN1 since this enzyme plays a major role in mRNA decay, appears to coordinate transcription rates with RNA decay rates, and is localized to the cytoplasm and thus readily accessible to cytoplasmic RNA viruses. We have previously shown that many members of Flaviviridae (e.g. Dengue, West Nile, Hepatitis C and Bovine Viral Diarrhea viruses) use RNA structures in their 5' or 3' untranslated regions (UTRs) to stall and repress XRN1. This results in the stabilization of viral RNAs while also causing significant dysregulation of cellular RNA stability (and thus dysregulation of overall cellular gene expression). In this dissertation we first extend this observation to another member of the Flaviviridae, Zika virus, by demonstrating that structures in the 3' UTR of the viral genomic RNA can stall and repress XRN1. Significantly, we also demonstrate that the 3' UTR of the N mRNA of the ambisense segment of Rift Valley Fever virus, as well as two other phleboviruses of the Phenuiviridae, also can effectively stall and repress XRN1. This observation establishes XRN1 stalling in an additional family of RNA viruses, in this case in the order Bunyavirales. We have mapped the region responsible for XRN1 stalling to a G-rich core of ~50 nucleotides and provide evidence that the formation of a G-quadruplex is contributing to stalling of XRN1. In addition to phleboviruses, we also detected RNA regions that stall XRN1 in the non-coding regions of two other virus families. The 3' UTRs of all four ambisense transcripts of Junin virus, an arenavirus, stall and repress XRN1. This observation was extended to two additional arenaviruses, suggesting that XRN1 stalling may be a conserved property of the 3' UTRs in the Arenaviridae. Finally, we demonstrate that the non-coding RNA from beet necrotic yellow vein virus RNA segment 3 is produced by XRN1 stalling and requires a conserved sequence called the coremin motif. Collectively, these observations establish XRN1 stalling and repression as a major strategy used by many virus families to effectively interface with the cellular RNA decay machinery during infection. We performed two proof of principle studies to extend the significance of the observation of XRN1 stalling during RNA virus infections. First, since XRN1 stalling may be associated with successful viral gene expression as well as cytopathology, we explored whether we could identify a small molecule compound that could interfere with the knot-like three helix RNA junction structure that stalls XRN1 in the 3' UTR of flaviviruses. We tested several triptycene-based molecules, compounds that have been previously shown to intercalate into three helix junctions and identified four triptycene derivatives that interfere with XRN1 stalling. Lastly, we explored whether there might be a cellular exoribonuclease that could navigate through the well-characterized flavivirus structure that effectively stalls XRN1. Our efforts focused on the mammalian Dom3z/DXO enzyme which contains both 5' decapping and 5'-3' exoribonuclease activity. Interestingly, recombinant Dom3z/DXO enzyme did not stall on RNAs containing the 3' UTR of either Dengue virus or the Rift Valley Fever Virus N mRNA. This may suggest that there is a molecular arms race of sorts between the cell and the virus for supremacy of regulating the 5'-3' decay of RNA during infection.
Open Access
Analysis of T cell subsets induced in response to Mycobacterium tuberculosis infection
(Colorado State University. Libraries, 2009) Henao-Tamayo, Marcela I., author; Orme, Ian, advisor
Tuberculosis, an ancient disease, still kills more people each year than does any other bacterial infection. The global epidemic of tuberculosis (TB) results in eight million new tuberculosis cases per year and two million deaths; 98% of these occur in developing countries. At present the only available vaccine against tuberculosis, M. bovis Bacillus Calmette-Guerin (BCG), has proven unreliable and only minimally protects against pulmonary tuberculosis in adults. The reasons why the BCG vaccine is not fully protective are still very unclear, and even though novel vaccines are being developed, there is not a clear understanding of what kind of immune response they should elicit in order to provide maximum protection. The overall aim of these studies, therefore, was to investigate the T cell subsets generated by M. tuberculosis [including clinical strains] as well as further analysis of those generated by BCG, in order to explore their role in protection against M. tuberculosis infection.
