The sun is the primary source of all chemical energy on the planet. Starch granules have evolved as storage deposits for captured light energy. Many complex biological functions take place at the starch granule surface, including starch granule metabolism and defense. The starch granule-associated protein puroindoline is a known antimicrobial with unique functional and biological properties, attributed to the presence of a unique tryptophan-rich domain. To test puroindoline's tight association, puroindoline removed from the starch granule surface during water-washing was assessed. Washing more than eight times failed to further reduce puroindoline content of starch granules, suggesting a strong association of puroindoline with the starch granule surface. To identify the tryptophan-rich domain tightly associated with the starch granule surface, we used a combination of in situ tryptic digestion and mass spectrometry. We identified the tryptophan-rich domain of puroindoline directly bound to the starch granule surface of wheat. This is the first instance of the tryptophan-rich domain directly observed at the starch granule surface. In addition, using mass spectrometry, we determined that during development and maturation, wheat seeds appear to have resisted infection and lysed the pathogens where, upon desiccation, the molecular evidence remained fixed at the starch granule surface. Proteins with known antimicrobial activity were identified, as well as several proteins from the plant pathogens Agrobacterium tumefaciens, Pectobacterium carotovorum, Fusarium graminearum, Magnaporthe grisea, Xanthomonas axonopodis, and X. oryzae. Future characterization may reveal previously unknown host-pathogen interactions. Finally, we have demonstrated that puroindoline, when expressed in the seeds of transgenic corn, will localize and associate with the starch granule surface in a pattern similar to the puroindoline expression pattern observed in wheat. Surprisingly, puroindoline expression in transgenic corn is correlated with an increase in total seed oil content.

The sun is the primary source of all chemical energy on the planet. Starch granules have evolved as storage deposits for captured light energy. Many complex biological functions take place at the starch granule surface, including starch granule metabolism and defense. The starch granule-associated protein puroindoline is a known antimicrobial with unique functional and biological properties, attributed to the presence of a unique tryptophan-rich domain. To test puroindoline's tight association, puroindoline removed from the starch granule surface during water-washing was assessed. Washing more than eight times failed to further reduce puroindoline content of starch granules, suggesting a strong association of puroindoline with the starch granule surface. To identify the tryptophan-rich domain tightly associated with the starch granule surface, we used a combination of in situ tryptic digestion and mass spectrometry. We identified the tryptophan-rich domain of puroindoline directly bound to the starch granule surface of wheat. This is the first instance of the tryptophan-rich domain directly observed at the starch granule surface. In addition, using mass spectrometry, we determined that during development and maturation, wheat seeds appear to have resisted infection and lysed the pathogens where, upon desiccation, the molecular evidence remained fixed at the starch granule surface. Proteins with known antimicrobial activity were identified, as well as several proteins from the plant pathogens Agrobacterium tumefaciens, Pectobacterium carotovorum, Fusarium graminearum, Magnaporthe grisea, Xanthomonas axonopodis, and X. oryzae. Future characterization may reveal previously unknown host-pathogen interactions. Finally, we have demonstrated that puroindoline, when expressed in the seeds of transgenic corn, will localize and associate with the starch granule surface in a pattern similar to the puroindoline expression pattern observed in wheat. Surprisingly, puroindoline expression in transgenic corn is correlated with an increase in total seed oil content.

The sun is the primary source of all chemical energy on the planet. Starch granules have evolved as storage deposits for captured light energy. Many complex biological functions take place at the starch granule surface, including starch granule metabolism and defense. The starch granule-associated protein puroindoline is a known antimicrobial with unique functional and biological properties, attributed to the presence of a unique tryptophan-rich domain. To test puroindoline's tight association, puroindoline removed from the starch granule surface during water-washing was assessed. Washing more than eight times failed to further reduce puroindoline content of starch granules, suggesting a strong association of puroindoline with the starch granule surface. To identify the tryptophan-rich domain tightly associated with the starch granule surface, we used a combination of in situ tryptic digestion and mass spectrometry. We identified the tryptophan-rich domain of puroindoline directly bound to the starch granule surface of wheat. This is the first instance of the tryptophan-rich domain directly observed at the starch granule surface. In addition, using mass spectrometry, we determined that during development and maturation, wheat seeds appear to have resisted infection and lysed the pathogens where, upon desiccation, the molecular evidence remained fixed at the starch granule surface. Proteins with known antimicrobial activity were identified, as well as several proteins from the plant pathogens Agrobacterium tumefaciens, Pectobacterium carotovorum, Fusarium graminearum, Magnaporthe grisea, Xanthomonas axonopodis, and X. oryzae. Future characterization may reveal previously unknown host-pathogen interactions. Finally, we have demonstrated that puroindoline, when expressed in the seeds of transgenic corn, will localize and associate with the starch granule surface in a pattern similar to the puroindoline expression pattern observed in wheat. Surprisingly, puroindoline expression in transgenic corn is correlated with an increase in total seed oil content.

