Microbiology and Immunology Department Theses and Dissertations
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Item Characterization of Normal and Preleukemic Hematopoietic Stem Cell Responses to Physiologic and Extra-Physiologic Oxygen Tension(2022-08) Aljoufi, Arafat; Kaplan, Mark H.; Zhang, Chi; Srour, Edward F.; Kapur, ReubenHematopoietic stem and progenitor cells (HSCs/HPCs) transplantation is a curative treatment for a variety of hematologic and non-hematologic diseases. Successful HSC transplantation requires infusing patients with a sufficient number of long-term engrafting HSCs. As a result, research efforts have focused on optimizing the collection process. Previous work established that harvesting mouse bone marrow HSCs under low oxygen tension similar to that reported for the bone marrow niche in situ (physioxia), results in enhanced HSC recovery and function. However, collecting bone marrow cells under physioxia is not a clinically viable approach. Here, I demonstrated that the collection and processing of peripheral blood mobilized with G-CSF alone or G-CSF and Plerixafor under physioxia resulted in a greater number of phenotypically defined long-term engrafting HSCs. Using high-resolution single cell sequencing to explore the molecular programs governing HSCs under physioxia, I identified increased expression of genes involved in HSC self-renewal and maintenance. In contrast, HSCs under ambient air upregulated genes implicated in HSC differentiation, apoptosis, and inflammatory pathways. Furthermore, wild-type HSCs under physioxia revealed a significant reduction in gene expression and activity of the epigenetic modifier Tet2. Consequently, I evaluated the phenotyping, engraftment potential and gene expression of preleukemic Tet2-/- bone marrow cells under physioxia and ambient air. Unlike wild-type HSCs, Tet2-/- HSCs/HPCs were unresponsive to changes in oxygen tension. Notably, we observed similar phenotypes, functions, and self-renewal and quiescence gene expression in wild-type HSCs under physioxia and Tet2- /- HSCs under physioxia or ambient air. These findings imply that the preserved stemness and enhanced engraftment of HSCs under physioxia may in part be a result of Tet2 downregulation. Understanding the mechanisms regulating wild-type and preleukemic HSCs under physioxia will have therapeutic implications for optimizing HSC transplantation and mitigating the growth advantage of preleukemic stem cells.Item The Role of Mammalian Lipid Transport Protein ORP1 During Coxiella Burnetii Infection(2022-05) Schuler, Baleigh Elizabeth; Gilk, Stacey D.; Arrizabalaga, Gustavo; Spinola, Stanley; Harrington, Maureen; Day, RichardCoxiella burnetii is an intracellular bacterium that causes the human disease Q fever. C. burnetii is transmitted from infected animals to humans through inhalation of infectious droplets. Acute Q fever is a flu-like illness lasting 10-14 days. Patients often have respiratory symptoms and present with pneumonia. Patients with suppressed immune systems or valvular heart disease can develop chronic Q fever, which causes endocarditis and vasculitis long after initial infection. Chronic Q fever is difficult to treat, and if untreated, is typically fatal. Currently, the United States lacks any vaccine for Q fever. In order to better prevent and treat this disease, it is important to understand how C. burnetii interacts with mammalian cells. Within the host cell, C. burnetii forms a large, acidic Coxiella-containing vacuole (CCV) and uses a Type 4B secretion system (T4SS) to secrete effector proteins into the host cell cytoplasm. While the CCV membrane is rich in sterols, cholesterol accumulation in the CCV is bacteriolytic, suggesting that C. burnetii regulation of lipid transport is critical for infection. The mammalian lipid transport protein ORP1L localizes to the CCV membrane and mediates CCV-ER membrane contact sites. ORP1L functions in lipid transport, including cholesterol efflux from late endosomes/lysosomes. Its sister isoform ORP1S binds cholesterol but localizes to the cytoplasm and nucleus. In ORP1- null cells, we found that CCVs were smaller than in wildtype cells, highlighting the importance of ORP1 in CCV development. CCVs in ORP1-null cells had higher cholesterol content than CCVs in wildtype cells, suggesting ORP1 functions in cholesterol efflux from the CCV. ORP1-null MH-S cells do not accumulate lipid droplets upon C. burnetii infection, supporting our hypothesis that ORP1 promotes cholesterol transfer from the CCV to the ER, as lipid droplets form from neutral lipids in