Open Access
Analysis of virus-derived small RNAs reveals that the RNA silencing response to flavivirus infection differs dramatically between C6/36 and Aag2 mosquito cell lines
(Colorado State University. Libraries, 2010) Scott, Jaclyn Christine, author; Blair, Carol D., advisor; Olson, Kenneth Edward, committee member; Wilusz, Carol J., committee member; Peersen, Olve, committee member
The exogenous small RNA pathway has been shown to be an important antiviral defense in mosquitoes against arboviruses such as dengue virus (DENV), but little is known about how the pathway and the virus interact in the cell. The studies described in this dissertation examine the how small RNA pathways interact with DENV and a mosquito-only flavivirus, cell-fusing agent virus (CFAV), in mosquito cell cultures. Deep sequencing of virus-specific small RNAs in Aedes aegypti Aag2 cells indicates that DENV2 is targeted by the exogenous RNA interference (RNAi) pathway in this cell line, which is consistent with the DENV2-specific small RNAs seen in DENV2-infected A. aegypti mosquitoes. When the DENV2-specific small RNAs from the Aedes albopictus C6/36 cell line were analyzed, the size and polarity of the small RNAs was not consistent with the exogenous small interfering RNA (siRNA) pathway. Further molecular analysis of the C6/36 cell line indicated that it appears to lack functional Dicer2 processing of long double-stranded RNA (dsRNA). CFAV small RNAs were also discovered in the Aag2 cell line during the deep sequencing analysis. It appears that this cell line is persistently infected with this mosquito-only flavivirus, and the virus is also targeted by the exogenous siRNA pathway in the cells. Sequence comparisons between CFAV and DENV2 RNA did not show long regions of sequence identity between the two viruses, indicating that a sequence-specific mechanism for virus-derived small RNAs from one virus to interfere with replication of the other virus during dual infections seems unlikely. The C6/36 cell line was inadvertently infected with CFAV, but the CFAV-specific small RNAs in C6/36 cells did not appear to be generated from the exogenous siRNA pathway, consistent with the DENV2-specific small RNAs in this cell line. The larger sized, mostly positive sense virus-specific small RNAs found in the C6/36 cells suggest that virus infections may be targeted by another small RNA pathway (such as the piwi-interacting pathway) in this cell line. These studies provide a better understanding of the interactions of DENV2 with the mosquito antiviral RNAi pathway in infected mosquito cells and have revealed a dysfunctional RNAi pathway in the C6/36 cell line. This work also provides a basis for further studies examining the interactions between mosquito-only flaviviruses, arboviruses and the antiviral RNAi pathway.
Open Access
Analyzing the role of the Aedes triseriatus inhibitor of apoptosis 1 gene in transovarial transmission of La Crosse virus
(Colorado State University. Libraries, 2007) Beck, Eric Thomas, author; Beaty, Barry J., advisor
Aedes triseriatus is the primary vector of La Crosse virus (LACV) in North America. The following studies were performed using field collections to elucidate the role of the Ae. triseriatus inhibitor of apoptosis 1 gene (AtIAP1) in conditioning TOT and to compare LACV ovarian titers in field collected mosquitoes with several laboratory Ae. triseriatus strains.
