Repository logo

Browsing by Author "Belisle, John, advisor"

Now showing 1 - 7 of 7
  • Thumbnail Image
    ItemEmbargo
    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.
  • Thumbnail Image
    ItemOpen Access
    Characterization and quantification of urinary metabolic biomarkers for early response to anti-tuberculosis treatment
    (Colorado State University. Libraries, 2016) Fitzgerald, Bryna, author; Belisle, John, advisor; Crick, Dean, committee member; Dobos, Karen, committee member; Cohen, Robert, committee member
    Development of new anti-tuberculosis (TB) therapies remains a major priority to combat this infectious disease and to prevent continued transmission of the causative agent Mycobacterium tuberculosis (Mtb). However, newly developed therapies require large, lengthy clinical trials to determine the number of treatment failures and relapses for evaluation of treatment efficacy. Biomarkers for the prediction of treatment outcome in TB patients at early time points would facilitate movement of new therapies through clinical trials. Previously, liquid chromatography-mass spectrometry (LC-MS) based metabolomics experiments identified potential biomarkers for early response to anti-TB treatment. The research presented in this dissertation involves experiments needed for the progression of these compounds towards a clinically useful biosignature. A major impediment to metabolomics-based biomarker discovery is metabolite identification, as approximately 50% of detectable products do not match structures in existing databases. In concordance with this, several of the potential small molecule biomarkers of anti-TB treatment response lacked structural identification. This research resulted in the structural characterization of three of these compounds as a core 1 O-glycosylated SerLeu peptide, N-acetylisoputreanine, and N1, N12-diacetylspermine. Both the core 1 O-glycosylated SerLeu peptide and N-acetylisoputreanine are novel compounds that had not been previously detected in human urine. Characterization of these compounds indicated a potential alteration of polyamine catabolism and the complement and coagulation pathways during anti-TB treatment. Another key aspect in biomarker discovery is defining the processes involved in formation of potential biomarkers. In order to determine whether these compounds were formed by processes upregulated during active disease, the abundances of these compounds were assessed in active TB patients and household contacts as well as in Mtb infected and uninfected Balb/c mice. The core 1 O-glycosylated SerLeu peptide and N1, N12-diacetylspermine were increased in the urine of index patients demonstrating a potential link between Mtb infection, associated disease pathology, and the formation of these compounds. N-acetylisoputreanine, however, was not increased in TB patient urine or infected mouse tissue indicating that this compound may be formed due to off target drug interactions. These experiments not only provided insights into the mechanisms behind alteration of these compounds during anti-TB treatment, but also highlighted those compounds that may be better biomarkers for anti-TB treatment response. Assessment of these compounds using an independent set of patient samples is needed to validate them as biomarkers for early anti-TB treatment response. Unlike the untargeted experiments used for discovery of potential biomarkers, validation typically employs targeted assays. This research describes the development of a targeted multiple reaction monitoring (MRM) assay which enabled accurate and precise quantitation of compounds previously detected in an untargeted metabolomics experiment. This targeted assay will be used for validation of these compounds in a larger set of patient samples representing a variety of different treatment outcomes. Overall these experiments confirmed the identity of three metabolites that decrease with anti-TB treatment response. Two of these metabolites are novel compounds and their characterization adds to metabolite databases expanding the number of metabolites available to other metabolomics researchers. Assessment of these compounds in samples representative of active TB disease confirmed two of them as promising biomarkers for anti-TB treatment response and highlighted another as a potential result of unintended drug effects. The development of a MRM assay for the quantification of these compounds enables their validation and confirmation as biomarkers of anti-TB treatment response. The work presented in this dissertation describes the advancement of metabolites identified during biomarker discovery towards application in clinical trials.
