Browsing by Author "Absalon, Sabrina"

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    A Human Pluripotent Stem Cell-Derived In Vitro Model of the Blood-Brain Barrier in Cerebral Malaria
    (2024-01) Gopinadhan, Adnan; John, Chandy C.; Nelson, David E.; Bauer, Margaret E.; Absalon, Sabrina; Tran, Tuan M.
    Blood-brain barrier (BBB) disruption is a central feature of cerebral malaria (CM), a severe complication of Plasmodium falciparum (Pf) infections. In CM, sequestration of Pf-infected red blood cells (Pf-iRBCs) to brain endothelial cells combined with inflammation, hemolysis, microvasculature obstruction and endothelial dysfunction mediates BBB disruption, resulting in severe neurologic symptoms including coma and seizures, potentially leading to death or long-term sequelae. In vitro models have advanced our knowledge of CM-mediated BBB disruption, but the physiological relevance remains uncertain. I aimed to develop a novel in vitro model of the BBB in CM using human induced pluripotent stem cell-derived brain microvascular endothelial cells (hiPSC-BMECs) that mimic a near in vivo barrier phenotype. hiPSC-BMECs were co-cultured with HB3var03 strain Pf-iRBCs up to 9 hours. Barrier integrity was measured using transendothelial electrical resistance (TEER). Localization and expression of tight junction (TJ) proteins, occludin and zona occludin-1 (ZO-1), and endothelial marker, intercellular adhesion molecule 1 (ICAM-1) was determined using immunofluorescence imaging (IF) and western blotting (WB). Expression of angiogenic and cell stress markers were also measured. hiPSC-BMECs showed improved barrier integrity and localization of TJ proteins compared to immortalized BMECs. After 6-hours of co-culture with Pf-iRBCs, hiPSC-BMECs showed reduced TEER and disruption of TJ protein localization compared to co-culture with uninfected RBCs (RBCs), but no change in TJ protein expression was observed by WB in the Pf-iRBCs co-cultures. Expression of ICAM-1 on hiPSC-BMECs co-cultured with Pf-iRBCs was higher compared to co-culture with RBCs. In addition, there was an increase in expression of the angiogenin, platelet factor 4, and phospho-heat shock protein-27 in the Pf-iRBCs co-cultures compared to co-cultures with RBCs. These findings demonstrate the physiological relevance of our hiPSC-BMEC-based in vitro model of the BBB, as determined by elevated TEER and appropriate TJ protein localization. In co-culture with Pf-iRBCs, breakdown in the barrier integrity, changes in TJ protein localization, increase in expression of ICAM-1, and of markers of angiogenesis and cellular stress, all point towards a more relevant in vitro model, suitable for investigating pathogenic mechanisms underlying BBB disruption in CM.
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    An Essential Adaptor for Apicoplast Fission and Inheritance in Malaria Parasites
    (Research Square, 2025-05-05) Blauwkamp, James; Rajaram, Krithika; Staggers, Sophia R.; Harrigan, Oliver; Doud, Emma H.; Prigge, Sean T.; Sun, Stella Y.; Absalon, Sabrina; Pharmacology and Toxicology, School of Medicine
    Blood-stage Plasmodium falciparum parasites rely on a non-photosynthetic plastid, the apicoplast, for survival, making it an attractive target for antimalarial intervention. Like the mitochondrion, the apicoplast cannot be generated de novo and must be inherited by daughter parasites during cell division. This inheritance relies on coordinated apicoplast positioning and fission, but the molecular mechanisms controlling these processes remain poorly understood. Here, we identify a previously uncharacterized P. falciparum protein (Pf3D7_0613600), which we name PfAnchor, as a key regulator of apicoplast fission. Using Ultrastructure Expansion Microscopy (U-ExM), we show that PfAnchor localizes to the apicoplast throughout the asexual blood-stage. Conditional depletion disrupts apicoplast fission, leading to incomplete cytokinesis and parasite death. Notably, loss of the apicoplast's elongated branched structure via azithromycin treatment rescues these defects, underscoring Anchor's specific role in apicoplast fission. Immunoprecipitation identified an interaction with the dynamin-like GTPase PfDyn2, a key mediator of both apicoplast and mitochondrial fission, establishing PfAnchor as the first apicoplast-specific dynamin adaptor protein. Our findings define PfAnchor as an essential factor for apicoplast fission and inheritance in P. falciparum blood-stage parasites, highlighting parasite-specific organelle division as a potential vulnerability for therapeutic intervention.
