IU Indianapolis ScholarWorks :: Browsing by Author "Arrizabalaga, Gustavo"

Browsing by Author "Arrizabalaga, Gustavo"

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A positive feedback loop mediates crosstalk between calcium, cyclic nucleotide and lipid signalling in calcium-induced Toxoplasma gondii egress
(Public Library of Science, 2022-10-20) Nofal, Stephanie D.; Dominicus, Caia; Broncel, Malgorzata; Katris, Nicholas J.; Flynn, Helen R.; Arrizabalaga, Gustavo; Botté, Cyrille Y.; Invergo, Brandon M.; Treeck, Moritz; Pharmacology and Toxicology, School of Medicine
Fundamental processes that govern the lytic cycle of the intracellular parasite Toxoplasma gondii are regulated by several signalling pathways. However, how these pathways are connected remains largely unknown. Here, we compare the phospho-signalling networks during Toxoplasma egress from its host cell by artificially raising cGMP or calcium levels. We show that both egress inducers trigger indistinguishable signalling responses and provide evidence for a positive feedback loop linking calcium and cyclic nucleotide signalling. Using WT and conditional knockout parasites of the non-essential calcium-dependent protein kinase 3 (CDPK3), which display a delay in calcium inonophore-mediated egress, we explore changes in phosphorylation and lipid signalling in sub-minute timecourses after inducing Ca2+ release. These studies indicate that cAMP and lipid metabolism are central to the feedback loop, which is partly dependent on CDPK3 and allows the parasite to respond faster to inducers of egress. Biochemical analysis of 4 phosphodiesterases (PDEs) identified in our phosphoproteomes establishes PDE2 as a cAMP-specific PDE which regulates Ca2+ induced egress in a CDPK3-independent manner. The other PDEs display dual hydrolytic activity and play no role in Ca2+ induced egress. In summary, we uncover a positive feedback loop that enhances signalling during egress, thereby linking several signalling pathways.
AP2IX-4, a cell cycle regulated nuclear factor, modulates gene expression during bradyzoite development in toxoplasma gondii
(2017-01-10) Huang, Sherri Y.; Arrizabalaga, Gustavo; Sullivan, William J., Jr.; Lu, Tao; Takagi, Yuichiro; Zhang, Jian-Ting
Toxoplasma gondii is a ubiquitous, protozoan parasite contributing significantly to global human and animal health. In the host, this obligate intracellular parasite converts into a latent tissue cyst form known as the bradyzoite, which is impervious to the immune response. The tissue cysts facilitate wide-spread transmission through the food chain and give rise to chronic toxoplasmosis in immune compromised patients. In addition, they may reactivate into replicating tachyzoites which cause tissue damage and disseminated disease. Current available drugs do not appear to have appreciable activity against latent bradyzoites. Therefore, a better understanding of the molecular mechanisms that drive interconversion between tachyzoite and bradyzoite forms is required to manage transmission and pathogenesis of Toxoplasma. Conversion to the bradyzoite is accompanied by an altered transcriptome, but the molecular players directing this process are largely uncharacterized. Studies of stage-specific promoters revealed that conventional cis-acting mechanisms operate to regulate developmental gene expression during tissue cyst formation. The major class of transcription factor likely to work through these cis-regulatory elements appears to be related to the Apetala-2 (AP2) family in plants. The Toxoplasma genome contains nearly 70 proteins harboring at least one predicted AP2 domain, but to date only three of these T. gondii AP2 proteins have been linked to bradyzoite development. We show that the putative T. gondii transcription factor, AP2IX-4, is localized to the parasite nucleus and exclusively expressed in tachyzoites and bradyzoites undergoing division. Knockout of AP2IX-4 had negligible effect on tachyzoite replication, but resulted in a reduced frequency of bradyzoite cysts in response to alkaline stress induction – a defect that is reversible by complementation. Microarray analyses revealed an enhanced activation of bradyzoite-associated genes in the AP2IX-4 knockout during alkaline conditions. In mice, the loss of AP2IX-4 resulted in a modest virulence defect and reduced brain cyst burden. Complementation of the AP2IX-4 knockout restored cyst counts to wild-type levels. These findings illustrate the complex role of AP2IX-4 in bradyzoite development and that certain transcriptional mechanisms responsible for tissue cyst development operate across parasite division.