  • Thumbnail Image
    ItemOpen Access
    Complex regulation of BpeEF-OprC mediated drug efflux in Burkholderia pseudomallei
    (Colorado State University. Libraries, 2016) Rhodes, Katherine, author; Belisle, John, advisor; Schweizer, Herbert, advisor; Dow, Steven, committee member; Stargell, Laurie, committee member
    Burkholderia pseudomallei (Bp) is a Gram-negative bacillus and the etiologic agent of melioidosis, a multifaceted syndrome causing high mortality in tropical regions of the world. The bacteria is classified as a Tier-1 Select Agent due to the seriousness of infection, low infectious dose, lack of effective vaccine, and difficulty of treatment. Bp’s many acquired and intrinsic antimicrobial resistance determinants make the study of these factors vital to improving the efficacy of bi-phasic treatment currently used to treat melioidosis. This study examines one factor in particular: the BpeEF-OprC efflux pump, a member of the resistance-nodulation and cell division family of efflux proteins, and capable of extruding both trimethoprim and sulfamethoxazole. A combination of these compounds (co-trimoxazole) is the first line of eradication phase therapy, making BpeEF-OprC the most clinically important efflux pump encoded by Bp. In spite of this, little is understood of the regulation of bpeEF-oprC, other than it is controlled in part by two LysR family proteins, BpeS, and BpeT. We hypothesized that these regulatory proteins 1) exert their action(s) by interacting with bpeEF-oprC at a specific site within the bpeT-llpE-bpeEF-oprC intergenic region, 2) are capable of influencing transcription of additional operons, and 3) that mutations to these proteins altered ability to form multimers, thereby influencing their function as observed by increased co-trimoxazole resistance and bpeF transcript levels. In Aim I of the study, we identified the cis regulatory regions by which these proteins interact within the bpeT-llpE-bpeE intergenic region using a combination of 5’ deletion assays, S1 nuclease protection, fluorescent-linked oligo extension and electrophoretic mobility assays. With this information we were able to locate bpeT transcriptional start sites and promoter regions as well as binding sites for both BpeT and BpeS. In Aim II, we examined the function of BpeT and BpeS as trans regulatory factors of BpeEF-OprC through mutation and deletion of both genes in part I, and as global regulatory factors in part II. Through overexpression and qRT-PCR or MIC analysis of wild type and mutant forms of both genes, we observed that while BpeT is a direct transcriptional activator of bpeEF-oprC, BpeS is not. Additionally, mutation position in BpeS seems to play a role in the expression phenotype of bpeEF-oprC. However, these mutations do not influence the ability of BpeS or BpeT to form multimers, as we observed no change between wild type and mutant protein oligomer formation through low-pressure gel chromatography and native gel electrophoresis. These same mutations also appeared to have no deleterious effect on the ability of the protein to bind their consensus region within the IR. Additionally, the loss of both genes did not interrupt the ability of bpeEF-oprC to be induced by substrates of BpeEF-OprC, suggesting an additional regulatory factor is at play. In Part II, RNA sequencing analysis and confirmation of select transcriptionally altered operons by RT-qPCR revealed that BpeS might influence expression of the Bsa Type 3 Secretion System (T3SS), while BpeT seems only to target bpeEF-oprC. This may have implication in the pathogenesis of Bp, and must be confirmed in in-vivo cell models using Select Agent excluded strain Bp82 in order to solidify the link between efflux and T3SS during infection. Ultimately, more work is needed to identify the missing regulatory factors in play during expression of bpeEF-oprC, understand how mutations to BpeT and BpeS alter their function, and confirm the relevance of a putative link between co-regulation of efflux and virulence during Bp infection.
  • Thumbnail Image
    ItemOpen Access
    Defining interactions between Mycobacterium leprae and Langerhans cells
    (Colorado State University. Libraries, 2021) Fletcher, Darcy, author; Belisle, John, advisor; Henao-Tamayo, Marcela, committee member; Hess, Ann, committee member; Zabel, Mark, committee member
    Leprosy is a chronic infection that affects the skin and peripheral nerves. Written accounts of the disease date back to at least 600 BC. Mycobacterium leprae, the causative agent of leprosy was first discovered by Dr. Gerhard Armauer Hansen in 1873. Leprosy remains a major health problem in several low- and middle-income countries including Brazil, India, and Indonesia. There are numerous clinical presentations of the disease which presents many challenges for controlling the disease including diagnosis, treatment regimen and duration, and occasional instances of drug resistant cases. Further challenges exist in studying the disease, knowledge of the intricate interactions with innate immune cells has made advances in some cell subsets but is limited in others leaving an incomplete picture of the disease. These gaps limit advances in disease management. M. leprae is an obligate, intracellular pathogen that grows preferentially between 33-35° C and selectively invades peripheral nerves and skin-resident innate immune cells including macrophages. Numerous host cells including macrophages and Schwann cells have been studied to understand their interaction with M. leprae, but other skin-resident immune cells like dendritic cells, specifically Langerhans cells, have not been studied as extensively. The findings that M. leprae antigens can be presented via CD1a on Langerhans cells has spurred interest in understanding how Langerhans cells interact and uptake M. leprae leading to downstream effects on T cell activation and overall immune