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    Apicoplast Dynamics During Plasmodium Cell Cycle
    (Frontiers Media, 2022-04-29) Elaagip, Arwa; Absalon, Sabrina; Florentin, Anat; Pharmacology and Toxicology, School of Medicine
    The deadly malaria parasite, Plasmodium falciparum, contains a unique subcellular organelle termed the apicoplast, which is a clinically-proven antimalarial drug target. The apicoplast is a plastid with essential metabolic functions that evolved via secondary endosymbiosis. As an ancient endosymbiont, the apicoplast retained its own genome and it must be inherited by daughter cells during cell division. During the asexual replication of P. falciparum inside human red blood cells, both the parasite, and the apicoplast inside it, undergo massive morphological changes, including DNA replication and division. The apicoplast is an integral part of the cell and thus its development is tightly synchronized with the cell cycle. At the same time, certain aspects of its dynamics are independent of nuclear division, representing a degree of autonomy in organelle biogenesis. Here, we review the different aspects of organelle dynamics during P. falciparum intraerythrocytic replication, summarize our current understanding of these processes, and describe the many open questions in this area of parasite basic cell biology.
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    Atlas of Plasmodium falciparum intraerythrocytic development using expansion microscopy
    (eLife Sciences, 2023-12-18) Liffner, Benjamin; Cepeda Diaz, Ana Karla; Blauwkamp, James; Anaguano, David; Frolich, Sonja; Muralidharan, Vasant; Wilson, Danny W.; Dvorin, Jeffrey D.; Absalon, Sabrina; Pharmacology and Toxicology, School of Medicine
    Apicomplexan parasites exhibit tremendous diversity in much of their fundamental cell biology, but study of these organisms using light microscopy is often hindered by their small size. Ultrastructural expansion microscopy (U-ExM) is a microscopy preparation method that physically expands the sample by ~4.5×. Here, we apply U-ExM to the human malaria parasite Plasmodium falciparum during the asexual blood stage of its lifecycle to understand how this parasite is organized in three dimensions. Using a combination of dye-conjugated reagents and immunostaining, we have cataloged 13 different P. falciparum structures or organelles across the intraerythrocytic development of this parasite and made multiple observations about fundamental parasite cell biology. We describe that the outer centriolar plaque and its associated proteins anchor the nucleus to the parasite plasma membrane during mitosis. Furthermore, the rhoptries, Golgi, basal complex, and inner membrane complex, which form around this anchoring site while nuclei are still dividing, are concurrently segregated and maintain an association to the outer centriolar plaque until the start of segmentation. We also show that the mitochondrion and apicoplast undergo sequential fission events while maintaining an association with the outer centriolar plaque during cytokinesis. Collectively, this study represents the most detailed ultrastructural analysis of P. falciparum during its intraerythrocytic development to date and sheds light on multiple poorly understood aspects of its organelle biogenesis and fundamental cell biology.