the ER. While the absence of ORP1 led to a C. burnetii growth defect in MH-S cells, there was no growth defect in HeLa cells. Together, our data demonstrate that C. burnetii uses the host sterol transport protein ORP1 to promote CCV development, potentially by using ORP1 to facilitate cholesterol efflux from the CCV to diminish the bacteriolytic effects of cholesterol.Item The Role of PfEMP1 Expression and Immunity in Ugandian Children with Severe Malaria(2022-05) Fernander, Elizabeth M.; John, Chandy; Bauer, Margaret; Gilk, Stacey; Tran, TuanSevere malaria, primarily caused by Plasmodium falciparum infection, is among the leading causes of childhood mortality globally. A key virulence factor and source of antigenic variation and immune evasion during infection is P. falciparum erythrocyte membrane protein 1 (PfEMP1). Encoded for by approximately 60 var genes, this complex protein mediates cytoadherence of infected erythrocytes to the host endothelium and is a prominent immune target for the anti-malarial immune response in children. During severe malaria, specific domains of PfEMP1 that bind to endothelial protein C receptor (EPCR) and intercellular adhesion molecule-1 (ICAM-1) on host endothelial cells, are more prevalently expressed. The interaction of these proteins and infected erythrocytes mediates the sequestration of infected erythrocytes and plays a role in severe malaria pathogenesis. Antibodies to these domains develop over time with exposure to the parasite and are thought to contribute to immunity against severe malaria in children. In this study, whole blood samples from children with different forms of severe malaria, enrolled in two observational prospective cohort studies were used to quantify the expression of PfEMP1 domains using RT-qPCR and to measure the antibody response to PfEMP1 domains via a bead-based multiplex immunoassay. Using these samples, we demonstrated that although the expression of var transcripts encoding PfEMP1 domains was generally similar across children with different forms of severe malaria, the expression of variants encoding specific EPCR-binding domains was associated with thrombocytopenia and severe anemia. The antibody response to PfEMP1 domains in children with severe malaria was highest in children with SMA and children with asymptomatic parasitemia, but not associated with decreased risk of additional malaria episodes. Overall, the results of this study suggest that PfEMP1 is acting similarly across different forms of severe malaria but that it can be related to pathogenesis and severe malaria immunity.Item The Kinetics of Antibody Responses to Plasmodium Vivax Vaccine Candidate Antigens in Brazilians with Acute Vivax Malaria(2022-05) Tashi, Tenzin; Tran, Tuan M.; Bauer, Margaret E.; Hoang, Quyen Q.Plasmodium vivax malaria is geographically widespread and remains a significant public health burden in the Americas, Southeast Asia, and the western Pacific. In order to achieve the end goal of malaria eradication, a highly effective vaccine targeting P. vivax is urgently needed. Unlike pre-erythrocytic vaccines that aim to confer sterile immunity that prevents malaria infection altogether, Plasmodium vivax blood-stage vaccines aim to confer clinical immunity that protects against malarial disease by controlling parasitemia and mitigating the symptomatic manifestations of malaria after infection. To design an effective P. vivax blood-stage vaccine, it is essential to understand the acquisition and longevity of natural humoral immune responses against promising P. vivax blood-stage vaccine candidate antigens. We hypothesize that acute vivax malaria induces differential humoral immune responses against P. vivax antigens that exhibit antigen-specific kinetic and compositional profiles, which can be used to identify vaccine candidates that elicit durable humoral responses. Therefore, we compared the kinetic profiles and half-lives of naturally acquired IgG antibodies reactive against nine promising P. vivax blood-stage vaccine candidate antigens up to 180 days post-infection in Brazilians with acute vivax malaria. Naturally acquired IgG antibodies against these antigens have previously been associated with a reduced risk of vivax malaria. Among the P. vivax antigens evaluated, the merozoite antigen Pv12 elicited the most durable IgG antibodies, whereas the DBP-FL elicited the most short-lived responses. Neither patient age nor prior malaria exposure significantly correlated with the magnitude and durability of IgG responses to any P. vivax antigen. Seropositivity, against Pv12, was