Due to recent declines regarding pollinators, The Swedish Civil Contingencies Agency launched a project investigating possible biological threats towards wild bumblebees. This led to an extensive collection of bumblebees from areas in southern Sweden during the summer of 2018. The purpose of this project was to investigate the prevalence of ten bumblebee pathogens and see if there were any difference in prevalence in areas with and areas without commercial bumblebees. This study also aimed to investigate if the prevalence of the pathogens tended to fluctuate during the season. Of the collected bumblebees (Bombus spp) the ones from the late summer season were analyzed in this project, and data from the already analyzed samples from the early summer season was used. To screen the bumblebees for the pathogens a laboratory process was followed with dissections – homogenization – DNA and RNA extractions – conversion of RNA to cDNA and lastly real-time quantitative PCR. The results showed significant differences between areas with and without commercial bumblebees for some pathogens, but the results were only consistent between early and late season for Apicystis bombi and Black queen cell virus. Apicystis bombi had a higher prevalence (2,0 and 3,0 for late and early season) in areas with commercial bumblebees. Black queen cell virus had a higher prevalence (1,3 and 1,6 for late and early season) in areas without commercial bumblebees. For the seasonal fluctuation a significant difference between early and late season could be seen for the pathogens – Crithidia bombi, Apicystis bombi, Locustacarus buchneri and Sphaerularia bombi.

Study of cell signalling pathways affected by pathogen entry comprises a fundamental aspect of understanding host-pathogen interactions. In this respect, the current study attempted to ascribe novel roles to Bone Morphogenesis Protein (BMP) signaling during infection. BMP pathway has been majorly studied in context of development where it plays an imperative role and its contribution to immunity has been poorly documented. Subsequent narrative talks about the perturbation of BMP signaling in context of specific signaling networks and its collaboration with other molecular players of host innate armamentarium. There is a pressing need to develop effective chemotherapy against Mycobacterium tuberculosis, the causative agent of tuberculosis, which has garnered the world’s attention as a leading cause of public health emergency. The tyrosine kinase, c-Abl was previously reported to be activated in murine bone marrow derived macrophages infected with mycobacteria. Yet, the identities of host signaling players and mechanisms exploited by mycobacteria in association with c-Abl lacked identification. Here, we deciphered an intricate signaling mechanism linking tyrosine kinase c-Abl, chromatin modifier, lysine acetyl transferase KAT5 and transcription factor, TWIST1 acting at Bmp2 and Bmp4 promoters. This molecular circuitry was observed to affect mycobacterial survival. Emerging studies suggest repurposing of c-Abl inhibitor, Imatinib, as an adjunct to existing anti-tuberculosis therapy. Through the use of Imatinib in an established model of tuberculosis, we demonstrated the ability of c-Abl inhibitors in potentiating innate immune responses. Distinctive instances report the cross regulation among Pattern Recognition Receptors (PRRs). Interestingly, TLR3 signaling cascade induced in response to its cognate ligand was dampened through c-Abl-BMP induced miR27a. TLR3 is known to activate immune surveillance upon viral infections; however, recent studies also suggest its role in tumour regression and induction of apoptosis. Our observation of mycobacteria elicited down regulation of TLR3 pathway corroborated with increased incidences of lung cancer among TB patients and mycobacterial evasion of a well characterized form of cell-death i.e. apoptosis. Further, we utilized a panel of such Mtb mutants associated with virulence and questioned their relevance in the activation of c-Abl-dependent BMP signaling. We found that nitric oxide, hypoxia and carbon monoxide-responsive mycobacterial WhiB3 and DosR, but not the sec-dependent protein secretion pathway, orchestrate mycobacteria driven c-Abl-BMP signaling. Continuing with the theme of exploring roles for BMP signaling during infection, we identified an important role for the C-type Lectin Receptor (CLR), Dectin-2, in activating Candida albicans-driven BMP signaling. Mounting evidences suggest BMP antagonists promote repair and regeneration in cells of varied lineages. We observed a role for BMP signaling in aggravating MMP2 and MMP9, factors that result in chronic non-healing wounds. Wounds are now increasingly recognized as being colonized with fungi along with bacteria. We propose a role for C. albicans orchestrated BMP signaling in contributing to enriched repressive methylation at Egf, Pdgf and Tissue Inhibitors of Matrix Metalloproteases (Timp2/3/4) promoters. Repressive H3K27me3 at these loci impedes the reparative tissue homeostasis, resulting in C. albicans endorsed impaired wound healing. Altogether, we uncovered hitherto unknown roles of BMP signaling during mycobacterial and fungal infections, enabling a better understanding of lesser studied pathways in mediating pathogenesis.

