Theses and Dissertations from UMD
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Item Meta-Transcriptomic Profiling of Human Cutaneous Leishmaniasis(2018) Christensen, Stephen Michael; Mosser, David M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Our understanding of the spectral nature of the neglected tropical disease leishmaniasis, and of host-parasite interactions in general, remains incomplete. In this work, we used high throughput RNA-sequencing (RNA-seq) to analyze human host and Leishmania gene expression in cutaneous leishmaniasis patients. Skin biopsies were taken from a total of 25 localized cutaneous leishmaniasis (LCL), 6 diffuse cutaneous leishmaniasis (DCL), and 10 healthy patients. LCL separated into groups that lacked detectable parasite transcripts in lesions (PTNeg) and a group in which parasite transcripts were readily detected (PTPos). These groups exhibited substantial differences in host responses to infection, including B lymphocyte presence, B and T cell activation, and immunoglobulin production. Analysis of DCL lesions revealed distinct differences in host responses relative to LCL, including atypical B lymphocyte accumulation, diminished cytotoxic T lymphocyte responses, and an altered macrophage activation state. Surprisingly, neither localized nor diffuse forms of the disease could be correlated with any indication of a Th2 immune response that had previously been implicated in mouse models of L. major susceptibility. The presence of low levels of parasite transcripts in the majority of LCL patients made it difficult to obtain a comprehensive analysis of the parasite transcriptome in LCL. However, high levels of parasite transcripts in DCL afforded a unique opportunity to examine parasite gene expression in this disease. Despite differences in age, gender, and illness duration, there was a remarkable uniformity in parasite gene expression in all 6 DCL patients. We identified transcripts that were highly expressed by all 6 DCL patients, and then curated a subset of conserved genes highly expressed in multiple Leishmania species. These subsets of genes emerge as targets for further research on host-pathogen interactions and a better understanding of Leishmania infection.. In summary, RNA-seq allowed us to fully examine host and parasite transcriptomes, characterize host responses in localized and diffuse cutaneous leishmaniasis lesions, and determine factors that define the variations in disease manifestation. New approaches to modify host immune responses in this disease and new parasite targets for drug development may emerge from this work.Item Transcriptomic profiling of Leishmania parasites and host macrophages during an infection(2015) Dillon, Laura Anne Liefer; El-Sayed, Najib M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Leishmania parasites cause leishmaniasis, a group of diseases that range in manifestations from skin lesions to fatal visceral disease. The parasite's life cycle is divided between its insect vector and its mammalian host, where it resides primarily inside of macrophages. Once intracellular, Leishmania parasites must avoid being killed by the innate and adaptive immune responses. We performed transcriptomic profiling using RNA-seq to simultaneously identify global changes in gene expression in Leishmania parasites across multiple lifecycle stages and in infected macrophages from both murine and human hosts. Using a novel approach based on a dual statistical test to identify genes that were differentially expressed relative to both uninfected macrophages and macrophages that had ingested inert particles, we were able to filter out genes that were differentially regulated as part of a general phagocytic response and thereby select genes specific to Leishmania infection. The most substantial and dynamic Leishmania-specific differential expression responses were observed during early infection, while changes observed later were common to phagocytosis more generally. An evaluation of RNA processing events within the parasite revealed precise UTR boundaries for a majority of genes and widespread alternative trans-splicing and polyadenylation. Collection of data from multiple biological replicates, the use of matched host control samples, careful statistical analysis of variation, and removal of batch effects enabled the detection of biological differences between samples and timepoints with high confidence and sensitivity. Pathway and gene ontology analyses provided insights into the higher level processes activated across parasite developmental stages and during intracellular infection to reveal signatures of Leishmania differentiation and