Open Access
Applications of feline immunodeficiency virus as a model to study HIV pathogenesis
(Colorado State University. Libraries, 2018) Miller, Craig Andrew, author; VandeWoude, Susan, advisor; Hoover, Edward, committee member; Han, Sushan, committee member; Webb, Craig, committee member
Feline immunodeficiency virus (FIV) is a naturally-occurring retrovirus that infects domestic and non-domestic feline species, and produces progressive immune depletion that eventually results in an acquired immunodeficiency syndrome (AIDS). While it is accepted that FIV is primarily transmitted by biting, few studies have evaluated FIV oral infection kinetics and transmission mechanisms over the last 20 years. Modern quantitative analyses applied to natural FIV oral infection could significantly further our understanding of lentiviral oral disease and transmission. In this Chapter 1 of this dissertation, I characterized FIV salivary viral kinetics and antibody secretions to more fully document oral viral pathogenesis. The results of this research demonstrate that (i) oral lymphoid tissues serve as a site for enhanced FIV replication, resulting in accumulation of FIV particles and FIV-infected cells in saliva, and (ii) failure to induce a virus-specific oral mucosal antibody response, and/or viral capability to overcome inhibitory components in saliva may perpetuate chronic oral cavity infection. Most importantly, these results provide a model of oral FIV pathogenesis and suggest alternative diagnostic modalities and translational approaches to study oral HIV infection. Feline immunodeficiency virus and human immunodeficiency virus (HIV) utilize parallel modes of receptor-mediated entry. The FIV surface glycoprotein (SU) is an important vaccine target for induction of virus neutralizing antibodies, and autoantibodies to the FIV binding receptor (CD134) block FIV infection ex vivo; highlighting the potential for immunotherapies which utilize anti-receptor antibodies to block viral infection. In Chapter 2 of this dissertation, I immunized cats with soluble CD134, recombinant FIV-SU protein, and/or CD134+SU complexes prior to challenge with FIV to determine if vaccination with CD134-SU complexes could induce protection against FIV infection. Immunization induced production of anti-CD134 and anti-SU antibodies in vaccinated cats, and purified anti-CD134 and anti-SU antibodies significantly inhibited FIV infection in vitro. However, no vaccine combination protected cats from FIV infection in vivo and vaccination induced high titers of antibodies directed at vaccine by-products relative to target antigens. The results of this research reinforce the need to monitor components of vaccine preparations, and emphasize that vaccination may induce proliferation of susceptible target cells and enhancement of heat-labile serum components that counteract neutralizing antibodies. Feline immunodeficiency virus induces lifelong infection in cats and may result in a spectrum of immunodeficiency-related diseases. Both prednisolone and cyclosporine A (CsA) are commonly used clinically to treat lymphoproliferative and immune-mediated diseases in cats, but the impact of these compounds on FIV infection has not been well documented, and their understanding immunomodulatory effects on FIV replication and persistence is critical to guide safe and effective use of these therapies in FIV infected cats. In Chapter 3 of this dissertation, I administered immunosuppressive doses of prednisolone or CsA to cats chronically infected with FIV and monitored alterations in hematological parameters and FIV viral/proviral loads in response to therapy. Interestingly, both treatments caused (i) acute increases in CD4+ lymphocytes, (ii) increased FIV viremia, and (iii) significant alterations in cytokine expression that favored a shift toward a Th2 response. The results of this research highlight the potential for immunosuppressive drug-induced perturbation of FIV replication and underscores the need for consideration of chronic viral infection status when prescribing immunomodulatory medications. Mucosal immune dysfunction, bacterial translocation, systemic immune activation, and chronic inflammation are well-documented features of chronic HIV infection. Despite the success of combinational antiretroviral therapy (cART) in diminishing HIV viral replication and prolonging immune function, a multitude of systemic and local manifestations of HIV infection persist, including the development of chronic inflammation (periodontitis and gingivitis). Commonly used animal models for studying HIV pathogenesis, including SIV/SHIV infections of non-human primates (NHPs) or HIV infections in humanized mice, do not reliably incite oral lesions. In contrast, gingivitis and periodontitis are primary clinical signs associated with untreated natural and experimental FIV-infection, and are principal attributes of this model that may be exploited to investigate pathogenic mechanisms involved in the perturbation of the oral immune system and microbial environment. Therefore, in Chapter 4, I outline the future directions and research goals for my career, and I present preliminary research results obtained thus far in my studies of the pathogenic mechanisms of HIV-induced oral disease. By assessing FIV-associated changes in clinical status, oral microbiota, local and systemic viral burden, and immune profile under such treatment protocols, future studies implementing the feline model of lentiviral-induced oral disease may provide a cornerstone to expand our understanding of the complex interactions between HIV infection, oral immune dysfunction, and the perturbations to the oral microbiota that occur in the context of HIV infection.