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    Big Discoveries in Small Parasites: Expansion Microscopy Reveals Plasmodium's Hidden Biology
    (2025-04) Blauwkamp, James Alan; Absalon, Sabrina; Arrizabalaga, Gustavo; Bidwell, Joseph; Wek, Ronald; Yeh, Elizabeth
    Malaria, caused by the intracellular Plasmodium parasites, remains a global health crisis, affecting an estimated 263 million people and causing 597,000 deaths in 2023. The efficacy of current antimalarial strategies is diminishing due to rising resistance to all major drugs developed over the past six decades and the absence of a highly effective, affordable vaccine. My work employs Ultrastructure Expansion Microscopy (U-ExM) to investigate critical aspects of organelle biogenesis, spatial organization, and dynamics during the Plasmodium blood-stage life cycle. Through U-ExM, I have gained insights into the biogenesis, fission, and spatial organization of the mitochondrion and the apicoplast, two essential metabolic organelles. Further, analysis of invasion organelles revealed the function of PfRON11 in the formation of rhoptry organelles. Investigating the mode of action of a novel antimalarial candidate, GNF179, revealed that treated parasites display an expansion of ER folds and a mislocalization of the Golgi Apparatus, leading to rapid parasite death. U-ExM analysis showed similar ER expansion in PfSEY1 knockdown parasites, suggesting that GNF179 targets this protein. Further, I analyzed the assembly and dynamics of the essential nuclear pore complexes (NPCs). U-ExM enabled the visualization and quantification of NPCs during the blood-stage life cycle. Employing a recombination-induced tag exchange (RITE) system, I demonstrated the dynamics of NPC assembly and recycling. Finally, I characterized a previously unstudied protein, PfAnchor, establishing its essential role in apicoplast fission during the blood stage. Using U-EXM and biochemical assays, I demonstrated that PfAnchor associates with the cytoplasmic side of the apicoplast membrane. Live microscopy of PfAnchor-deficient parasites revealed daughter parasites remained clustered together, and U-ExM analysis confirmed a failure in apicoplast fission. Importantly, treatment with azithromycin disrupted the apicoplast's branching structure, resulting in a vesiculated morphology, which rescued growth defects in PfAnchor-deficient parasites. This finding underscores PfAnchor's essential role in apicoplast fission and its impact on parasite replication. Protein pulldown assays showed an interaction between PfAnchor and PfDyn2, recently implicated in apicoplast fission. Collectively, my work advances our understanding of Plasmodium cell biology and establishes U-ExM as a transformative tool for malaria research. These findings can open new avenues for therapeutic interventions against Plasmodium parasites.
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    Characterizing the Formation and Functionality of Immune Memory Cells in Response to Plasmodium Infection
    (2025-05) Fusco, Elizabeth Michelle; Schmidt, Nathan W.; Absalon, Sabrina; Bauer, Margaret; Longtin, Krista; Luo, Wei; Richer, Martin
    Malaria is an infectious disease caused by Plasmodium parasites. Over 40% of the world lives in malaria endemic regions, and children under the age of 5 in Sub-Saharan Africa face the highest burden of this disease. The clinical symptoms of malaria are caused by the cyclical infection and rupture of red blood cells by Plasmodium, and these parasites are cleared from the blood by the immune system. Plasmodium infection does not induce sterilizing immunity; however, individuals can generate clinical immunity to malaria after repeated exposures, but the factors that regulate this process are poorly understood. An emerging modulator of the immune response is the gut microbiome. We previously identified that the composition of the gut microbiome correlates with the outcome of Plasmodium infections in African children and impacts ability of the immune system to fight a Plasmodium infection in mice. We next investigated how the gut microbiome impacts the immune memory response in mice. We determined that the gut microbiome influences the formation of memory B cells and memory T cells during primary Plasmodium yoelii infection. Furthermore, the gut microbiome governs the ability of these immune memory cells to mount a secondary germinal center (GC) response to a Plasmodium berghei ANKA challenge. Curiously, the gut microbiome did not affect the accumulation of plasma cells (PCs) in the bone marrow following P. yoelii infection, and we observed that antigen-specific PC accumulation was poor. It is hypothesized that PCs are important for protection against reinfection with Plasmodium due to their ability to secrete high-affinity antibodies. We next characterized how P. yoelii infection impacted the generation and maintenance of PCs. We discovered that P. yoelii infection impairs the ability of the GC to produce long-lived PCs (LLPCs). Additionally, P. yoelii alters the composition of the bone marrow, negatively impacting the ability of PCs to engraft in the bone marrow as LLPCs. These defects in the generation and maintenance of P. yoelii-induced LLPCs likely impairs the ability of the immune system to protect against future Plasmodium infections.