infection.Item CHARACTERIZATION OF HEME ACQUISITION IN LEISHMANIA(2014) Renberg, Rebecca; Andrews, Norma W; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Heme is an essential co-factor for many critical cellular processes. The protozoan parasite Leishmania amazonensis is a heme auxotroph and must acquire heme from the environment. One pathway of heme acquisition is through Leishmania Heme Response 1 (LHR1), a heme transporter localized to the plasma membrane and acidic intracellular compartments. In this work we further characterize LHR1 and the mechanism by which it promotes heme uptake. We show that overexpression of LHR1 in Leishmania amazonensis increases the total parasite intracellular heme pool, and that expression in Saccharomyces cerevisiae promotes uptake of the heme analog Zinc Mesoporphyrin IX (ZnMP). Our results indicate that heme binding to LHR1 is pH independent, whereas heme transport by the parasites is more efficient under acidic conditions. To examine the molecular mechanisms responsible for LHR1 heme transport, we performed a mutagenesis analysis of LHR1. We show that three key tyrosines residues, Tyr-18, Tyr-80, and Tyr-129, located in predicted transmembrane domains near the cytoplasmic leaflet of the plasma membrane, are important for heme transport. Although the mutant proteins appear to not affect promastigote growth, they have a profound inhibitory effect on intracellular amastigote replication in macrophages, and are necessary for virulence in vivo. Finally, we also examine the differential regulation of LHR1 expression in a visceralizing species, Leishmania chagasi, compared to L. amazonensis, a species that causes cutaneous lesions. L. chagasi has higher amounts of LHR1 transcripts than Leishmania amazonensis under heme-depleted conditions, and uptakes ZnMP faster and to a greater extent than Leishmania amazonensis. This differential regulation of LHR1 may be due to differences in the gene 3’ Untranslated Regions (UTRs) between the two species. This works adds to our understanding of the critical process of heme transport and its role in Leishmania virulence.Item CHARACTERIZATION OF THE ROLE OF MAPKS IN LEISHMANIA INFECTED MACROPHAGES.(2009) Yang, Ziyan; Mosser, David M.; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In the current study, we examined the role of the Mitogen Activated Protein Kinases (MAPKs) on the biological responses of macrophages infected with Leishmania. The first section examined the role of MAPK/ERK in IL-10 production by Leishmania-infected macrophages. The macrophage-derived IL-10 has been shown to exacerbate Leishmaniasis. However, the molecular mechanisms whereby Leishmaniasis prompts IL-10 induction are poorly understood. A combination of two signals was necessary for IL-10 induction by the Leishmania amastigotes-infected macrophages. The first signal is mediated by TLR ligation whereas the second signal is mediated by FcgammaR ligation, which yields a population of regulatory macrophages that produce high levels of IL-10. Infection of macrophages with L. amazonensis amastigotes from the lesion sites sparked MAPK/ERK activation, which was required, but not sufficient for IL-10 induction. In combination with an inflammatory stimulus, LMW-HA from the extracellular matrix, these parasites triggered the macrophages to highly produce IL-10. MAPK/ERK activation initiated an epigenetic modification of chromatin at the IL-10 locus, which allowed for transcription factor Sp1 binding to drive IL-10 transcription and subsequent production. U0126, an inhibitor of MAPK/ERK activation, decreased lesion progression in Leishmania infected mice. The second section examined the role of MAPK/p38 in cytokine production and vaccination against Leishmaniasis. TLR agonists activate macrophages to produce pro-inflammatory cytokines and reactive oxygen intermediates. Inhibition of MAPK/p38 reciprocally increased IL-12 but decreased TNFa production from LPS-stimulated macrophages, which also occurred following stimulation by a variety of other TLR agonists, and using different APCs. MAPK/p38 inhibition induced IL-12p40 mRNA accumulation mainly due to enhanced mRNA stability, which was independent of IL-10. Similar results were observed by knocking down MAPK/p38 using specific siRNAs or by targeted deletion of MKK3. IL-12 production following the inhibition of MAPK/p38 skewed antigen-specific T cells to produce more IFN-gamma and less IL-4 in vitro. A MAPK/p38 inhibitor was applied as an adjuvant to vaccine mice against L. major, which resulted in smaller lesions with fewer parasites. Our findings reveal an important role of MAPKs in the Leishmania pathogenesis, and suggest