Assessing Knowledge and Perceptions Related to Preventive Methods and Treatment of Malaria in the Local Endemic Area of Trujillo, Honduras
(Springer, 2015) Campodonico, Joanna; Sevilla-Martir, Javier; Arrizabalaga, Gustavo; Kochhar, Komal; Department of Family Medicine, IU School of Medicine
Malaria in Honduras is endemic and accounts for 40% of the total cases in Central America. Our goal was to assess knowledge of preventive methods and current treatment of malaria among the affected community of Trujillo, Honduras. A cross-sectional survey was administered to 71 individuals. Most respondents had a good understanding about common malaria symptoms but not about the complications associated with severe cases. More important, we found that less than 20% of the respondents recognized indoor residual sprays and insecticide-treated nets as effective preventive measures, which are the most efficient preventive methods. Our study highlights the perceptions the people of Trujillo have about malaria. From our observations, we put forward recommendations to implement a comprehensive campaign to educate the Trujillo population about malaria preventive methods and to recruit local and international efforts to distribute insecticide-treated nets.
Biochemical and pharmacological characterization of the Atg8 conjugation system in toxoplasma gondii
(2017-06-28) Varberg, Joseph M.; Arrizabalaga, Gustavo; Sullivan, William J., Jr.; Mosley, Amber; Safa, Ahmad; Vasko, Michael R.
Toxoplasma gondii is an important human pathogen that infects millions of people worldwide and causing severe and potentially lethal disease in immunocompromised individuals. Recently, a homologue for the autophagy protein Atg8 (TgAtg8) was identified in Toxoplasma that is required for both canonical and noncanonical processes essential for parasite viability. Importantly, TgAtg8 functionality requires its conjugation to phosphatidylethanolamine through the activity of the Atg8 conjugation system. In this thesis, we characterized the proteins that interact with TgAtg8 and TgAtg3, a component of the Atg8 conjugation system, to further define their functions in Toxoplasma and identify opportunities for targeted inhibition of Atg8-related processes. We previously identified that TgAtg8 is acetylated at lysine 23 (K23) and assessed the role of this modification in this thesis. Using mutagenesis, we showed that K23 acetylation did not modulate the interaction with TgAtg3, but appeared to promote TgAtg8 protein stability. Additionally, endogenous mutation of K23 to the nonacetylatable amino acid arginine resulted in severe impairment of parasite replication and spontaneous differentiation into bradyzoites. To gain insight into the role of TgAtg8 in Toxoplasma biology, we next characterized TgAtg8 and TgAtg3 interacting proteins using affinity purification and mass spectrometry. We identified a novel group of interacting proteins that are unique to Toxoplasma, including the dynamin-related protein DrpC. Functional characterization of DrpC identified a potential role of TgAtg8 in trafficking of membrane from the Golgi to the nascent daughter parasites during replication. Lastly, we examined a group of small molecules recently identified as Atg3-Atg8 inhibitors in Plasmodium falciparum and assessed their activity against Toxoplasma. Although the compounds effectively inhibited Toxoplasma replication, they did so through novel mechanisms of action unrelated to the disruption of the TgAtg3-Atg8 interaction. Together, this work provides insight into the function of the Atg8 conjugation system in Toxoplasma that will help guide the future development of novel therapeutics targeting Atg8-related processes.
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, Frank
Toxoplasma’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.
