Browsing by Author "Jackson, Mary, committee member"
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Item Open Access Antibacterial growth effects and speciation of several vanadium salts and complexes(Colorado State University. Libraries, 2023) Arhouma, Zeyad Kamal, author; Crans, Debbie C., advisor; Crick, Dean, committee member; Roess, Deborah, committee member; Jackson, Mary, committee memberVanadium (V) is a first-row transition metal ion that acts as a phosphatase inhibitor with a wide variety of biochemical and physiological functions. The ability of vanadium to form stable polyoxovanadates (POVs) and organometallic complexes has attracted attention for studying the properties and effects of these compounds in various biological systems. In my research, I used the bacterium species Mycobacterium smegmatis (M. smeg), which has undergone reclassification and is now classified as Mycolicibacterium smegmatis. Despite the taxonomic change, both the previous and current classifications use the same abbreviation, M. smeg. I also carried out some studies in Mycobacterium tuberculosis (M. tb). In my work, I explored the properties of several types of vanadium compounds including salts, oxometalates, and coordination complexes to investigate how they impact cellular growth. The first chapter of this dissertation focuses on determining the growth inhibitory effects of decavanadate (V10) and rapidly exchanging oxovanadates on the growth of two mycobacterial species: M. tb and M. smeg. Speciation analysis, utilizing 51V NMR spectroscopy, was employed to document that one specific oxometalate exhibits greater potency as a growth inhibitor for these mycobacterial species compared to other oxovanadates, indicating selectivity in its cellular interaction. Oxometalates have been involved in numerous applications in biological and medical studies, including their ability in addressing the phase-problem in X-ray crystallography of the ribosome. This study investigated the effect of different vanadate salts on the growth of M. smeg and M. tb, highlighting the critical role of speciation in the observed growth inhibition. Specifically, the large orange-colored sodium decavanadate (V10O286−) anion was found to be a stronger growth inhibitor for these bacteria compared to the colorless oxovanadate derived from sodium metavanadate. The 51V NMR spectroscopy and speciation calculations were employed to monitor the vanadium(V) speciation in the growth media and its conversion among species under growth conditions. Our results show that the decavanadate was 200-20 times more potent in inhibiting growth dependent the consideration of molecules or total vanadium content. The findings presented in this work are particularly important in the context of the numerous applications of polyoxometalates in biological and medical studies. The second chapter focuses on investigating the inhibitory effects of two monosubstituted decavanadates (V10): monoplatino(IV)nonavanadate(V) ([H2PtIVV9O28]5−, V9Pt), and by MoIV in monomolybdo(VI)nonavanadate(V) ([MoVIV9O28]5−,V9Mo) on the growth of M. smeg. The inhibitory effects of V9Pt and V9Mo were examined against the growth of M. smeg with EC50 values of 0.0048 mM and 0.015 mM, respectively. These values were compared to the reported inhibitory value of decavanadate ([V10O28]6−/[HV10O28]5−, V10) on M. smeg (EC50 = 0.0037 mM). Time-dependent 51V NMR spectroscopic studies were carried out for all three polyanions in aqueous solution, biological medium (7H9), and heated and non-heated supernatant. These studies aimed to evaluate their stability in their respective media, monitor their hydrolysis over time to form different oxovanadates, and calculate the corresponding EC50 values. The results presented in this study indicate that the two related derived decavanadate derivatives (V9Pt and V9Mo) and V10 exhibited greater potency as growth inhibitors of M. smeg, compared to monomeric vanadate (V1). The spectroscopic characterization conducted in the growth medium led to the conclusion that both the decavanadate structure and its properties play significant roles in their growth effects. In the third chapter in this dissertation, we investigated the growth effects of an anticarcinogenic non-toxic Schiff base oxidovanadium(V) complex (N-(salicylideneaminato)-N'-(2-hydroxyethyl) ethane-1,2-diamine) coordinated to the 3,5-di-tert-butylcatecholato ligand on a representative bacterium, M. smeg.. In addition, we synthesized a series of the Schiff base V-complexes based on previously reported methods and examined the effect of complexes as well as the free catecholates on the bacterial growth. To determine the inhibition activity of these complexes on M. smeg., the biological studies