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<p>Malaria, caused by the apicomplexan parasite Plasmodium, is a disease which affects up to 500 million people each year. Historically, malaria infection has been combated both through the control of its vector, the Anopheles mosquito, and use of a variety of drugs, such as quinine (1800s) and chloroquine (1900s). However, with the evolution of resistance to the majority of available anti-malarial drugs, current approaches have settled upon combinatorial therapies. The most effective of these currently are ACTs (Artemisinin Combination Therapies - Artemisinin derivatives combined with a number of other drugs). However reports of Artemisinin resistance are continuing to emerge, suggesting that new approaches and increased understanding of the Plasmodium parasite is required.</p><p> Beginning with the complete sequencing of Plasmodium falciparum genome and continuing with comprehensive profiling of both the parasite's proteome and transcriptome, various genomic approaches applied in the study of malaria have led to significant new insights into the underlying biology of this parasite. While these new findings have greatly increased our understanding of genetic regulation within the malaria parasite, they largely have not yet translated into new therapeutic approaches. For this reason, considerable attention has been paid to the study of human genetic disorders which convey resistance to malaria, in the hopes that elucidating the mechanisms behind these resistances might lead to increased understanding of the parasite's biology and thus novel therapeutic approaches.</p><p> Sickle cell (HbS) erythrocytes are well known to resist malaria infection. However, the molecular basis of this resistance, long been recognized as multifactorial, contains elements which remain poorly understood. Here we show that the dysregulated erythrocytic microRNA composition, present in both HbAS and HbSS erythrocytes, is a significant determinant of resistance against the malaria parasite Plasmodium falciparum. During the intraerythrocytic lifecycle of P. falciparum, a subset of erythrocyte microRNAs translocate into the parasite. Two microRNAs, miR-451 and let-7i, were highly enriched in HbAS and HbSS erythrocytes and these miRNAs, along with miR-223, negatively regulated parasite growth. Surprisingly, we found that miR-451 and let-7i integrated into essential parasite mRNAs and, via impaired ribosomal loading, resulted in translational inhibition of the target mRNA. Hence, sickle cell erythrocytes exhibit cell-intrinsic resistance to malaria in part through an atypical microRNA activity which may present a novel host defense strategy against complex eukaryotic pathogens. In addition, the formation of these chimeric transcripts even in normal host erythrocytes illustrates a unique parasitic post-transcriptional adaptation to the host-cell environment.</p> / Dissertation

Invasive plants may leave enemies behind when they colonize a new habitat, allowing selection to favor increased investment in growth and/or reproduction over defensive traits. Previous studies have identified reduced diversity of potential bacterial pathogens and evolutionary increases in growth and reproduction in invading populations of yellow starthistle (Centaurea solstitialis). This study leverages a recently developed high-throughput assay of immune function to test for evidence of a trade-off between increased growth and defense against bacterial pathogens in yellow starthistle's invasion of California (USA). Seven bacterial strains were cultured from infected leaf tissue in the native range. Healthy leaf tissue from five native European collections and six invading collections were exposed to these native bacterial strains. A standardized assay of peroxidase activity was used measure the oxidative burst immune response to pathogen recognition by the leaf. Immune responses were compared to plant growth within and between ranges to assess evidence for a trade-off. Plant genotypes from the native range demonstrated a higher immune response to bacterial strains than did invading genotypes, consistent with a trade-off with plant growth across regions. The same trade-off was also apparent across genotypes from the native range, but not across genotypes from the invaded range. Our results provide evidence that increased growth in a highly invasive plant species may come at a cost to immune function, consistent with the hypothesis that escape from enemies can provide opportunities for shifts in resource allocation that favor the proliferation of non-native species.

Drosophila melanogaster has been extensively used as a model organism to study diverse facets of biology, including host-pathogen interactions and the basic biology of its pathogens. I have used the fruit fly as a model to study elementary aspects of Nora virus biology, such as the role of the different proteins encoded by the virus genome. Nora virus, an enteric virus transmitted via the feca-oral route, does not cause any obvious pathology in the fly, although the infection is persistent. Nora virus genome consists of a positive strand RNA that is translated in four open reading frames (ORF).  Since sequence homology studies did not yield much information about the different Nora virus proteins, I have used the cDNA clone of the virus to construct mutants to identify the specific function of each protein. My results have shown that, 1) The protein(s) encoded by ORF 1 are crucial for the replication of the virus genome. 2) The C-terminus of the ORF 1-encoded protein (VP1), is an inhibitor to the RNAi pathway. 3) The transmembrane domain in the N-terminus of the ORF2-encoded protein (VP2) is important for the formation of Nora virus virions. 4) The ORF 3-encoded protein (VP3) forms α-helical trimers and this protein is essential for the stability of Nora virus capsid.                                                     I have also performed RNA sequencing to investigate the transcriptional response of D. melanogaster in response to Nora virus infection and my results indicate that,                        5) The upregulation of genes related to cellular stress and protein synthesis and the downregulation of basal digestive machinery, together with the induction of upd3, implies major gut epithelium damage and subsequent regeneration.