generally maintained for at least 30 days after acute vivax malaria. These findings suggest that a blood-stage vaccine targeting Pv12 may benefit from boosting IgG antibodies against this antigen after natural vivax “breakthrough” infections. Further studies will be needed to determine the Pv12-specific memory B cell response as well as the functional role for naturally acquired Pv12-specific antibodies in reducing parasitemia and/or clinical disease. In summary, the current study has provided insight into the longevity of IgG antibody responses to important P. vivax antigens after an acute malaria episode.Item Proteomic Analysis of Nuclear HBV rcDNA Associated Proteins Identifies UV-DDB as a Host Factor Involved in cccDNA Formation(2022-01) Marchetti, Alexander Lloyd; Guo, Haitao; Yu, Andy; Androphy, Elliot J.; Robinson, ChristopherDespite the lifecycle of the hepatitis B virus (HBV) being extensively investigated and described, there remains a significant gap in our knowledge of arguably one of the most crucial steps in the HBV lifecycle, the formation and maintenance of a covalently closed circular DNA (cccDNA) reservoir. Advancements in our understanding of host factors and pathways involved in cccDNA formation have been made through hypothesis driven studies and shRNA/siRNA screenings. We sought to create a targeted-unbiased assay to directly observe host factor-rcDNA interactions. This was achieved through an rcDNA Co-Immunoprecipitation paired Mass Spectrometry (rcDNA-CoIP/MS) assay. We created a DNA oligo complimentary to the open portion of the HBV rcDNA, labeled with biotin, to facilitate easy precipitation of nuclear rcDNA and complexed proteins. Proteins precipitated were analyzed through liquid chromatography paired mass spectrometry (LC/MS). Along with previously reported host factors, several factors of DNA damage repair pathways/complexes were also identified. A component of the UV-DDB complex, DDB1, surfaced as a hit. UV-DDB/rcDNA binding was confirmed through ChIP-qPCR. DDB2, the DNA damage binding component of the UV-DDB complex was knocked out in HepG2-NTCP and HepAD38 cells. This resulted in a significant decrease in the formation of cccDNA in DDB2 knockout cell lines following infection or induction. The subsequent reduction of downstream indicators of cccDNA formation such as viral RNA and proteins, HBcAg and HBeAg, showed a consistent decrease with cccDNA levels. Ectopic expression of DDB2 in the knockout cell lines rescued HBV phenotypes of cccDNA levels and its downstream indicators. Inactive mutant DDB2 plasmids were also transfected into the DDB2 K/O cell lines and failed to rescue cccDNA indicators. We therefore showed through a novel assay that we can discover novel viral rcDNA-host interactions, such as the UV-DDB complex recruiting DNA repair pathways to “repair” rcDNA to cccDNA.Item The Role of Chlamydia Protein TC0600 in Gastrointestinal Tract Infection(2021-12) Alrebdi, Waleed; Nelson, David; Bauer, Margaret; Yang, X. FrankChlamydia is the most frequently reported bacterial sexually transmitted infection in the world. Most urogenital chlamydia infections in men and women are asymptomatic, but these infections can lead to irreparable damage in the reproductive system and other tissues. Apart from the urogenital chlamydial infections, we know that chlamydia infects the gastrointestinal tract (GIT) in humans and can colonize the GIT for extended intervals without eliciting pathology. We are interested in investigating tissue tropism determinants in Chlamydia spp. because these could be targeted to development live-attenuated vaccines. Recently, we generated mutagenized isolates of the mouse pathogen Chlamydia muridarum, a close relative of the human pathogen Chlamydia trachomatis which causes chlamydia. One mutant that we isolated is significantly attenuated in murine gastrointestinal tissues compared to wild type, but retains its pathogenicity in the murine urogenital tract. Using novel genetic techniques, whole-genome sequencing, and complementation using newly developed vector systems we identified a chromosomal factor, tc0600, that we believe mediates the altered tissue tropism phenotype of this mutant in mice. Notably, the Chlamydia trachomatis ortholog of tc0600 has been linked to chlamydial GIT tropism in humans.Item Inhibition of TFEB activation promotes Coxiella burnetii growth(2021-05) Das Ghatak, Piya; Gilk, Stacey D.; Bauer, Margaret E.; Robinson, Christopher M.Coxiella burnetii is the etiologic agent of Q fever, a zoonotic disease characterized by flu-like sickness in acute