were complemented by spectroscopic studies using UV-Vis spectrophotometry and NMR spectroscopy. These spectroscopic studies determine which complexes remained intact under biologically relevant conditions. In this work, we examine (1) the growth effects of Schiff base oxidovanadium complexes coordinated to a catechol, (2) the growth effects of the respective free catecholates on M. smeg., and (3) the effects of the scaffold. These studies allowed us to demonstrate that some metal coordination complex exhibited greater potency than the ligand alone under biological conditions, whereas others showed greater effects of the free catecholate ligand and in one case the effects were similar of complex and catecholate ligand. The findings from these studies revealed that the observed effects of the Schiff base V-catecholate complex were influenced by the properties of the catechol, including toxicity, hydrophobicity, and steric factors. Finally, the fourth chapter presents preliminary research data on the antimicrobial effects of two pseudospherical mixed-valence polyoxovanadates (MV-POVs), namely K(NH4)4[H6PVIV2VV12O42]·11H2O (V14) and (Me4N)6[VIV8VV7O36(Cl)] (V15) on the growth of M. smeg. These MV-POVs showed complex effects on cell growth, as many of these systems are not stable under biological conditions. To investigate the vanadium(V) speciation in aqueous solutions and growth media, as well as to monitor any conversion among species under growth conditions, 51V NMR spectroscopy was employed. The 51V NMR spectra revealed some hydrolysis and more extensive oxidation of vanadium(IV) in V14 compared to V15 in both aqueous solutions and media. The studies show that both MV-POVs are effective growth inhibitors. The combined findings from the studies described in all the chapters of this dissertation indicate that the stability of the vanadium compound and its structure plays a significant role in the ability of the vanadium complexes to inhibit bacterial growth. These studies highlight the importance of speciation in the biological activity of vanadium complexes.Item 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 memberA 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.Item Open Access Characterization of Mycobacterium leprae diguanylate cyclases(Colorado State University. Libraries, 2016) Rotcheewaphan, Suwatchareeporn, author; Belisle, John T., advisor; Borlee, Brad R., committee member; Brennan, Patrick J., committee member; Jackson, Mary, committee member; Prenni, Jessica E., committee memberMycobacterium leprae is the causative agent of leprosy, which is still a major health problem in several developing countries. Management of leprosy has been challenging because of the long incubation period of the disease and the development of a spectrum of clinical manifestations. Leprosy treatment is further complicated by the development of drug resistance. Knowledge of infection mechanisms and pathogenesis of leprosy is still limited. These fundamental gaps significantly limit the development of disease management, including treatment and prevention. Although M. leprae is an obligate intracellular pathogen, this bacterium must possess mechanisms to adapt to different host defenses or cell types. The discovery of cyclic diguanylate monophosphate (c-di-GMP) and its potential roles in bacteria as a second messenger to regulate several cellular activities responding to environmental stimuli have stimulated an interest on c-di-GMP studies in Mycobacterium spp., especially M. leprae which has massive gene decay but still harbors several potential proteins functioning as diguanylate cyclases. The hypothesis of this study is that M. leprae has the ability to synthesize c-di-GMP. This study evaluated M. leprae’s potential to synthesize c-di-GMP. Bioinformatics analyses were performed to identify proteins that are involved in c-di-GMP synthesis (diguanylate cyclase, DGC) and turnover (phosphodiesterase, PDE). Bioinformatics revealed that M. leprae harbors a putative DGC-PDE protein (ML1750c) and two putative DGC proteins (ML1419c and ML0397c). Interestingly, homologues of ML1419c and ML0397c are not encoded by Mycobacterium tuberculosis. The M. leprae genes ml1419c, ml0397c, and ml1750c were cloned and expressed in Pseudomonas aeruginosa PAO1 and Escherichia coli BL21(DE3) pLysS. Assays for well-described phenotypes of c-di-GMP production (colony morphology, macromolecule synthesis, and biofilm formation) were performed with the recombinant clones. Direct measurement of c-di-GMP levels was accomplished by LC-MS. RNA was extracted from M. leprae infected mouse footpads, and expression of ml1419c and ml0397c was measured by droplet digital PCR. DGC proteins produced by M. leprae in armadillo