responses. The hypothesis of this study is that M. leprae interacts with Langerhans cells via various cell surface receptors that influence a Th1 or Th2 immune response. This study interrogates the complex interactions between Mycobacterium leprae and Langerhans cells via multiple cell surface receptors. In Chapter 2, an ex vivo optical tissue clearing method was modified for fragile skin samples to analyze innate cell recruitment to the site of infection. Colocalization between Langerhans cells and a closely related mycobacterial spp. to M. leprae, M. haemophilum, was observed in a 3D optically cleared tissue. These observations indicate that wholistic insight of bacteria/innate immune cell interactions can be gleaned using experimentally infected tissues or human skin biopsies. Chapter 3 presents the contributions from multiple cell surface receptors present on Langerhans cells in recognizing and binding M. leprae. Langerin was found to play a role in binding M. leprae, however, was not the only cell surface receptor involved in recognition of M. leprae. CD5+ Langerhans cells can be separated into CD5high and CD5low LCs that have differences in binding capacity for M. leprae. This study builds the foundation to explore the wholistic contributions of Langerhans cells interactions and uptake of M. leprae. Further work should be conducted to identify M. leprae ligand(s) for CD5 and downstream effects on cytokine secretion and T cell activation.
  • Thumbnail Image
    ItemOpen Access
    Identification of small extracellular RNA fragments of Mycobacterium tuberculosis
    (Colorado State University. Libraries, 2014) Sheldon, Sarah Winter, author; Belisle, John, advisor; Jackson, Mary, committee member; Goodridge, Lawrence, committee member
    In 2012, the World Health Organization reported 8.6 million estimated incident cases of tuberculosis, 1 million deaths among HIV-negative people, and 0.3 million deaths from HIV-associated tuberculosis. The Stop TB Partnership has a 2015 goal of reducing the 1990 prevalence rates by half. In order to accomplish this goal, there is a large effort to develop new vaccines, diagnostics, treatment, and therapeutics. Understanding how the pathogen, Mycobacterium tuberculosis, interacts with the host is critical to the development of these goals. An emerging area of interest is how host cells respond to bacterial nucleic acids; there are several bacteria that produce nucleic acids that impact pathogenesis through recognition by host pattern recognition receptors. Previous work by Obregón-Henao et al. found that the culture filtrate (CF) of M. tuberculosis was able to induce apoptosis in monocytes, and the material was identified as small stable RNAs. Through cloning, the M. tuberculosis small RNA present in the CF was found to predominantly consist of tRNA and rRNA with lengths between 30 and 70 bases. The goal of this work was to further understand the composition of the small, stable, extracellular RNA of M. tuberculosis. The first step in further elucidating the extracellular RNA population was to develop an RNA isolation method, allowing for the reliable purification of RNA from the CF of M. tuberculosis H37Rv. The method developed previously was not optimized for RNA purification, and a more streamlined method was needed. Available commercial kits did not fit the specific needs of the project as a method was needed to isolate small RNAs from large volumes of CF. The method developed resulted primarily in small RNAs and allowed for isolation of extracellular RNA free of contaminants that could interfere with biological assays, including DNA, protein, LAM, and LPS. The kinetics of RNA release into the CF was examined, comparing the rate of RNA release to that of protein. The RNA and protein were found to have parallel release rates, which could indicate active release rather than passive release of the RNA. Once a reliable RNA isolation method was developed, the composition of the extracellular RNA was interrogated utilizing Next Generation Sequencing as a high-throughput method. A pilot study was developed to determine the optimal concentration of extracellular RNA for sequencing. The Next Generation Sequencing provided a better understanding of the components of the secreted or released RNA. Ribosomal RNA and transfer RNA fragments were found to be present in the extracellular RNA, correlating to what was found by Obergón-Henao et al. A third group of small RNAs were also identified in this study, many of which corresponded to small RNAs previously reported in the literature, however novel small RNA sequences were also identified. The possibility of bias in the sequencing technology was investigated using synthesized tRNA DNA oligonucleotides (stDNA oligos) added at specific concentrations. The quantitation bias study indicated that some bias occurs, although the cause is unknown. All of the stDNA oligos in the sample were identified, giving some confidence in the qualitative nature of this technology. However, based on the possibility of bias, it may be too generous to state that the technology is truly quantitative. Based on these studies, it is possible to say with confidence that what is identified is present, but not that things are not missed. The long-term goals of this work are to fully understand how the extracellular RNA interacts with the host at a molecular level and to understand the mechanism of RNA release. In order to accomplish these goals, it will be necessary to evaluate more M. tuberculosis extracellular RNA using Next Generation Sequencing. A time course study with Next Generation Sequencing should also be done to see if the RNA composition changes over time, as well as for comparison to intracellular small RNAs. It would also be important to develop an assay to confirm fragments found using the Next Generation Sequencer, as well as to evaluate selective release from M. tuberculosis.