Toxoplasma gondii, Neospora caninum et Plasmodium falciparum sont des parasites protozoaires intracellulaires obligatoires, respectivement responsables de la toxoplasmose, de la néosporose et du paludisme. Les différents traitements mis en œuvre reposent sur une association médicamenteuse. Cependant, des échecs thérapeutiques et des résistances aux traitements ont été décrits. Notre travail a porté sur l’identification de molécules actives isolées par Chomatographie de Partage Centrifuge (CPC) à partir d’extraits d’écorces d’Anogeissus leiocarpus, un arbre d’Afrique de l’ouest connu pour son activité antipaludique, et de dix arbres de la région Champagne-Ardenne. Nous nous sommes penchés, dans un premier temps, sur l’activité antiparasitaire des fractions obtenues à partir d’extrait d’écorce d’A. leiocarpus. La trachélospérogénine E et l’extrait global sans tanin se sont révélés actifs, notamment en inhibant l’invasion des cellules hôtes par T. gondii. Cet extrait a également préservé la survie des souris atteintes de toxoplasmose chronique. Les mêmes composés naturels ont eu un effet contre N. caninum et P. falciparum. Dans une seconde partie, 30 extraits d’écorces de dix arbres de la région Champagne-Ardenne ont été testés sur T. gondii et N. caninum. Les composés responsables de l’activité antiparasitaire présents chez Alnus glutinosa semblent être la bétuline et ses dérivés. Dans la dernière partie, nous nous sommes intéressés à l’activité de 400 molécules de synthèse de la Pathogen Box. Huit d’entre elles ont eu un effet significatif contre T. gondii, dont trois avec une sélectivité importante. Des expérimentations sont toutefois à réaliser pour N. caninum. / Toxoplasma gondii, Neospora caninum and Plasmodium falciparum are mandatory intracellular protozoan parasites and are responsible for toxoplasmosis, neosporosis and malaria, respectively. The different treatments used are based on drug combination. However therapeutic failures and drug resistances have been described. Our work focused on the identification of active compounds isolated by Centrifugal Partition Chromatography (CPC) from crude barks extracts from Anogeissus leiocarpus, a West African tree known for its antimalarial activity, and ten trees from the Champagne-Ardenne region. First we studied the activity of the fractions obtained from the crude bark extract from A. leiocarpus. Trachelosperogenin E and the global extract without tannin showed a good activity by inhibiting host cell invasion by T. gondii. The latter was able to preserve mice survival toward chronic toxoplasmosis. These extracts were also active on N. caninum and P. falciparum. In a second part 30 crude barks extracts from ten trees located in the Champagne-Ardenne region were screened on T. gondii and N. caninum. Compounds responsible for the antiparasitic activity found in Alnus glutinosa were especially betulin and its derivatives. In the last part of this study we focused on the antiparasitic activity of 400 synthetic molecules from the Pathogen Box. Eight out of them were significantly efficient against T. gondii, among which three showed an important selectivity. Further experiments must be completed in the case of N. caninum.