tissue were also monitored with protein-specific polyclonal antibodies. Phenotypic studies revealed that recombinant expression of ml1419c in P. aeruginosa altered colony morphology, motility, and biofilm formation, and the recombinant expression of ml0397c increased curli and cellulose production of E. coli. These phenotypes were consistent with increased DGC activity and c-di-GMP production. LC-MS analyses confirmed increased c-di-GMP production by ML1419c and ML0397c. In vivo gene expression studies revealed that ml1419c, ml0397c, and ml1750c are expressed by M. leprae during infection. Additionally, ML1419c and ML1750c proteins were clearly identified in whole cell sonicate of armadillo derived M. leprae. This study demonstrated that M. leprae has significant potential to produce c-di-GMP. ML1419c and ML0397c were confirmed as functional DGCs. This study is significant because it provides evidence that M. leprae has the ability to produce c-di-GMP. Furthermore, these studies will pave the way for future research to characterize the biological roles of c-di-GMP in M. leprae and the pathogenesis of leprosy. Continued studies to elucidate the biological roles and the environmental signals for ML1419c, ML0397c, and ML1750c are being performed. These efforts are directed at defining the function of c-di-GMP in M. leprae. It is anticipated that these future efforts along with the data in this dissertation will shed light on the signaling mechanisms that respond to environmental changes experienced by M. leprae.Item Open 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 memberIn 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.Item Open Access Inhalational antibiotic therapy for treatment of chronic pulmonary Mycobacterium abscessus disease in mice(Colorado State University. Libraries, 2019) Pearce, Camron, author; Gonzalez-Juarrero, Mercedes, advisor; Jackson, Mary, committee member; Volckens, John, committee memberMycobacterium abscessus (M. abscessus) is a nontuberculous mycobacterium that causes chronic pulmonary infections. Due to M. abscessus's intrinsic antibiotic resistance, treatment is often complex with low cure rates. Tigecycline, a glycylcycline class antibiotic, demonstrates bactericidal effects against M. abscessus without eliciting bacterial resistance mechanisms, however, this antibiotic requires intravenous administration and causes significant side effects that limit its use. Here, we tested the hypothesis that tigecycline administered via inhalation has the potential to maximize the bactericidal effect while reducing side effects. GM-CSF knockout mice with pulmonary M. abscessus infection were treated by intrapulmonary tigecycline aerosols in 0.25 mg, 1.25 mg, and 2.50 mg doses for 28 days. Assessment of pulmonary bacterial burden after full treatment duration shows that inhaled tigecycline is highly effective, dose-dependent, and well tolerated. We concluded that inhaled tigecycline represents a viable treatment option for M. abscessus pulmonary disease. Future studies should address the pharmacokinetics, and ultimately, translation into clinical trials.Item Open Access Potentiation of beta-lactam antibiotics against Mycobacterium tuberculosis by 2-aminoimidazoles: investigation into the mechanism of action and its relevance to mycobacterial bioenergetics(Colorado State University. Libraries, 2017) Jeon, Albert Byungyun, author; Basaraba, Randall J., advisor; Borlee, Brad, committee member; Gustafson, Daniel, committee member; Jackson, Mary, committee member; Melander, Christian, committee member; Obregón-Henao, Andrés, committee memberTuberculosis, caused by Mycobacterium (M.) tuberculosis, is a global health problem still causing morbidity and mortality due in part to the emergence of drug-resistance and the lack of new antimicrobial agents to treat the disease. While infection with drug-sensitive M. tuberculosis has cure rates between 90-95% with the conventional multidrug-regimen comprised of four different first-line anti-tuberculosis drugs, administered for a minimum of 6 months. In the event where premature termination of the treatment or poor patient compliance occurs, the disease may progress into latent tuberculosis, which holds the risk of reoccurring disease or even leads to development of drug-resistant strains that are refractory to first line anti-tuberculosis drugs. This persistence is a major hurdle in global tuberculosis control and warrants the development of a new class of anti-tuberculosis drugs or novel strategies to target persisting bacilli. However, the current anti-tuberculosis drug pipeline does not suggest an immediate solution required for the successful control of global tuberculosis epidemic. In sum, there is an urgent need for a new strategy to complement current tuberculosis chemotherapy. 