IU Indianapolis ScholarWorks :: Browsing by Author "Absalon, Sabrina"

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.
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.
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.
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.
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.
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.
Expansion Microscopy Reveals Plasmodium falciparum Blood-Stage Parasites Undergo Anaphase with A Chromatin Bridge in the Absence of Mini-Chromosome Maintenance Complex Binding Protein
(MDPI, 2021-11) Liffner, Benjamin; Absalon, Sabrina; Pharmacology and Toxicology, School of Medicine
The malaria parasite Plasmodium falciparum undergoes closed mitosis, which occurs within an intact nuclear envelope, and differs significantly from its human host. Mitosis is underpinned by the dynamics of microtubules and the nuclear envelope. To date, our ability to study P. falciparum mitosis by microscopy has been hindered by the small size of the P. falciparum nuclei. Ultrastructure expansion microscopy (U-ExM) has recently been developed for P. falciparum, allowing the visualization of mitosis at the individual nucleus level. Using U-ExM, three intranuclear microtubule structures are observed: hemispindles, mitotic spindles, and interpolar spindles. A previous study demonstrated that the mini-chromosome maintenance complex binding-protein (MCMBP) depletion caused abnormal nuclear morphology and microtubule defects. To investigate the role of microtubules following MCMBP depletion and study the nuclear envelope in these parasites, we developed the first nuclear stain enabled by U-ExM in P. falciparum. MCMBP-deficient parasites show aberrant hemispindles and mitotic spindles. Moreover, anaphase chromatin bridges and individual nuclei containing multiple microtubule structures were observed following MCMBP knockdown. Collectively, this study refines our understanding of MCMBP-deficient parasites and highlights the utility of U-ExM coupled with a nuclear envelope stain for studying mitosis in P. falciparum.
Function of a Unique Dually Localized EF-Hand Domain Containing Protein, TgEFP1, During the Lytic Cycle of the Human Parasite Toxoplasma Gondii
(2022-08) Dave, Noopur Kirti; Arrizabalaga, Gustavo; Absalon, Sabrina; Fehrenbacher, Jill; Gilk, Stacey; Jerde, Travis; Mastracci, Teresa
The pathogenesis associated with toxoplasmosis is attributed to repeated rounds of the parasite lytic cycle, which has been shown to be regulated by calcium fluxes. However, little is known about the calcium homeostatic mechanisms utilized by T. gondii. Recently, our lab has identified a novel protein-TgEFP1 (TGGT1_255660), which is predicted to bind Ca2+ through its two EF-hand domains. Interestingly, TgEFP1 showed a unique dual localization at the PLV/ELC and the PV of the parasite. Previous work showed that the PLV/ELC harbors other ion binding and conducting proteins that are important for parasite survival and propagation. However, the function of this compartment in the parasite is unknown. Therefore, I hypothesize that the PLV/ELC, through the function of TgEFP1, plays a key role in calcium homeostasis of T. gondii. To test this hypothesis, we sought to characterize the function of TgEFP1 during the parasite lytic cycle and determine TgEFP1 interacting proteins that also localize to the PLV/ELC. Partial permeabilization and ultrastructure expansion microscopy techniques confirmed the dual localization of TgEFP1 at the PLV/ELC and the PV. TgEFP1 knockout parasites exhibited several phenotypic defects including a faster lytic rate, shorter intracellular cycle, and were more sensitive to calcium ionophore treatment. Signal peptide deletion led to a mislocalization of TgEFP1 as cytosolic puncta, while mutations at key calcium coordinating residues lead to exclusive localization of TgEFP1 at the PV. Lastly, immunoprecipitation assays followed by LC-MS/MS identified a novel lectin-like protein- TgLectin (TGGT1_258950) as a direct interactor of TgEFP1-HA. Collectively, these findings support that through the function of TgEFP1, the PLV/ELC, plays a key role in calcium-dependent processes during the lytic cycle of the parasite.
Global Release of Translational Repression Across Plasmodium’s Host-to-Vector Transmission Event
(bioRxiv, 2024-03-16) Rios, Kelly T.; McGee, James P.; Sebastian, Aswathy; Moritz, Robert L.; Feric, Marina; Absalon, Sabrina; Swearingen, Kristian E.; Lindner, Scott E.; Pharmacology and Toxicology, School of Medicine