Embargo
Assessing antimicrobial mechanisms in Langerhans cells during a Mycobacterium leprae infection
(Colorado State University. Libraries, 2024) Fischbacher, Linda, author; Belisle, John, advisor; Gries, Casey, committee member; Tesfaye, Dawit, committee member
Langerhans cells are essential immune cells in the skin that maintain homeostasis and clear pathogens. Despite their importance, much is unknown about Langerhans cells, including their innate antimicrobial mechanism. Single-cell sequencing of leprosy skin lesions identified genes upregulated in Langerhans cells of reversal reaction lesions that may be associated with antimicrobial activity. CCL22, MPEG1, and IDO1 were selected to study further as potential effectors in Langerhans cells for killing Mycobacterium leprae. We hypothesized that altered metabolic processes in Langerhans cells impact antimicrobial effects on M. leprae. An in vitro model was developed to induce antimicrobial gene expression in Langerhans cell-like dendritic cells (LCDCs). IL-1β was identified as the best inducer of CCL22 and MPEG1, and IFN-γ as the best inducer of IDO1. Induction was measured by gene expression and protein production, as well as enzyme activity for IDO1 by measuring metabolites. The antimicrobial effect of IDO1 on intracellular M. leprae in LCDCs was assessed by inducing IDO1 with IFN-γ or inhibiting IDO1 activity with 1-methyltryptophan. Stimulation by this agonist or this antagonist modulated IDO1 expression and activity but did not affect M. leprae viability. The changes of intracellular tryptophan catabolites in non-stimulated and M. leprae-infected LCDCs were measured. The M. leprae infection increased two kynurenine pathway catabolites after 24 and 48 hours, 3-hydroxyanthranilic acid and quinolinic acid. These data indicate that while M. leprae did not induce IDO1 expression, it did increase IDO1 and kynurenine pathway activity. Neither metabolite has reported antimicrobial properties, but quinolinic acid may benefit M. leprae for synthesizing nicotinamide adenine dinucleotide. A different tryptophan catabolite pathway leads to serotonin production. In M. leprae-infected LCDCs, serotonin was decreased, but 5-hydroxyindoleacetic acid, a breakdown product of serotonin, was increased. The implication of these changes for an M. leprae infection of LCDCs is unknown. 5-hydroxyindoleacetic acid is also increased in Mycobacterium tuberculosis patients. This metabolite may benefit these mycobacteria as it is reported to increase PPARγ activity, which is known to support M. leprae and M. tuberculosis in macrophages. The main antimicrobial mechanism of IDO1 is depleting tryptophan from tryptophan auxotrophic pathogens. Whether the tryptophan biosynthesis pathway in M. leprae is functional was assessed using 13C-tracing, to determine if tryptophan depletion by IDO1 could kill M. leprae. In axenic media, M. leprae did not synthesize tryptophan from 13C-glucose and 13C-palmitic acid nor synthesize tryptophan from intracellular 13C-glucose. In vitro, M. leprae only synthesized tryptophan from an intermediate, anthranilic acid. Using the same method, M. tuberculosis synthesis of tryptophan from 13C-glucose was confirmed as a control. The functionality of the tryptophan biosynthesis pathway in M. leprae could not be confirmed. However, because of the homology between the M. leprae and M. tuberculosis genes for tryptophan biosynthesis, this pathway likely is functional, and M. leprae would not be killed by IDO1-mediated tryptophan depletion. These findings indicate that IDO1 is not associated with antimicrobial activity towards M. leprae in LCDCs. Instead, increased IDO1 activity induced by M. leprae infection resulted in increased tryptophan catabolites likely to benefit rather than kill M. leprae in LCDCs. M. leprae likely evades the primary killing mechanism of IDO1, tryptophan depletion, by possessing an intact pathway for tryptophan biosynthesis. Further studies to elucidate the importance of quinolinic acid and 5-hydroxyindoleactic acid for M. leprae and validate that the M. leprae tryptophan biosynthesis pathway is functional will aid in identifying essential pathways for M. leprae that can be targeted with therapeutics. Other potential antimicrobial effectors in LCDCs, including CCL22 and MPEG1, will need to be assessed to study this innate mechanism in Langerhans cells further.