that the manipulation of these kinases may provide novel therapeutics for potential clinical applications.Item The development and characterization of transgenic Leishmania major expressing murine CD40L(2007-05-18) Field, Ann Elizabeth; Mosser, David M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Leishmanization is the inoculation of live Leishmania into the host to vaccinate against subsequent infections. This approach has been largely discontinued due to safety concerns. We have previously shown that combining CD40L with Leishmania antigen preferentially induces a type 1 immune response and provides some protection to vaccinated mice. In the present study, we developed transgenic L. major which express and secrete the extracellular portion of CD40L (L. major CD40LE). We hypothesized that these organisms would be less virulent but more immunogenic than wild-type organisms, and therefore be more effective at leishmanization. Transgenic parasites expressing CD40L mRNA and protein were developed. These parasites had similar growth characteristics to wild-type organisms. Susceptible BALB/c mice infected with these parasites developed significantly smaller lesions containing fewer parasites than animals infected with wild-type organisms. Infection of C57BL/6 CD40L-/- mice with transgenic L. major resulted in significantly smaller lesions than infection with wild-type L. major, indicating in vivo biological activity of the transgenic protein. Infection of resistant C57BL/6 mice with low doses of transgenic parasites induced a significant amount of protection against subsequent high dose infection with wild-type organisms. These results demonstrate that transgenic organisms expressing CD40L are less virulent than wild-type organisms while retaining full immunogenicity. The implications of this study are that parasites expressing immune-modulatory molecules may be improved alternatives to traditional leishmanization.Item The Influence of Parasite-derived Chemokines in Leishmaniasis(2006-12-13) Conrad, Sean Martin; Mosser, David M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Transgenic chemokine-secreting parasites were generated and used to actively recruit immune cells into Leishmania lesions. It was hypothesized that the chemokine induced cell migration would influence the magnitude and character of the immune response and thereby effect the outcome of disease. Two different transgenic chemokine-secreting parasites were engineered. One transgenic parasite secretes murine MCP-1, a CC chemokine primarily responsible for macrophage recruitment. The other transgenic parasite secretes murine IP-10, a CXC chemokine known to attract activated T-cells. Both transgenic parasites transcribed murine chemokine mRNA, translated murine chemokine protein, and infected and replicated inside resting peritoneal macrophages similar to wild-type parasites. However, the two transgenic parasites caused diverse phenotypes in infected mice. The MCP-1 secreting parasites caused little or no detectable lesions, while the IP-10 secreting parasites caused lesions that were significantly larger than the wild-type infected mice. The healing phenotype caused by MCP-1 secreting parasites was further analyzed. Infection of BALB/c, C57BL/6, or MCP-1 knockout (KO) mice with MCP-1 secreting parasites resulted in minimal lesion development compared to mice infected with wild-type parasites. MCP-1 secreting parasites caused substantial lesions with relatively high numbers of parasites in CCR2 KO mice indicating that the parasites are viable and healthy, and that the lack of lesion development is CCR2- dependent. The enumeration of transgenic MCP-1 parasites in lesions demonstrated a significant reduction in parasite numbers, which coincided with an increase in CCR2-positive macrophage migration on day 7. CCR2-positive macrophages isolated from ears of mice infected with transgenic MCP-1 parasites contained virtually no parasites, whereas infection with wild-type parasites resulted in heavily-infected macrophages in lesions. The lack of parasite survival in mice infected with MCP-1 secreting parasites suggests that parasite-derived MCP-1 is recruiting a population of CCR2-positive macrophages to the lesion that efficiently kill Leishmania parasites. In-vitro studies revealed that optimal parasite killing required the recruitment of CCR2-positive macrophages followed by stimulation with a combination of both MCP-1 and IFN-g. This work suggests that the parasite-derived MCP-1 can recruit a restrictive population of CCR2-positive macrophages into lesions that can be optimally stimulated by MCP-1 and IFN-g to efficiently kill Leishmania parasites.