Malaria parasites must be able to respond quickly to changes in their environment, including during their transmission between mammalian hosts and mosquito vectors. Therefore, before transmission, female gametocytes proactively produce and translationally repress mRNAs that encode essential proteins that the zygote requires to establish a new infection. This essential regulatory control requires the orthologues of DDX6 (DOZI), LSM14a (CITH), and ALBA proteins to form a translationally repressive complex in female gametocytes that associates with many of the affected mRNAs. However, while the release of translational repression of individual mRNAs has been documented, the details of the global release of translational repression have not. Moreover, the changes in spatial arrangement and composition of the DOZI/CITH/ALBA complex that contribute to translational control are also not known. Therefore, we have conducted the first quantitative, comparative transcriptomics and DIA-MS proteomics of Plasmodium parasites across the host-to-vector transmission event to document the global release of translational repression. Using female gametocytes and zygotes of P. yoelii, we found that nearly 200 transcripts are released for translation soon after fertilization, including those with essential functions for the zygote. However, we also observed that some transcripts remain repressed beyond this point. In addition, we have used TurboID-based proximity proteomics to interrogate the spatial and compositional changes in the DOZI/CITH/ALBA complex across this transmission event. Consistent with recent models of translational control, proteins that associate with either the 5' or 3' end of mRNAs are in close proximity to one another during translational repression in female gametocytes and then dissociate upon release of repression in zygotes. This observation is cross-validated for several protein colocalizations in female gametocytes via ultrastructure expansion microscopy and structured illumination microscopy. Moreover, DOZI exchanges its interaction from NOT1-G in female gametocytes to the canonical NOT1 in zygotes, providing a model for a trigger for the release of mRNAs from DOZI. Finally, unenriched phosphoproteomics revealed the modification of key translational control proteins in the zygote. Together, these data provide a model for the essential translational control mechanisms used by malaria parasites to promote their efficient transmission from their mammalian host to their mosquito vector.
Investigation of the Knockout of LMF1 on the Transcriptome of Toxoplasma gondii
(2024-01) Thibodeau, Katherine E.; Arrizabalaga, Gustavo; Absalon, Sabrina; Fehrenbacher, Jill; Flak, Jonathan; Schmidt, Nathan
Toxoplasma gondii is an obligate intracellular apicomplexan parasite that infects one third of the global population. There are limited treatments for Toxoplasmosis, however a potential drug target for Toxoplasma is its mitochondrion. While much is known about the function of this organelle in Toxoplasma, little is known about the mechanisms that regulate mitochondrial structure and division. The shape of the mitochondrion changes throughout the life cycle of the parasite. When inside a host cell, the mitochondrion is in a lasso shape, stretching around the periphery of the parasite, while in extracellular parasites it is collapsed towards the apical end of the parasite. While in a lasso shape the mitochondrion shows areas of contact with the parasite pellicle. We have determined that the proteins LMF1 (associated with the outer mitochondrial membrane) and IMC10 (inner membrane complex) interact and form a reversible tether that maintains the lasso shape of the mitochondrion. When either of these proteins are knocked out, the mitochondrion collapses. To elucidate the biological relevance of the interaction between the mitochondrion and the pellicle we explored the consequence of disrupting the interaction on the transcriptome of the parasite. RNA sequencing of the LMF1 knockout strain showed a disruption in the expression of genes involved in nucleotide metabolism and Coenzyme A biosynthesis, which might be an adaptation mechanism to the disruption of mitochondrial morphology. Current work focuses on investigating the connection between mitochondrial tethering and these pathways as well as a potential role for the mitochondrion/pellicle connection in metabolite transport.

Browsing by Author "Absalon, Sabrina"

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