cases; endocarditis may occur and turn deadly if not treated correctly in chronic patients. Coxiella, an obligate intracellular bacterium, requires establishment of a replicative niche in the host cell. After being phagocytosed by the eukaryotic cell, the bacterium resides in a tight-fitting nascent phagosome which matures through the host canonical endocytic pathway, acquiring endosomal/lysosomal markers as well as acidic pH. Initial acidification of the Coxiella containing vacuole (CCV) is central to the bacterium’s pathogenesis because translocation of bacterial effector proteins into the host cell by the type 4B secretion system (T4BSS) initiates only after it senses the acidic environment. The effector proteins are required for subverting different host cell functions in favor of Coxiella growth, CCV maturation and are crucial for bacterial virulence. Contrary to the belief that since CCV matures through the host endocytic pathway, CCV is as acidic as lysosome, we found that CCV is significantly less acidic (pH~5.2) than lysosomes (pH~4.8) and inducing further CCV acidification causes Coxiella lysis. Furthermore, increasing lysosomal biogenesis in the host cell is detrimental for Coxiella growth. So, we hypothesized that Coxiella blocks lysosomal biogenesis in host cells to maintain the CCV pH just optimal for its growth. Lysosomal biogenesis is regulated by the master transcription factor EB (TFEB). Its ability to act as a transcription factor depends on its subcellular localization, which relies on its phosphorylation state. TFEB, when phosphorylated is cytosolic and inactive, whereas dephosphorylated TFEB translocates to the nucleus and is active, binding to promoter regions of lysosomal genes of the CLEAR network, thus controlling lysosome biogenesis. Therefore, we hypothesized that Coxiella blocks TFEB translocation to the nucleus, thus inhibiting lysosome biogenesis. We determined that Coxiella grows significantly better in TFEB-KO cells than they do in parentals. Also, using a torin-induced TFEB translocation model, we observed remarkably decreased TFEB activation in the Coxiella infected cells as was evident by less TFEB translocation to nucleus. Overall, data obtained from this work suggest that Coxiella inhibits lysosome biogenesis by blocking TFEB nuclear translocation.Item The Development and Function of IL-9-Secreting T Helper Cells During Chronic and Allergen Recall-Induced Allergic Airway Disease(2021-04) Ulrich, Benjamin Joseph; Kaplan, Mark H.; Dent, Alexander L.; Srour, Edward F.; Tepper, Robert S.Asthma is a chronic inflammatory lung disease with intermittent flares predominately mediated through memory T cells. The majority of the T cells in tissues such as the lung are tissue-resident memory (Trm) cells, defined as cells that maintain long-lasting presence in the tissue and have rapid functional recall following challenge. Allergen-specific CD4 T helper cells that secrete the cytokine IL-9 have been shown to be a necessary component of asthma pathogenesis. However, the precise characterization and function of IL-9-secreting CD4+ cells (Th9 cells) are unknown. Here we demonstrate that IL-9 production is progressively lost in Th9 cells over several rounds of culture and that environmental cues dictate the instability or effector function of the Th9 phenotype. We show Th9 cells are long-lived tissue-resident cells with the capacity to rapidly respond to secondary allergen challenge causing allergic airway disease (AAD). We found in a memory model of Aspergillus fumigatus challenge, Th9 cells maintain tissue residency throughout a 12-week period of antigen-free rest. Additionally, we demonstrated increased frequency of IL-9-producing cells and quantity of IL-9 upon rechallenge, characteristic of a secondary response. Antibody blockade of IL-9 immediately prior to the recall challenge significantly reduced overall allergic lung inflammation, suggesting that IL-9 plays an obligate role in the allergic memory response following pulmonary allergen challenge. The protection afforded by IL-9 antibody blockade was not seen in a chronic model asthma-like disease demonstrating IL-9 has a specific role in allergic memory responses. Interestingly, IL-9-secreting cells have a polyfunctional multi-cytokine phenotype demonstrating a highly pathogenic state that we reproduced in culture. These observations suggest that IL-9 from Trm cell populations and Th9 cells play a novel role in allergen recall responses and are potential therapeutic targets for patients suffering from chronic intermittent asthma.Item Leptin Receptor, a