2-aminoimidazoles and their derivatives have been shown to be effective inhibitors of bacterial biofilms. Not only does this class of small molecules inhibit the formation of or disperse biofilms, but they also exhibit a clinically relevant feature of potentially abrogating antibiotic resistance in important pathogenic bacteria. From the studies characterizing persistent M. tuberculosis bacilli after anti-tuberculosis therapy in animal models, it has been suggested that this subpopulation of bacilli share similarities with bacterial biofilms. Our group developed an in vitro culture system where M. tuberculosis can be cultured in biofilm-like surface-attached communities with host-derived macromolecules and showed they express extensive drug-tolerance to one of the first-line anti-tuberculosis drug, isoniazid. Based on the previous effects of 2-aminoimidazoles on biofilms and drug-resistant bacteria, we hypothesized that 2-aminoimidazoles could reverse phenotypic drug-tolerance expressed by M. tuberculosis in our model and demonstrated that, indeed, derivatives of 2-aminoimidazoles effectively resensitized drug-tolerant bacilli to isoniazid. Additionally, a fortuitous but critical observation was made in which one of the potent 2-aminoimidazole derivatives potentiated the effect of ß-lactam antibiotics against M. tuberculosis. As repurposing ß-lactams in tuberculosis treatment regimen has potential therapeutic value, which are described throughout this dissertation. In chapter 2, 2-aminoimidazole compounds are shown to be effective at potentiating multiple ß-lactam antibiotics. Minimum inhibitory concentrations, as well as bactericidal concentrations, of ß-lactams were dramatically reduced when combined with 2-aminoimidazoles. Through a transcriptional analysis of M. tuberculosis treated with 2B8, one of our lead 2-aminoimidazoles induced cell envelope related stress responses and suppressed mycolic acid biosynthesis. Thereafter, it was hypothesized that 2-aminoimidazoles disrupts one or more factors conferring M. tuberculosis ß-lactam resistance, which we shown in chapter 3 is in large part due to a reduction in secretion of the enzyme ß-lactamase and by increasing cell envelope permeability. 2B8 treated M. tuberculosis exhibited significantly lower ß-lactamase activity in culture supernatant, which was due to a general protein secretion defect, and not from direct inhibition of ß-lactamase enzyme activity by 2-aminoimidazole compounds. As expected from the transcriptional analysis, 2B8 induced alterations in cell envelope lipid composition highlighted by the accumulation of trehalose monomycolate, the reduction of trehalose dimycolate, as well as a decrease in mycolic acid biosynthesis. Additionally, increased sensitivity to the detergent SDS, increased permeability to multiple nucleic acid staining dyes, and increased bindings of peptidoglycan-targeting antibiotics were observed when with 2B8 treatment. Based on major findings from chapter 3, it was hypothesized that the underlying mechanisms of 2-aminoimidazoles are the disruption of proton motive force and the disturbance of mycobacterial bioenergetics. In chapter 4, the collapse of proton motive force with additional dose-dependent block of mycobacterial electron transport chain is highlighted. Through a series of assays, we determined that 2B8 blocks the M. tuberculosis electron transport chain downstream of complex I and II, but upstream of complex IV. Taken together, these results collectively extend our current understanding of the various effects 2-aminoimidazole treatment has on M. tuberculosis susceptibility to ß-lactam antibiotics through perturbation of mycobacterial bioenergetics which can provide a profound impact in improving current tuberculosis therapy. Furthermore, this study offers valuable information for the construction of the next generation of potent 2-aminoimidazoles to improve efficacy against M. tuberculosis as well as other compounds that may be developed as a new anti-TB drug targeting bioenergetics.Item Open Access The interaction of free-living amoeba with rice bacterial and fungal pathogens(Colorado State University. Libraries, 2018) Long, Jia Jun, author; Leach, Jan E., advisor; Jahn, Courtney E., advisor; Jackson, Mary, committee memberFree-living amoebae are ubiquitous microbes found in the soil and water across the globe. Amoebae live a predominantly heterotrophic lifestyle – preying on a variety of organisms including bacteria, fungi and even other protists. Although extensively studied, their potential as a biocontrol for agricultural pathogens is largely unexplored. As many pathogens occupy the same habitat as amoeba, we