Characterization of a Putative Acid Phosphatase in Toxoplasma Gondii and Its Role in Parasite Propagation
(2020-11) Blakely, William James; Arrizabalaga, Gustavo; Gilk, Stacey; Meroueh, Samy; Wek, Ronald
The parasite Toxoplasma gondii infects approximately one-third of people worldwide. Infection can lead to severe disease in those with a compromised immune system and primary infection during pregnancy can lead to severe birth defects or miscarriage. Treatment options are limited, have significant side effects, and are ineffective for all infection stages. Imperative to the discovery of novel therapeutic targets is a thorough understanding of how Toxoplasma propagates within a host. To replicate, the parasite must enter the cells of an infected organism where, during the invasion process, it surrounds itself with host cell membrane to form a parasitophorous vacuole (PV), within which it freely divides. To endure the intracellular environment of a host cell, Toxoplasma secretes a large repertoire of proteins beyond the PV to manipulate important host cellular functions. How these Toxoplasma proteins transit from parasites to host cell is not well understood. Protein translocation into the host cell is mediated by three proteins hypothesized to function as a putative translocon complex inside the PV, but whether other proteins are involved in the structure or regulation of this putative translocon remains unknown. The secreted protein GRA44, which contains a putative acid phosphatase domain, has been discovered to interact with members of this translocon and is required for downstream alteration of host cells. GRA44 was found to be post-translationally cleaved in a region homologous to sequences targeted by protozoan proteases of the secretory pathway with both major cleavage products secreted to the PV. Conditional knockdown of GRA44 resulted in loss of host cell cMyc upregulation, a phenotype also seen in translocon member disruption. Therefore, the putative acid phosphatase GRA44, in association with the translocon complex, is critical for host cell manipulation during infection, a process Toxoplasma relies upon for successful propagation as an intracellular pathogen.
Characterization of Plasmodium Atg3-Atg8 Interaction Inhibitors Identifies Novel Alternative Mechanisms of Action in Toxoplasma gondii
(American Society for Microbiology, 2018-01-25) Varberg, Joseph M.; LaFavers, Kaice A.; Arrizabalaga, Gustavo; Sullivan, William J., Jr.; Pharmacology and Toxicology, School of Medicine
Protozoan parasites, including the apicomplexan pathogens Plasmodium falciparum (which causes malaria) and Toxoplasma gondii (which causes toxoplasmosis), infect millions of people worldwide and represent major human disease burdens. Despite their prevalence, therapeutic strategies to treat infections caused by these parasites remain limited and are threatened by the emergence of drug resistance, highlighting the need for the identification of novel drug targets. Recently, homologues of the core autophagy proteins, including Atg8 and Atg3, were identified in many protozoan parasites. Importantly, components of the Atg8 conjugation system that facilitate the lipidation of Atg8 are required for both canonical and parasite-specific functions and are essential for parasite viability. Structural characterization of the P. falciparum Atg3-Atg8 (PfAtg3-Atg8) interaction has led to the identification of compounds that block this interaction. Additionally, many of these compounds inhibit P. falciparum growth in vitro, demonstrating the viability of this pathway as a drug target. Given the essential role of the Atg8 lipidation pathway in Toxoplasma, we sought to determine whether three PfAtg3-Atg8 interaction inhibitors identified in the Medicines for Malaria Venture Malaria Box exerted a similar inhibitory effect in Toxoplasma While all three inhibitors blocked Toxoplasma replication in vitro at submicromolar concentrations, they did not inhibit T. gondii Atg8 (TgAtg8) lipidation. Rather, high concentrations of two of these compounds induced TgAtg8 lipidation and fragmentation of the parasite mitochondrion, similar to the effects seen following starvation and monensin-induced autophagy. Additionally, we report that one of the PfAtg3-Atg8 interaction inhibitors induces Toxoplasma egress and provide evidence that this is mediated by an increase in intracellular calcium in response to drug treatment.
The common parasite Toxoplasma gondii induces prostatic inflammation and microglandular hyperplasia in a mouse model
(Wiley, 2017-07) Colinot, Darrelle L.; Garbuz, Tamila; Bosland, Maarten C.; Wang, Liang; Rice, Susan E.; Sullivan, William J., Jr.; Arrizabalaga, Gustavo; Jerde, Travis J.; Pharmacology and Toxicology, School of Medicine
BACKGROUND: Inflammation is the most prevalent and widespread histological finding in the human prostate, and associates with the development and progression of benign prostatic hyperplasia and prostate cancer. Several factors have been hypothesized to cause inflammation, yet the role each may play in the etiology of prostatic inflammation remains unclear. This study examined the possibility that the common protozoan parasite Toxoplasma gondii induces prostatic inflammation and reactive hyperplasia in a mouse model. METHODS: Male mice were infected systemically with T. gondii parasites and prostatic inflammation was scored based on severity and focality of infiltrating leukocytes and epithelial hyperplasia. We characterized inflammatory cells with flow cytometry and the resulting epithelial proliferation with bromodeoxyuridine (BrdU) incorporation. RESULTS: We found that T. gondii infects the mouse prostate within the first 14 days of infection and can establish parasite cysts that persist for at least 60 days. T. gondii infection induces a substantial and chronic inflammatory reaction in the mouse prostate characterized by monocytic and lymphocytic inflammatory infiltrate. T. gondii-induced inflammation results in reactive hyperplasia, involving basal and luminal epithelial proliferation, and the exhibition of proliferative inflammatory microglandular hyperplasia in inflamed mouse prostates. CONCLUSIONS: This study identifies the common parasite T. gondii as a new trigger of prostatic inflammation, which we used to develop a novel mouse model of prostatic inflammation. This is the first report that T. gondii chronically encysts and induces chronic inflammation within the prostate of any species. Furthermore, T. gondii-induced prostatic inflammation persists and progresses without genetic manipulation in mice, offering a powerful new mouse model for the study of chronic prostatic inflammation and microglandular hyperplasia.