[No abstract available] / xi, 176 leaves : ill. (chiefly col.) ; 29 cm

Salmonellen spp. sind invasive, intrazelluläre Pathogene, die in einem membranumhüllten Kompartiment innerhalb der infizierten Wirtszelle überleben. Wie auch andere intrazelluläre Pathogene repliziert Salmonella in dieser intrazellulären Nische, obwohl es anscheinend von sowohl extra- als auch intrazellulären Nährstoffquellen isoliert ist. Wir zeigen hier, dass intrazelluläre Salmonella den Proteinabbau des Wirts ausnutzen, um Aminosäuren in Form von Peptiden zu erhalten. Dieser spezielle, auch als Chaperon-vermittelte Autophagie bekannte, Abbauweg spielt eine Rolle im Transport zytosolischer Proteine zum Abbau im Lysosom. Ein Salmonellenmutant, der nur in Anwesenheit von Peptiden im Medium als Aminosäurenquelle wächst, wies intrazellulär eine Wachstumsrate auf, die der des Wildtyps ähnlich war. Dies deutet darauf hin, dass Peptide intrazellulär für Salmonella zugänglich sind. Wir fanden heraus, dass die Salmonella-enthaltende Vakuole (SCV, Salmonella containing vacuole) die Wirtproteine LAMP-2A und Hsc73, Kernkomponenten von CMA, anzieht, jedoch nicht lysosomale Proteine wie LAMP-2B und LIMP-2. Im Gegensatz zum Salmonellawildtyp zeigte der peptidabhängige Mutantentstamm stark verringertes Wachstum, wenn die Wirtszellen mit CMA-Inhibitoren behandelt wurde. Diese Ergebnisse zeigen einen neuen Mechanismus auf, durch den ein intrazelluläres Pathogen vom membranumhüllten Kompartiment aus Zugriff auf Cytosol der Wirtzelle zur Beschaffung von Nährstoffen hat. Wir schlagen vor, dass diese Ergebnisse eine Erklärung für die Rückfälle von persistenten Salmonellainfektionen liefern können. Des Weitern schlagen wir diesen Mechanismus als moegliches Ziel antibakterieller Therapeutika zur Bekämpfung intrazellulärer Pathogene vor. / Salmonella spp. are invasive, intracellular pathogens which survive and proliferate within a membrane-bound compartment inside infected host cells. Like other intracellular pathogens, Salmonella replicates within this intracellular niche, despite its apparent isolation from both extra- and intracellular sources of nutrients. Here, we show that intracellular Salmonella acquire amino acids in the form of peptides by co-opting the host protein degradation pathway known as chaperone-mediated autophagy (CMA) involved in the transport of cytosolic proteins to the lysosome for degradation. A mutant of Salmonella strictly dependent upon peptides in growth media as a source of amino acids, showed intracellular growth similar to the wild-type strain in host cells, indicating intracellular access to peptides. We found that the Salmonella-containing vacuole (SCV) acquires the host cell proteins LAMP-2A and Hsc73, key components of CMA, but excludes lysosomal proteins such as LAMP-2B and LIMP-2. In contrast to wild-type Salmonella, the peptide-dependent mutant strain showed a severe reduction in growth when host cells were treated with inhibitors of CMA.. These results reveal a novel means whereby an intracellular pathogen can access the host cell cytosol to acquire nutrients from within its membrane-bound compartment. We suggest these results may provide an explanation for relapse infections resulting from persistent Salmonella infections, and suggest a possible means of targeting antibacterials against intracellular pathogens.