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    Decreased Natural Killer Cell Function in Pediatric Severe Malaria in Areas of Higher Transmission
    (2025-05) Turyasingura, Grace; John, Chandy C.; Kaplan, Mark H.; Schmidt, Nathan W.; Absalon, Sabrina
    Natural killer (NK) cells inhibit Plasmodium falciparum parasite growth through antibody-dependent cellular cytotoxicity (ADCC) in vitro. Research conducted in malaria-endemic regions has demonstrated that memory-like NK cells are elevated in individuals exposed to malaria, exhibit enhanced ADCC activity, and correlate with reduced parasitemia and protection against uncomplicated malaria. However, the role of NK cells in pediatric severe malaria (SM) is not known. To evaluate the NK cell phenotype and function in SM, we used flow cytometry to evaluate CD56 bright, CD56 dim, and CD56 neg NK cell subsets in Ugandan children 6 months – 4 years of age with SM (cerebral malaria (CM), n=11), severe malarial anemia (SMA), n=10) and asymptomatic community children as controls (CC, n=19). Children were enrolled from sites of moderate (Jinja) and low (Kampala) malaria transmission. Analysis revealed that children with SM had a lower proportion of total NK cells and CD56 bright NK cells; however, absolute counts of NK cells per ml did not differ. In addition, LILRB1, an inhibitory receptor, was the only phenotypic marker whose proportions were significantly increased in children with SM compared to CC. Functionally, children with SM had a higher proportion of degranulating (CD107a+, IFN-γ-) memory-like NK cells. However, memory-like NK cell subsets from children with SM had a lower proportion of interferon-γ only (CD107a-, IFN-γ+)-production than CC. In addition, when comparing malaria transmission intensities with NK cell function, NK cells of children with SM in moderate transmission area exhibited a lower proportion of degranulation compared to the area of low transmission. Conversely, in low malaria transmission areas, NK cells of children with SM demonstrated a higher proportion of degranulation compared to CC. These findings elucidate distinct functional differences in NK cells among children with SM in areas of low versus moderate malaria transmission.
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    Depletion of the mini-chromosome maintenance complex binding protein allows the progression of cytokinesis despite abnormal karyokinesis during the asexual development of Plasmodium falciparum
    (Wiley, 2021) Absalon, Sabrina; Dvorin, Jeffrey D.; Pharmacology and Toxicology, School of Medicine
    The eukaryotic cell cycle is typically divided into distinct phases with cytokinesis immediately following mitosis. To ensure proper cell division, each phase is tightly coordinated via feedback controls named checkpoints. During its asexual replication cycle, the malaria parasite Plasmodium falciparum undergoes multiple asynchronous rounds of mitosis with segregation of uncondensed chromosomes followed by nuclear division with intact nuclear envelope. The multi-nucleated schizont is then subjected to a single round of cytokinesis that produces dozens of daughter cells called merozoites. To date, no cell cycle checkpoints have been identified that regulate the Plasmodium spp. mode of division. Here, we identify the Plasmodium homologue of the Mini-Chromosome Maintenance Complex Binding Protein (PfMCMBP), which co-purified with the Mini-Chromosome Maintenance (MCM) complex, a replicative helicase required for genomic DNA replication. By conditionally depleting PfMCMBP, we disrupt nuclear morphology and parasite proliferation without causing a block in DNA replication. By immunofluorescence microscopy, we show that PfMCMBP depletion promotes the formation of mitotic spindle microtubules with extensions to more than one DNA focus and abnormal centrin distribution. Strikingly, PfMCMBP-deficient parasites complete cytokinesis and form aneuploid merozoites with variable cellular and nuclear sizes. Our study demonstrates that the parasite lacks a robust checkpoint response to prevent cytokinesis following aberrant karyokinesis.