  • Thumbnail Image
    ItemOpen Access
    Investigation into Mycobacterium bovis phenolic glycolipid as a potential biomarker of bovine tuberculosis in urine
    (Colorado State University. Libraries, 2019) Morphet, Stephanie Marie Little Thunder, author; Belisle, John, advisor; Crick, Dean, committee member; Wilusz, Carol, committee member; Henry, Chuck, committee member
    Mycobacterium bovis, the etiological agent of bovine tuberculosis (bTB) is reported to cause disease in man and animal alike on every continent aside from Antarctica. Although coordinated efforts have been made for over a century in the US to cease transmission of this pathogen, outbreaks still occur. It has been posited that the failure to eliminate transmission of this pathogen is partially due to the diagnostic in use, which lacks critical sensitivity and specificity. To address this gap, we investigated a potential new method of identifying infected animals that is through the detection of a pathogen-derived biomarker. M. bovis phenolic glycolipid (PGL) is a species-specific, highly abundant, and unique glycolipid that comprises up to 2.5% of the dry cell mass. Coupling an LC-ESI-TOF-MS, method with a solid phase extraction, we successfully detected PGL derived from the urine of naturally-infected cattle. With this knowledge, we aimed to generate a detector of PGL that could be applied in a rapid and field friendly diagnostic platform. Using phage display technology, we selected M13 bacteriophage capable of binding M. bovis PGL with specificity that differentiated between M. bovis PGL and M. Canetti PGL, as well as between total lipid fractions of various species of Mycobacterium and other lipids with similar biochemical properties to PGL. These M. bovis PGL specific phage were able to differentiate between unspiked cattle urine and urine spiked with PGL. Lastly, we assessed the relative stability of PGL, specifically contrasted to another highly abundant mycobacterial glycolipid, phosphatidylinositol mannoside (PIM). We found that PGL was the more stable molecule when testing thermal and chemical stability, as well as when treated with protease K. However, when treating these glycolipids with whole cell lysates derived from fresh bovine organs, accurate detection on an LC-ESI-TOF-MS platform was lost. Further studies will be required to probe the stability of these molecules in vivo. Overall, this potential methodology to assess for infection status may be beneficial in improving the control of M. bovis if further developed, as this new approach has the potential to be more specific and sensitive than the currently used diagnostic.
  • Thumbnail Image
    ItemOpen Access
    Mining the Francisella tularensis proteome for vaccine candidates
    (Colorado State University. Libraries, 2012) White, Kimberly G., author; Belisle, John, advisor; Schweizer, Herbert, committee member; Tjalkens, Ron, committee member
    Based on methodologies developed for the identification of T cell antigen of other intracellular bacterial pathogens, a proteomic approach was applied for the elucidation of T cell antigens of Francisella tularensis (Covert, 2001). Specifically, subcellular components (membrane and soluble) of F. tularensis LVS were resolved by size using preparative SDS-PAGE and fractions collected using a whole gel elution technique. A total of 16 soluble and 19 membrane-sized fractions were produced, each of which were assessed for antigen reactivity based on the ability to elicit IFN-γ; from splenocytes of F. tularensis LVS-infected mice. Of these 35 preparative SDS-PAGE fractions, seven yielded a dominant T cell response. These seven fractions were further investigated using tandem mass spectrometry (MS/MS) to identify individual proteins in each immunodominant fraction. A total of 40 and 31 proteins were identified with greater than 95% confidence from the immunodominant membrane and soluble fractions, respectively. Further, MS/MS analysis of different protein quantities (2.5 μg to 10 μg) allowed for identification of the most abundant proteins in each fraction, thus focusing the number of possible proteins to nine proteins of interest. These data provide the basis for production of recombinant proteins and further immunological evaluations to select suitable candidates for inclusion in a subunit vaccine against tularemia.