Ambient temperature is an important abiotic factor shaping the process of pathogen transmission because of its effects on hosts, pathogens, and interactions between them. However, most experimental studies demonstrating the effects of temperature on transmission remain correlative and often exclude endothermic taxa, which modify behavior and energy allocation strategies in colder environments in ways that could increase pathogen spread. Additionally, because many endotherms serve as important reservoirs for zoonotic diseases and are facing conservation threats due to disease, understanding how temperature influences transmission in these systems has downstream relevance to human and wildlife health. In this dissertation, I use three laboratory experiments to determine how temperature affects several mechanisms of transmission in a naturally occurring songbird-pathogen system. House finches (Haemorhous mexicanus) are small songbirds that rely on bird feeders to meet thermoregulatory demands during winter. However, interactions with other birds at the feeder and contact with contaminated feeder surfaces are important sources of transmission of the bacterial pathogen Mycoplasma gallisepticum (MG). These interactions likely contribute to the fall and winter outbreaks of mycoplasmal conjunctivitis, a disease characterized by severe conjunctival swelling and changes in behavior in house finches. In my first experiment, I simulated infection in house finches to determine how temperature (warm versus cold) affected contact-relevant sickness behaviors, and in turn, the potential for transmission. I found that ambient temperature had a complex effect on some but not all contact-relevant sickness behaviors in this system, which could have key implications for downstream pathogen spread. Next, I investigated how ambient temperatures influenced another mechanism of transmission, the viability and pathogenicity of MG harbored on bird feeder surfaces. I found that MG remained viable and pathogenic to birds significantly longer when incubated on feeder surfaces at colder versus warmer temperatures. In my final chapter, I determined how temperature influenced the pairwise-transmission of MG from an experimentally-inoculated "donor" bird to its susceptible "receiver" bird cagemate. Here I examined how temperature influenced host infectiousness and estimated exposure dose, as well as the behaviors of both sick and healthy birds. I found that donor birds in colder temperatures were slower to recover from infection, and thus remained infectious for longer, compared to donor birds in warmer temperatures. I also found that receiver birds had more contacts with bird feeders and higher estimated doses of MG in colder temperatures. Despite evidence suggesting that MG transmission could be more successful in colder versus warmer temperatures, overall transmission success did not differ by temperature treatment. My work highlights the complex and non-uniform effects of temperature on aspects of the MG transmission process and suggests ways that temperature could have major implications for seasonal disease dynamics in this system. More broadly, my dissertation provides a framework for testing how different abiotic factors could influence the spread of other directly-transmitted diseases, which will be needed now more than ever in the face of global climate change. / Doctor of Philosophy / Temperature can alter disease spread by changing how organisms interact with each other and their environment. Most scientific studies on this topic have focused on diseases in plants and cold-blooded animals, even though temperature can influence disease spread in warm-blooded animals as well. Warm-blooded animals must use large amounts of energy to stay warm in colder temperatures and will often change their behavior or how they spend their energy to save on energetic costs. In some cases, the way that warm-blooded animals respond to colder temperatures can also increase the risk of disease spread. Understanding how warm-blooded animals spread disease is important because many warm-blooded animals carry human diseases, and because climate change brings both conservation and disease threats. In this dissertation, I test how temperature influences factors that cause disease spread in a wild songbird. House finches (Haemorhous mexicanus) are social backyard birds that eat from bird feeders, particularly in winter months when ample food is needed to keep their bodies warm. However, busy bird feeders can cause sick and healthy birds to interact more frequently, and bird feeders themselves often carry the bacterium Mycoplasma gallisepticum (MG), which causes contagious pink-eye like symptoms in birds. Like many animals, house finches that are sick with MG save energy during infection by spending less time being active. Colder temperatures can be problematic for sick birds because they must spend energy to stay warm but save enough energy for fighting infection. In my first experiment, I examined this conflict between temperature and infection in birds, and in turn, how this conflict could shape disease spread. I found that temperature affected some but not all sickness-related behaviors in house finches, which could mean more disease spread at some temperatures, and less at others. My next experiment studied the bacterium itself, and how well it can survive outside of birds in winter versus summer temperatures. I found that not only was MG better at surviving on a bird feeder in colder temperatures, but it also caused worse disease symptoms in birds over time. In my last experiment, I infected one bird with MG and determined if disease was more likely to spread to its healthy cagemate in warmer or colder temperatures. This was important for studying the effects of temperature on two other factors related to disease spread: the ability of sick hosts to remain contagious to others and the approximate number of pathogens eventually picked up by healthy individuals. I found that in colder temperatures, sick hosts had a harder time recovering, remaining contagious for longer. I also found that healthy bird partners were more likely to spend time at bird feeders in colder temperatures, where they encountered more pathogens on feeder surfaces. Despite these findings, overall MG spread was not higher in colder temperatures. This study provided some of the first evidence showing the complicated relationship between temperature and MG spread in house finches and suggests how temperature could play a role in the seasonal outbreaks of MG seen in nature. My study also provides a blueprint for studying how other environmental factors, such as humidity and rainfall, could shape the spread of other infectious diseases, which will be more important now than ever in a rapidly changing climate.

Master of Science / Department of Clinical Sciences / Michael D. Apley / One hundred ninety four steers, bulls, and heifers weighing 182-318 kg were purchased at an Arkansas sale barn and shipped 12 hours to a northern Kansas feedlot. There was no previous history of treatment and the cattle had been delivered to the sale barn within the 24 hour period prior to the sale. The objectives of the study were to evaluate (1) bacterial pathogen isolates in different locations in the respiratory tract, (2) pathogen load in clinically ill and clinically normal calves, and (3) compare histological damage that may be a result of clinical disease. Fifteen calves were identified with signs of acute bovine respiratory disease (BRD) based on clinical score and a minimum rectal temperature of 40° C. An additional 5 calves with no clinical signs and rectal temperatures < 40° C were selected as controls. Cattle were humanely euthanized following recording of antemortem clinical observations. At postmortem, samples for microbiologic and histologic (hematoxylin and eosin stain) analysis were collected from grossly normal and/or consolidated tissue in each lung lobe. Samples were also collected from the tonsils and trachea. Quantification of the BRD pathogens per gram were determined for each positive site and then converted to total counts for each animal. Total colony forming units (CFU) of pathogens in the entire lung for cattle with identified pathogens ranged from 2x10[superscript]7 – 2x10[superscript]8 CFU for Pasturella multocida and 9x10[superscript]6 – 9x10[superscript]8 CFU for Mannheimmia haemolytica. Total visual estimated percent consolidation ranged from 0.0% to 45.0% of the total lung. Isolated pathogens from the upper and lower respiratory tract were compared and showed to have no significant agreement. Histology scores of 0-4 were assigned to the tissue samples and compared to the quantified BRD pathogens to test a possible association between the pathologic process and the total agents in that tissue sample. A significant difference in bacterial counts between histology scores of zero or 1 and a histology score of 4 was observed.