Surface Marker for a Subset of Highly Engrafting Long-Term Functional Hematopoietic Stem Cells(2021-04) Trinh, Thao Le Phuong; Broxmeyer, Hal E.; Srour, Edward F.; Kapur, Reuben; Utpal, DaveThe entire hematopoietic system rests upon a group of very rare cells called hematopoietic stem cells (HSCs). Due to this extraordinarily crucial role, after birth HSCs are localized to the deep bone marrow niche, a hypoxic environment inside the bone where HSCs are under well-orchestrated regulation by both cellular and humoral factors. Among the cellular components regulating hematopoiesis are Leptin Receptor (LEPR)-expressing mesenchymal/stromal cells and adipocytes; both have been demonstrated to have significant influence on the maintenance of HSCs under homeostasis and in stress-related conditions. It has been reported in early work by others that HSCs and hematopoietic progenitor cells (HPCs) express LEPR. However, whether LEPR+ HSCs/HPCs are functionally different from other HSCs/HPCs was unknown. In this study, I demonstrated for the first time that murine LEPR+ Lineage-Sca-1+cKit+ (LSK, a heterogenous population consisting of HSCs/HPCs) cells even though constituting a small portion of total LSK cells are significantly enriched for both phenotypic and functional self-renewing long-term (LT) HSCs as shown in primary and secondary transplants in lethally irradiated recipients. LEPR+LSK cells are also more enriched for colony-forming progenitor cells assessed by colony-forming unit (CFU) assays. In addition, LEPR+ HSCs (defined as LSKCD150+CD48-) exhibited robust repopulating potential as compared to LEPR-HSCs in long-term competitive transplantation assays. To elucidate the molecular pathways that may govern functional properties of LEPR+HSCs, bulk RNA-seq on freshly sorted cells was done. Gene set enrichment analyses (GSEA) revealed Interferon Type I and Interferon γ (IFNγ) Pathways were significantly enriched in LEPR+HSCs while mitochondrial membrane protein gene set was significantly enriched in LEPR-HSCs. Interestingly, proinflammatory signaling including IFNγ pathway has been suggested to be critical for the emergence of embryonic HSCs from the hemogenic endothelium. Altogether, our work demonstrated that LEPR+HSCs represent a small subset of highly engrafting adult BM HSCs. These results may have potential therapeutic implications in the field of hematopoietic transplantation as LEPR is highly conserved between mice and humans.Item Characterization of a Novel Fis1 Interactor Required for Peripheral Distribution of the Mitochondrion of Toxoplasma Gondii(2021-02) Jacobs, Kylie; Arrizabalaga, Gustavo; Gilk, Stacey; Graham, Brett; John, Chandy; Yang, FrankToxoplasma’s singular mitochondrion is extremely dynamic and undergoes morphological changes throughout the parasite’s life cycle. While intracellular‚ the mitochondrion is maintained in a lasso shape that stretches around the parasite periphery and is in close proximity to the pellicle‚ suggesting the presence of membrane contact sites. Upon egress‚ these contact sites disappear‚ and the mitochondrion retracts and collapses towards the apical end of the parasite. Once reinvaded‚ the lasso shape is quickly reformed‚ indicating that dynamic membrane contact sites regulate the positioning of the mitochondrion. We discovered a novel protein (TgGT1_265180) that associates with the mitochondrion via interactions with the fission related protein Fis1. Knockout of TgGT1_265180‚ which we have dubbed LMF1 for Lasso Maintenance Factor 1‚ results in a complete disruption of the normal mitochondrial morphology. In intracellular LMF1 knockout parasites, the mitochondrial lasso shape is disrupted‚ and instead it is collapsed as normally only seen in extracellular parasites. Additionally, proper mitochondrial segregation is disrupted‚ resulting in parasites with no mitochondrion and extra mitochondrial material outside of the parasites. These gross morphological changes are associated with a significant reduction of parasite propagation and can be rescued by reintroduction of a wildtype copy of LMF1. Co-immunoprecipitations and Yeast Two-Hybrid predict interactions with the parasite pellicle. Therefore, we hypothesize that LMF1 mediates contact between the mitochondrion and the pellicle in a regulatable fashion‚ and that the LMF1-dependent morphodynamics are critical for parasite propagation. Current studies are focused on characterizing the consequences of mitochondrial collapse and identifying proteins that interact with LMF1 to position the mitochondrion to the periphery of the parasite.