The Dually Localized EF-Hand Domain-Containing Protein TgEFP1 Regulates the Lytic Cycle of Toxoplasma gondii
(MDPI, 2022-05-21) Dave, Noopur; LaFavers, Kaice; Arrizabalaga, Gustavo; Pharmacology and Toxicology, School of Medicine
The propagation of the obligate intracellular parasite Toxoplasma gondii is tightly regulated by calcium signaling. However, the mechanisms by which calcium homeostasis and fluxes are regulated in this human pathogen are not fully understood. To identify Toxoplasma’s calcium homeostasis network, we have characterized a novel EF-hand domain-containing protein, which we have named TgEFP1. We have determined that TgEFP1 localizes to a previously described compartment known as the plant-like vacuole or the endosomal-like compartment (PLV/ELC), which harbors several proteins related to ionic regulation. Interestingly, partial permeabilization techniques showed that TgEFP1 is also secreted into the parasitophorous vacuole (PV), within which the parasite divides. Ultrastructure expansion microscopy confirmed the unusual dual localization of TgEFP1 at the PLV/ELC and the PV. Furthermore, we determined that the localization of TgEFP1 to the PV, but not to the PLV/ELC, is affected by disruption of Golgi-dependent transport with Brefeldin A. Knockout of TgEFP1 results in faster propagation in tissue culture, hypersensitivity to calcium ionophore-induced egress, and premature natural egress. Thus, our work has revealed an interplay between the PV and the PLV/ELC and a role for TgEFP1 in the regulation of calcium-dependent events.
Elucidating the Role of the Essential Kinase TgGSK in the Human Parasite Toxoplasma Gondii
(2025-02) Krueger, Amanda; Yeh, Elizabeth; Arrizabalaga, Gustavo; Sullivan, William; Nass, Richard; Aoki, Scott
Toxoplasma gondii is an intracellular parasite that infects nearly a third of the world’s human population. While infection is largely asymptomatic in an immunocompetent host, Toxoplasma infection in immunocompromised or immunosuppressed individuals can lead to toxoplasmosis, which can include brain lesions and lead to death. Similarly, toxoplasmosis can result in birth defects, brain swelling, and blindness of a developing fetus in the case of a congenital infection. With minimal treatments for toxoplasmosis available, it is crucial to study parasite-specific processes that could be potential drug targets for the treatment of toxoplasmosis. Toxoplasma gondii divides through a unique process known as endodyogeny, where two daughter parasites are formed within a mother. In this study, we investigated a novel protein called TgGSK that is crucial for proper parasite division. Experiments reveal that TgGSK changes its localization within the parasite dependent on the stage of division. Knockdown of TgGSK causes abnormal division phenotypes and causes Toxoplasma to be unable to complete its propagation cycle. We determined through microscopy and phosphoproteomics that TgGSK may play its role in parasite division through an interaction with the centrosome, an organelle which is a main feature of cell division in many organisms. Our findings suggest that TgGSK also regulates messenger RNA processing. Finally, our study suggests that TgGSK is regulated and stabilized through acetylation from the GCN5b lysine acetyltransferase complex. Taken together, we have performed an in-depth study of the functional role of the essential protein TgGSK in Toxoplasma gondii. This and future studies have potential to demonstrate that TgGSK is a parasite-specific drug target for the therapeutic treatment of toxoplasmosis.

Browsing by Author "Arrizabalaga, Gustavo"

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