investigated their interactions as a first step to determine if amoeba are possible biocontrol agents. Our research focuses on two important pathogens of rice, the bacteria Xanthomonas oryzae and the fungus Rhizoctonia solani. Much of this thesis centers on the interaction between amoebae and X. oryzae, which is explored in depth and presented in the first chapter. Experimentation involved five common amoebal species and two highly virulent X. oryzae pathovars. Microscopy and vitality assays of amoebae-bacteria co-cultures first established that X. oryzae does not grow or dies in the presence of our amoebae. On the other hand, amoebae are not adversely affected, with most cells remaining alive in the metabolically active trophozoite form. Although the bacteria are harmed in this interaction, it is likely not through phagocytosis, the most common and well-studied tactic that amoeba use to feed. Observations made through confocal microscopy revealed that X. oryzae was rarely detected inside amoebae. Furthermore, lysis of amoebae after exposure to bacteria did not yield any viable bacteria, suggesting that either bacteria are rarely internalized and/or that X. oryzae does not survive in the amoeba cell. Conversely, amoebal trophozoites have no impact on the biofilms of X. oryzae either. These data indicate that amoeba do not directly or physically interact with X. oryzae. Instead, our amoeba-conditioned media assays reveal that amoeba alter the media and render it harmful to X. oryzae. The most likely scenario is that amoeba secrete a bactericidal agent into their surroundings. At this time, we have yet to isolate or identify the compound, but its presence may prove to be a boon with a variety of applications. The dynamics between amoeba and R. solani were not studied as extensively, but the basic interaction is presented in the appendices of this thesis. Again, five amoeba species were incubated with mature R. solani mycelia. First, co-cultures were observed with a compound microscope. Acanthamoeba and Dictyostelium did not have any effect on the fungi. Acanthamoeba species physically associated with the mycelia, but also rapidly encysted – suggesting some antiprotozoal activity from R. solani. D. discoideum had no interaction with the fungi: the trophozoites did not attach to the mycelia and neither cysts nor spore-forming bodies were seen. V. vermiformis was the only amoeba with some effect on the fungi. In co-cultures, fungal mycelium developed a shriveled and wrinkled morphology. V. vermiformis was attached to the fungi and most amoebae remained as viable trophozoites. V. vermiformis and its interaction with R. solani was further examined using scanning electron microscopy, which further corroborated the light microscope observations. While the reason/effect of the shriveling is unknown, it is a potential avenue for further experiments. Also in the appendices are references to two published papers I co-authored. For more information, the two papers can be found in their respective open-access journals.Item Open Access The pathogenesis of diabetes-tuberculosis comorbidity(Colorado State University. Libraries, 2014) Podell, Brendan K., author; Basaraba, Randall J., advisor; Dow, Steven, committee member; Rovnak, Joel, committee member; Jackson, Mary, committee member; Ishii, Douglas, committee memberExposure to the bacterium, Mycobacterium tuberculosis, only leads to the active form of tuberculosis disease (TB) in 5-10% of infected individuals. The development of active TB, at any stage of infection, is often the result of a known TB risk factor, either intrinsic to the individual or acquired as a communicable or non-communicable disease. An association between diabetes and TB has long been recognized, but only recently was diabetes confirmed to increase the risk of developing active TB disease. The convergence of a growing diabetes epidemic on regions with endemic TB has positioned diabetes as an emerging global threat to TB control. Of particular importance is the rapidly growing incidence of type 2 diabetes, which accounts for up to 95% of the global diabetic population. Since the potential impact of this growing comorbidity has only been recently emphasized, little is known regarding the mechanisms of dysregulated immune function and metabolism by which diabetes predisposes to active TB disease. The current understanding of this comorbidity is further limited by the lack of appropriate animal models that replicate the pathogenesis of both human type 2 diabetes and TB. The guinea pig is a well-established model of TB that replicates human pathology and disease progression. This species was emphasized in this series of studies with the goal of better understanding the impact of type 