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    The Detection and Analysis of Pathogen-Reactive Immunoglobulins in the Urine of Men With Nongonococcal Urethritis
    (2023-05) Ryan, John D.; Nelson, David E.; Jordan, Stephen J.; Kaplan, Mark H.; Absalon, Sabrina
    Inflammation of the urethra—urethritis—is commonly diagnosed in men and women who have sexually transmitted infections (STI). Characteristic signs and symptoms of urethritis include urethral discharge and burning pain during urination (dysuria). However, these findings are non-specific and can be elicited by STI for which optimal treatment approaches differ. We wanted to investigate if immunoglobulins (antibodies) in the urine of men with acute urethritis could determine the etiologies of these cases. Previously, we conducted an observational case-control study of biological males to compare the urethral microbiota of participants with unambiguous, laboratory-confirmed urethritis (cases) and participants without urethral inflammation (controls). This revealed that nearly 2 in 5 men with nongonococcal urethritis tested negative for all common STI. We identified atypical urethral pathogens in approximately 1/3 of these STI-negative individuals using shotgun metagenomic sequencing. However, we did not detect microorganisms suspected to be urethral pathogens in the remaining 2/3 of STI-negative participants. We hypothesized that these men with “pathogen-negative” urethritis had persisting inflammation from a recent STI that already cleared spontaneously by the time of testing. We observed that urine IgA antibodies against Chlamydia trachomatis (Ctr) infectious particles were significantly more prevalent among men with pathogen-negative urethritis compared to controls. In contrast, we found that the prevalence of urine anti-Ctr IgA was similar between controls and urethritis cases with atypical infections. However, our efforts to detect antibodies against another common STI, Mycoplasma genitalium (Mgen), were complicated by low abundance in urine and the unexpected prevalence of Mgen-reactive antibodies among controls. Collectively, our results suggest that signs and symptoms of urethritis can continue after the causative STI(s) have been eliminated. Furthermore, male urine represents a practical, non-invasive source of pathogen-reactive antibodies that could be evaluated using point-of-care diagnostic tests to elucidate urethritis etiologies. Importantly, our results also suggest that sexual partners of men with pathogen-negative, nongonococcal urethritis are an unrecognized chlamydia reservoir.
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    Disruption of Plasmodium falciparum kinetochore proteins destabilises the nexus between the centrosome equivalent and the mitotic apparatus
    (Springer Nature, 2024-07-10) Li, Jiahong; Shami, Gerald J.; Liffner, Benjamin; Cho, Ellie; Braet, Filip; Duraisingh, Manoj T.; Absalon, Sabrina; Dixon, Matthew W. A.; Tilley, Leann; Pharmacology and Toxicology, School of Medicine
    Plasmodium falciparum is the causative agent of malaria and remains a pathogen of global importance. Asexual blood stage replication, via a process called schizogony, is an important target for the development of new antimalarials. Here we use ultrastructure-expansion microscopy to probe the organisation of the chromosome-capturing kinetochores in relation to the mitotic spindle, the centriolar plaque, the centromeres and the apical organelles during schizont development. Conditional disruption of the kinetochore components, PfNDC80 and PfNuf2, is associated with aberrant mitotic spindle organisation, disruption of the centromere marker, CENH3 and impaired karyokinesis. Surprisingly, kinetochore disruption also leads to disengagement of the centrosome equivalent from the nuclear envelope. Severing the connection between the nucleus and the apical complex leads to the formation of merozoites lacking nuclei. Here, we show that correct assembly of the kinetochore/spindle complex plays a previously unrecognised role in positioning the nascent apical complex in developing P. falciparum merozoites.