A fundamental challenge faced by all organisms is the risk of infection by pathogens that can significantly reduce their fitness. The evolutionary dynamic between hosts and pathogens is expected to be a coevolutionary cycle, as pathogens evolve by increasing their level of virulence and hosts respond by increasing their level of resistance. The factors that influence the dynamics of adaptation by pathogen and host in response to one another are not well understood. Social insects live in dense colonies in high-pathogen soil environments, making them an ideal model system to study the factors influencing the evolution of pathogen resistance. In this thesis work, I investigated several alternative hypotheses to explain patterns of host resistance to entomopathogenic fungi in the harvester ant genus Pogonomyrmex: that high resistance is associated with high environmental pathogen loads, that local adaptation leads to increased resistance to coevolved pathogen populations, that life history tradeoffs increase allocation to resistance in harsher environments, and that increased genetic diversity caused by interspecific hybridization enhances inherent resistance. First, I characterized patterns of spatial variation in abundance and diversity of fungal pathogens among habitats of Pogonomyrmex species. I found 17 genera of fungi in the soil, six of which were entomopathogenic. Lower precipitation habitats, where P. rugosus occurs, had the lowest diversity, while the highest was experienced by the H lineage, one of two hybrid populations. When actual infection rates of field-caught workers were compared, the mesic-habitat P. barbatus was infected significantly more often. These results suggest that habitat does plays a role in fungal diversity, and that species are exposed to more entomopathogens may be more likely to get infected. Second, I tested experimentally whether hybridization and or habitat differences play a role in pathogen resistance by testing the effect of soil type and species identity on infection rates in pupae of the two species and their hybrids. This experiment showed P. rugosus ants had the highest inherent resistance to infection, supporting the life history tradeoff hypothesis. This suggest that Pogonomyrmex ants species are allocating their resources differently according to their environment, with more stressful environment leading to less investment in reproduction and more in protection against pathogens. Overall our study shows that environment plays a role in differences in infection risk, while genetic effects such as hybridization may not play a role in pathogen resistance.

Naegleria fowleri, a free-living ameba, is the causative agent of Primary Amebic Meningoencephalitis. Highly-pathogenic mouse-passaged amebae (Mp) and weakly-pathogenic axenically-grown (Ax) N. fowleri were examined for peptidase activity. Zymography and azocasein peptidase activity assays demonstrated that Mp and Ax N. fowleri exhibited a similar peptidase pattern. Prominent for whole cell lysates, membranes and conditioned medium from Mp and Ax amebae were the presence of an activity band of approximately 58kDa and 100 kDa bands susceptible to the action of cysteine and metallopeptidase inhibitors, respectively. Further roles of the peptidases during the invasion process were examined by in vitro invasion assays in the presence of inhibitors and Cysteine and metallopeptidase inhibitors were found to greatly reduce invasion through the ECM. This study establishes a functional linkage of the expressed peptidases to the invasion process, and these peptidases may serve as a candidate target for therapeutic management of N. fowleri infection.

Surface waters containing fecal bacteria present significant public health risks. Understanding the sources of and factors affecting the distribution of fecal-indicating bacteria is necessary to predict potential illnesses more effectively. This thesis presents two studies on the distribution of fecal bacteria in the James River through Richmond, Virginia. Chapter 1 describes nearly 11 years of water quality, climate, and hydrologic data that occurred with changes in Escherichia coli concentrations, concluding that Richmond contributes significant quantities of fecal bacteria to the James River, and that the distribution of these bacteria varies seasonally. Chapter 2 details the development of Polymerase Chain Reaction (PCR)-based methods to identify four common pathogenic taxa of fecal bacteria, and indicates that the factors controlling the distribution of these pathogens may be taxa-specific. Both studies, taken together, indicate that urbanization increases the presence of fecal bacteria and pathogens in this system, and that recreational contact with the river presents potential health risks.