2 diabetes on TB progression and the mechanisms that may change the host response to M. tuberculosis infection. In Chapter 2, we investigated the impact of hyperglycemia alone, induced as post-prandial hyperglycemia through daily administration of sucrose, on TB disease progression in non-diabetic guinea pigs. Guinea pigs receiving daily sucrose developed both higher bacterial burdens in pulmonary and extrapulmonary tissue and also more severe pathology by day 60 of infection. This exacerbated disease manifestation was accompanied by the accumulation of advanced glycation end-products, which are inflammatory by-products of chronic hyperglycemia with known involvement in the development of diabetes-related complications. Interestingly, by monitoring glucose and lipid metabolism in these guinea pigs, we learned that TB alone leads to severe metabolic disturbances, manifesting as hyperglycemia and accumulation of circulating total free fatty acids. From this study, we were able to conclude that not only does mild post-prandial hyperglycemia worsen the course of TB disease in guinea pigs, but also, infection with M. tuberculosis alone induces metabolic disease resembling diabetes, similar to what has been previously reported in human TB. These conclusions rationalize the investigation of novel adjunctive therapies to restore metabolic homeostasis, which may improve the host response to infection, limit bacterial growth, and increase the efficacy of frontline antimicrobial drugs. In Chapter 3, we developed a novel model of type 2 diabetes in the guinea pig to be used in future investigations of type 2 diabetes-TB comorbidity. Previously, the guinea pig as a diabetic model has been described only in the context of β-cell cytotoxicity with the drug, streptozotocin (STZ), but with variable efficacy. In this study, we initially optimized the dose response and STZ preparation to achieve an induction of hyperglycemia that was uniform with limited mortality. This hyperglycemic response was transient but could be stabilized through continued β-cell stress, in the form of a high fat, high sugar diet. Feeding of this modified diet led to impaired glucose tolerance and a compensatory β-cell response that could be abrogated with the use of a single optimized dose of STZ. This novel model of type 2 diabetes develops both insulin resistance and β-cell failure, which replicate the typical progression of type 2 diabetes in humans, all within a reasonable experimental timeframe. From this study, two models emerged, a type 2 diabetic guinea pig as well as a model of impaired glucose tolerance, or prediabetes, that would be used to investigate the mechanisms of diabetes-TB comorbidity. In Chapter 4, the newly developed guinea pig models were used to investigate the overall impact of type 2 diabetes and impaired glucose tolerance on TB progression and the host immune response to M. tuberculosis infection. Although impaired glucose tolerance alone had limited impact on TB progression with exacerbation of disease only at chronic end points, M. tuberculosis infected type 2 diabetic guinea pigs closely resembled the reported manifestations of human diabetes-TB comorbidity including more severe TB disease, higher bacterial burdens, and a robust innate and cell-mediated immune response. Despite evidence of strong Th1 cell-mediated immunity, which is known to be critical for limiting bacterial growth and disease progression, diabetic guinea pigs were unable to control bacterial growth and developed damaging neutrophilic inflammation. To better understand the immune mechanisms leading to uncontrolled bacterial growth and severe disease, in Chapter 5, we investigated the innate and adaptive immune response over the course of early infection in type 2 diabetic guinea pigs. Diabetic guinea pigs were slow to develop early lesions with delayed bacterial transport to the lung draining lymph node, and a corresponding delay in antigen-specific Th1 immunity. Early alterations in cytokine expression were identified that may explain the delayed development of cell-mediated immunity and allow for substantial growth of M. tuberculosis in the lung of infected diabetic guinea pigs. These data indicate that not only does type 2 diabetes increase the severity of TB but also that the chronic inflammatory process associated with TB itself may worsen diabetes. This has important implications worthy of further investigation revolving around the diagnostic criteria for diabetes when associated with TB, the impact of active TB on medical management of diabetes, and the investigation of novel therapeutic targets, both metabolic and immunological, to enhance the host immune response to infection and limit TB disease severity in diabetics.