Open Access
Assessment of anopheles vectorial capacity metrics and malaria transmission factors within the Rimdamal II Study
(Colorado State University. Libraries, 2021) Gray, Lyndsey Irene, author; Foy, Brian, advisor; Kading, Rebekah, committee member; Mueller, Rachel, committee member; Stenglein, Mark, committee member
To view the abstract, please see the full text of the document.
Open Access
Assessment of mosquito and animal model factors in Aedes-borne arbovirus transmission and disease
(Colorado State University. Libraries, 2021) Miller, Megan Rae, author; Foy, Brian, advisor; Wilusz, Jeffrey, committee member; Kading, Rebekah, committee member; Montgomery, Tai, committee member; Magunda, Forgivemore, committee member
To view the abstract, please see the full text of the document.
Open Access
Avian immunity to West Nile virus
(Colorado State University. Libraries, 2008) Nemeth, Nicole M., author; Bowen, R. A., advisor; Spraker, Terry R., advisor
As West Nile virus (WNV) becomes endemic throughout much of North America, it continues to have detrimental effects on countless birds of various taxonomic groups. However, many birds survive infection, mounting an effective immune response. This dissertation focuses on the avian immune response to WNV, including naturally and experimentally-induced antibody duration and passive transfer of immunity. In addition, persistent WNV infection is a potential factor in altering pathogenesis if immunity were to wane. The duration and protection provided by anti-WNV antibodies was documented in house sparrows (Passer domesticus) and raptors for 3-4 years. Antibody levels were relatively stable over time, and protected against viremia in the former and recurrence of clinical disease in the latter. Passive transfer of WNV immunity from hen to eggs and chicks was characterized in domestic chickens (Gallus gallus domesticus). Eggs from both seropositive and seronegative hens were either sacrificed to test for WNV antibody in yolks or chicks artificially inoculated to examine viremic and serologic responses. Concurrently, age-associated differences in response to WNV infection were documented. The passive transfer experiment was repeated in house sparrows to explore this phenomenon in a passerine species; passive transfer was less prevalent in sparrow versus chicken chicks, was of shorter duration, and was less protective. Persistent WNV shedding, viremia, and tissue infection was examined in house sparrows, with juveniles sampled more intensively on a shorter time scale (30-65 days) and adults sampled at 1, 6, 12, 18, and 24 months post-infection. Infectious WNV was isolated from an oral swab, spleen, and kidney of several individuals at 30 DPI, but not from sera after 6 DPI or swabs after 15 DPI. However, WNV was detected in an oral swab by RT-PCR at 44 DPI and was in multiple tissues from most sparrows at 30 DPI, and from kidney and spleen of two individuals at 65 DPI. These findings suggest that WNV infection in tissues may persist beyond the acute stage of infection, while implications for natural transmission and avian health remain unknown.
Open Access
Avian influenza A virus transmission and the emergence of drug resistance
(Colorado State University. Libraries, 2011) Achenbach, Jenna Elizabeth, author; Bowen, Richard A., advisor; Landolt, Gabriele A., committee member; Avery, Anne C., committee member; Frye, Melinda A., committee member
As avian influenza A viruses (AIV) continue to circulate worldwide both naturally, within the reservoir host of wild waterfowl, and cross species barriers, eventually establishing itself in new host species, it is imperative to study the natural reservoir in respect to virus change and transmissibility. This dissertation will focus on the transmissibility of a mallard virus from mallards to other wild and domestic species as well as elucidate the possible outcomes of oseltamivir contamination in the environment and its effect on influenza A virus infected mallards. Low pathogenicity (LP) AIVs of the H5N2 and H7N3 subtypes were utilized to evaluate the ability of transmission of a mallard derived virus to other species present in a co-habitation (barnyard) scenario. Other species in contact with the mallards were chickens, blackbirds, rats, and pigeons. Viral replication was assessed directly from ducks in the barnyard with assessment of the other animals in the barnyard through sero-conversion. Additional animals of each species were directly inoculated with these two viruses and assessed for viral replication. The H5N2 virus was transmitted to other ducks and chickens in the barnyard through either direct or environmental contamination, but not to rats or blackbirds. The H7N3 virus was transmitted to other