Abstract ORIENTIA TSUTSUGAMUSHI ANKYRIN REPEAT-PROTEIN FAMILY TARGETING OF THE HOST ENDOPLASMIC RETICULUM By Lauren VieBrock, B.S. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University Virginia Commonwealth University, 2015 Director: Jason A. Carlyon, Ph.D. Professor Microbiology and Immunology Scrub typhus is an understudied, potentially fatal febrile illness, which poses threat to one billion people annually in the Asia-Pacific region. The host-pathogen interactions that facilitate the intracellular survival of the etiologic agent, Orientia tsutsugamushi, are not well understood. The Orientia tsutsugamushi genome encodes a large number of ankyrin repeat-containing proteins (Anks), key virulence factors for other intracellular pathogens, as well as components for Type I (T1SS) and Type 4 secretion systems (T4SS), commonly used to deliver them. We sought to characterize the roles of the Anks in O. tsutsugamushi infection. In this study, we demonstrated that O. tsutsugamushi expressed all 20 anks and the genes for the T1SS, for which they are substrates. Many ectopically expressed Anks displayed a tropism for the host endoplasmic reticulum (ER). These results suggest the importance of the Anks and the ER to Orientia tsutsugamushi pathobiology. We demonstrated that O. tsutsugamushi tightly associated with the ER and induced ER stress and defects in protein secretion of its host cells. Therefore, we hypothesized that the ER-tropic anks expressed during the initial hours of infection are critical for establishing infection and do so by interacting with specific host cell targets to modulate host cell function to benefit intracellular survival. ER-tropic Ank4 was detected as expressed early in infection and was further characterized for its contribution to the alterations of the ER during infection. Bat3 was identified as a target of Ank4, and Ank4 expression correlated with a decrease in Bat3 protein levels, induction of ER stress, and defects in protein secretion. These effects were Ank4 F-box dependent, implicating polyubiquitination and proteosomal degradation of Bat3. As Ank4 colocalized with Bat3, a chaperone component of ER-associated degradation (ERAD) of misfolded proteins, ERAD function was measured in cells expressing Ank4. In an F-box dependent manner, Ank4 expression resulted in decreased degradation of a model substrate and indicated inhibition of the ERAD pathway. Similarly, we demonstrated that in O. tsutsugamushi infection, Bat3 levels were significantly reduced early in infection and ERAD degradation was inhibited. After several days of infection however, Bat3 levels and ERAD degradation had both recovered, suggesting temporal modulation of ERAD in infection. Taken together, these data suggest that O. tsutsugamushi has a large capacity to disrupt the host ER, exemplified by Ank4 mediated ERAD dysfunction by depletion of host Bat3.

Pseudomonas aeruginosa (PA) infections in Cystic Fibrosis (CF) patients are not easily cleared due to the conversion from a nonmucoid to a mucoid phenotype. Alginate is an acetylated exopolysaccharide produced by mucoid PA that is responsible for increased resistance to antibiotics, host phagocytic killing, and propagating biofilm formation. Understanding the interaction between PA and host cells is critical to understanding chronic infection and inflammation in CF. In order to investigate this, we used A549 pulmonary epithelial cells and murine alveolar macrophages (MH-S) to examine host response to nonmucoid versus mucoid PA infection. Adhesion assays in A549 pulmonary epithelial cells revealed that mucoid PA mutants adhere poorly compared to their nonmucoid counterparts. Similarly, phagocytosis assays using MH-S infected with PA revealed that mucoid PA are increasingly resistant to phagocytosis. The alginate acetylation mutant FRD1175 is more susceptible to phagocytic killing than alginate+ FRD1. Adherence and phagocytosis of mucoid FRD1 was increased by increasing the multiplicity of infection (MOI) from 50:1 to 500:1. Furthermore, confocal microscopy revealed that mucoid PA are inherently less inflammatory than nonmucoid strains in both A549 and MH-S. Increasing the MOI of mucoid FRD1 from 50:1 to 500:1 significantly increased caspase-1 activation in MH-S but not in A549, revealing that intensity of inflammatory signaling by epithelial cells is likely independent of increased adherence. FRD1175 infection in both A549 and MH-S revealed that alginate acetylation plays a significant role in reducing inflammasome activation. Western analysis revealed that PA does not actively induce TGF-β secretion by A549 epithelial cells. Similarly, NF-κB expression was reduced in both A549 and MH-S when infected with mucoid FRD strains, but not PA from the PAO background, suggesting FRD strains have accumulated additional mutations facilitating escape of inflammation. MH-S treated with cytochalasin D to block phagocytosis were still able to activate NF-κB signaling, suggesting NF-κB activation is adherence but not phagocytosis dependent. These data increase our understanding of the various mechanisms in which mucoid PA is able to evade host immune defenses and provides insight into potential therapies to treat PA infections.