ducks, chickens, pigeons, and rats. Chickens and blackbirds directly inoculated with both virus strains shed significant amount of virus and seroconverted, but rats and pigeons (except for one pigeon) failed to shed virus but did develop antiviral antibodies. Knowing that both mallard viruses can directly transmit without adaptation, show the mallard to be a good model to further evaluate the outcome of oseltamivir contamination in the environment and its effect on AIV infected mallards. The environment has been shown to be contaminated with significant amounts of oseltamivir carboxylate (OC) in an area of high drug prescription use. We analyzed the outcomes of AIV in infected mallards when they have access to OC in their drinking water. Two separate LPAIV H5N2 viruses were tested for their ability to mutate under drug pressure. One H5N2 virus did not demonstrate any altered sequence after 7-10 days of drug access and infection. The other H5N2 virus did show mutations in the neuraminidase gene that led to an increase in resistance to oseltamivir caused by a specific mutation at E119V. This resistant virus was further evaluated for its ability to transmit between infected and naïve mallards. While the resistant virus did transmit duck to duck, the mutation at position 119 was not detected after challenge or transmission showing instability of this mutation. This could either be a reversion to wild-type or possibly the low level presence of wild-type present in the resistant strain stock that outcompeted with the mutant strain to succeed in the host. This shows, that in these duck experiments, the E119V mutation is not stable in the absence of drug pressure and unlikely to succeed in the host.
Open Access
Bats as reservoir hosts: exploring novel viruses in New World bats
(Colorado State University. Libraries, 2018) Malmlov, Ashley, author; Schountz, Tony, advisor; Bowen, Richard, committee member; Dinsmore, Page, committee member; Pabilonia, Kristy, committee member
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Open Access
Binding of MBNL1 to CUG repeats slows 5'-to-3' RNA decay by XRN2 in a cell culture model of type I myotonic dystrophy
(Colorado State University. Libraries, 2017) Zhang, Junzhen, author; Wilusz, Carol J., advisor; Wilusz, Jeffrey, advisor; Duval, Dawn, committee member; Di Pietro, Santiago, committee member; Yao, Tingting, committee member
Type I myotonic dystrophy (DM1) is a multi-systemic inherited disease caused by expanded CTG repeats within the 3' UTR of the dystrophia myotonica protein kinase (DMPK) gene. The encoded CUG repeat-containing mRNAs are toxic to the cell and accumulate in nuclear foci, where they sequester cellular RNA-binding proteins such as the splicing factor Muscleblind-1 (MBNL1). This leads to widespread changes in gene expression. Currently, there is no treatment or cure for this disease. Targeting CUG repeat-containing mRNAs for degradation is a promising therapeutic avenue for myotonic dystrophy, but we know little about how and where these mutant mRNAs are naturally decayed. We established an inducible C2C12 mouse myoblast model to study decay of reporter mRNAs containing the DMPK 3' UTR with 0 (CUG0) or ~700 (CUG700) CUG repeats and showed that the CUG700 cell line exhibits characteristic accumulation of repeat-containing mRNA in nuclear foci. We utilized qRT-PCR and northern blotting to assess the pathway and rate of decay of these reporter mRNAs following depletion of mRNA decay factors by RNA interference. We have identified four factors that influence decay of the repeat-containing mRNA – the predominantly nuclear 5' 3' exonuclease XRN2, the nuclear exosome containing RRP6, the RNA-binding protein MBNL1, and the nonsense-mediated decay factor, UPF1. We have discovered that the 5' end of the repeat-containing transcript is primarily degraded in the nucleus by XRN2, while the 3' end is decayed by the nuclear exosome. Interestingly, we have shown for the first time that the ribonucleoprotein complex formed by the CUG repeats and MBNL1 proteins represents a barrier for XRN2-mediated decay. We suggest that this limitation in XRN2-mediated decay and the resulting delay in degradation of the repeats and 3' region may play a role in DM1 pathogenesis. Additionally, our results support previous studies suggesting that UPF1 plays a role in initiating the degradation of mutant DMPK transcripts. This work uncovers a new role for MBNL1 in DM1 and other CUG-repeat expansion diseases and identifies the nuclear enzymes involved in decay of the mutant DMPK mRNA. Our model has numerous applications for further dissecting the pathways and factors involved in removing toxic CUG-repeat mRNAs, as well as in identifying and optimizing therapeutics that enhance their turnover.
Open Access
Biofilm dynamics and the response to N-oxides in Burkholderia pseudomallei
(Colorado State University. Libraries, 2019) Mangalea, Mihnea R., author; Borlee, Bradley R., advisor; Slayden, Richard A., committee member; Bowen, Richard A., committee member; Stenglein, Mark D., committee member; Charkowski, Amy O., committee member
Burkholderia pseudomallei is a saprophytic bacterium inhabiting wet soils in tropical regions and is the causative agent of melioidosis, an emerging infectious disease of high mortality. Although the incidence of melioidosis is more prevalent in the monsoonal wet season in Southeast Asia and Northern Australia, gardens and farms also serve as a reservoir for B. pseudomallei infection in the dry season, due to anthropogenic disturbances including irrigation and application of nitrogen (N)-based fertilizer use. Melioidosis is historically associated with rice farming in rural regions of the tropics where rain-fed lowland environments predominate and planting fields are often managed by the addition of N-based fertilizers to keep up with the demand for global rice consumption. In these oxygen-limiting environments, B. pseudomallei is a facultative anaerobic organism capable of growth in anoxic conditions by substituting nitrate (NO3-) as a terminal electron acceptor. B. pseudomallei is capable of complete denitrification, a step-wise enzymatic reaction that is carried out by four individual enzyme complexes or reductases, that reduce NO3- to N2. Denitrification among proteobacteria is regulated by sensing systems that depend on both the presence of substrate and hypoxic conditions, however little is known about this ecological and physiological phenomenon in B. pseudomallei. In hosts infected with B. pseudomallei, similar oxygen tensions are experienced by the organisms in abscesses, lesions, and during intracellular growth; however, little is known regarding the extent of anaerobic metabolism and defense from host-associated reactive nitrogen intermediates in B. pseudomallei. This study examines the predicted nitrate sensing and metabolism genes in a clinical isolate, B. pseudomallei 1026b, and specifically their role in regulating biofilm dynamics. We hypothesized that nitrate sensing and metabolism negatively regulate biofilm formation and aimed to describe the genetic and metabolic determinants of this phenotype in B. pseudomallei. In Aim I of this study, we characterized a dose-dependent biofilm inhibition model that responds to increasing concentrations of sodium nitrate and sodium nitrite, donors of the inorganic anions NO3- and NO2-, respectively. Based on in silico analyses of predicted nitrate sensing and metabolism loci, we screened transposon insertional mutants to identify candidates involved in the biofilm inhibitory response. We identified five mutants that no longer respond to nitrate-mediated biofilm inhibition in genes predicted to comprise key components of the denitrification pathway: the alpha and beta subunits of the dissimilatory nitrate reductase narGHJI-1, the narX-narL two-component regulatory system, and the nitrate/nitrite extrusion gene narK-1. Using LC-MS/MS, we quantified the intracellular concentration of the secondary metabolite cyclic-di-GMP, and observed a significant decrease of this key biofilm-associated molecule in response to sodium nitrate treatment. Furthermore, we evaluated the expression of cyclic-di-GMP regulatory enzymes to propose a mechanism for the nitrate-dependent biofilm inhibition phenotype in B. pseudomallei. In Aim II, we examined the functions of NarX and NarL in response to exogenous sodium nitrate and sodium nitrite and the biofilm inhibition model using separate in-frame deletion mutants. We characterized a disparity in biofilm inhibition that is dependent on nitrate but not nitrite in this two-component sensing system, before analyzing the global transcriptome of these mutants relative to the wild type in growth conditions supplemented with either N-oxide. Differential expression analysis of RNA sequencing reads revealed significant transcriptomic shifts in several gene clusters associated with biofilm formation, nitrate metabolism, general metabolism, antibiotic resistance, virulence, and secondary metabolite biosynthesis that responded similarly to both NO3- and NO2- supplementation. Additionally, we demonstrated that narX and narL mutants are deficient in intracellular survival in murine macrophages, providing a link between nitrate sensing and metabolism and B. pseudomallei host-pathogen interactions. These data suggest that denitrification is an important mechanism for biofilm dynamics and is also relevant to survival and pathogenicity in animal hosts during B. pseudomallei infection.
Open Access
Bioinformatic identification and characterization of cytokinetic regulators in Mtb
(Colorado State University. Libraries, 2013) Crew, Rebecca M., author; Slayden, Richard, advisor; Jackson, Mary, committee member; Hanneman, William, committee member
A fundamental lack of understanding of Mtb regulation during latent tuberculosis infections (LTBI), which comprises the vast majority of tuberculosis cases, has hindered global eradication efforts. To elucidate mechanisms associated with transition to the non-replicating persistent (NRP) state associated with LTBI, we set out to identify regulators involved in cell division control in Mtb. Bioinformatic analysis identified rv1708 as encoding a MinD-like protein putatively involved in septum placement, and rv2216 as encoding a potential SOS-associated cell division inhibitor, SulA. Bioinformatic-based assessments of orthology revealed a differential lineage than anticipated for the proteins encoded by both open reading frames (ORFs). We describe these two novel regulators in Mtb here for the first time. It was found that Rv1708 lacks regions vital for MinD function and shows greater similarity with the Soj protein from Bacillus sp. involved in the regulation of sporulation and timing of division. Due to these similarities we have re-named Rv1708 as SojMtb. Significantly, SojMtb shows potential as a cytokinetic and dormancy regulator both by homology, morphology, and growth kinetic analysis. Overexpression of sojMtb attenuates growth and elicits filamentation characteristic of a disruption in early division, similar to Soj activity in other organisms. Given the role of Soj in the control of dormancy phenotypes in Bacillus sp. we believe SojMtb serves as an important regulator during dormancy transitions in mycobacteria, as associated with the development of LTBI. Although Rv2216 was initially identified by homology to SulA proteins, analysis of orthology indicates greater similarity with a separate group of widely conserved yet poorly defined cell division regulatory proteins. Thusly, we have re-name rv2216 as cdr for cell division regulator. Cdr proteins share limited similarity to SulA: enough to be mis-identified in organisms lacking a true SulA but insufficient to infer similar functionalities. Cdr proteins are present in hundreds of organisms through different walks of life, yet this work presents the first characterization of their effects on cellular activity. Induction of the SOS response by Mitomycin C treatment did not induce cdr expression, supporting our classification Cdr proteins separate from SulA. Overexpression of cdr resulted in a bimodal increase in cell length without an apparent effect on growth kinetics, suggesting Cdr stimulation of cellular elongation relative to division. Profiling of cell cycle discriminant genes in response to cdr overexpression corroborates this hypothesis, showing an induction of late division events associated with the production of new plasma membrane and cell wall components. Sub-cellular localization studies using an inducible Cdr-GFP fusion protein revealed cell cycle-dependent localization to the inner membrane at sites involved in cell wall and plasma membrane growth and remodeling. Furthermore, global transcriptional analysis revealed a unique profile of adaptive programs associated with hypoxia-associated NRP, de novo lipid synthesis and phospholipid/triacylglycerol turnover. These processes are required for normal growth and promote homeostasis during times of stress by preventing and repairing oxidative damage to membrane constituents in diverse organisms. Importantly, Cdr represents a novel regulatory class of proteins with broad representation in all classifications of life, potentially involved in division and stress responses associated with dormancy, and is described here for the first time in Mtb. The foundation provided here, both for SojMtb and Cdr, provides insight into the regulatory mechanisms employed during NRP transitions associated with LTBI, and will aid in the development and implementation of more targeted studied in the future.