Biology subjects

Arrizabalaga, G.

Publications and source records attributed to Arrizabalaga, G..

11 recordsLinked to original sources

bioRxiv2026

CD8+ T CELLS ASSOCIATE WITH FORMING GLANDULAR NODULES IN TOXOPLASMA GONDII-INDUCED PROSTATIC HYPERPLASIA AND HUMAN BPH

Background: Chronic inflammation is the most common histological feature in Benign Prostatic Hyperplasia (BPH), and T cells are a key component of immune infiltrate. Advanced BPH is commonly associated with the formation of nodules, but it remains unclear whether a link exists among T cell infiltration, nodular development, and BPH progression. Using a Toxoplasma gondii (T. gondii) model and human specimens, we characterize the subtypes of T cells present during prostatic hyperplasia and their association with nodular development of the prostate. Methods: Male CBA/j mice were intraperitoneally infected with T. gondii parasites, and flow cytometry was performed on the prostate to quantify the number of CD4+ and CD8+ T cells. Histology was used to score microglandular hyperplasia (MGH), and immunofluorescence was used to quantify and examine the locality of CD4+ and CD8+ T cells and compared that to human BPH tissue. Results: We found that infecting male mice with T. gondii resulted in an increase of both CD4+ and CD8+ T cells in the prostate acutely and that CD8+ cells remained sustained at chronically. We also established the presence of glandular nodule formation at this timepoint through hematoxylin and eosin (H&E) staining. Immunofluorescence revealed that CD8+ cells were found proximal to forming glandular nodules relative to non-nodular glands. We also found more CD8+ cells localized to non-nodular glands in nodular BPH tissue versus non-nodular BPH tissue. Finally, we discovered a higher prevalence of CD8+ cells in T. gondii IgG+ patients than in IgG- patients. All T. gondii IgG+ patients exhibited nodular BPH, whereas all but one IgG- patient exhibited non-nodular BPH. Conclusions: This study is the first to investigate the presence and location of CD4+ and CD8+ T cells within nodular and non-nodular BPH glands. We found an association of the presence of CD8+ T cells with nodular progression. This association held true in human prostate tissue. Translationally, CD8+ T cells may enhance nodular BPH progression, and T. gondii infection may promote this CD8+ T cell-mediated response.

pathology↗

bioRxiv2026

Mitochondrion-IMC contact sites are critical for cofactor biosynthesis and egress signaling in Toxoplasma gondii

Toxoplasma gondii is a single-celled parasite belonging to the Apicomplexa phylum. Toxoplasmas single mitochondrion is highly dynamic, changing its morphology as the parasite undergoes egress and invasion. Recently, we have demonstrated that mitochondrial morphology is driven by a protein named Lasso Maintenance Factor 1 (LMF1). This protein interacts with IMC10, a protein present at the parasites inner membrane complex (IMC), mediating a unique membrane contact site between the IMC and mitochondrion. Interestingly, parasites lacking either LMF1 or IMC10 have abnormal mitochondrial morphology, cell division defects, and delayed propagation in tissue culture. Although both components of the tether were identified, the functions of this contact site remain unknown. In this work, we show that {Delta}lmf1 parasites exhibit upregulation of egress signaling and downregulation in folate metabolism and pantothenate biosynthesis. {Delta}lmf1 parasites exhibit increased intracellular calcium levels, leading to greater sensitivity to ionophore-induced egress and microneme secretion. We have confirmed that parasites have decreased levels of tetrahydrofolate and coenzyme A, showing a limitation in cofactor production. Interestingly, the {Delta}lmf1 parasites prefer glutamine instead of glucose as a catabolic substrate. Accordingly, we demonstrate for the first time that proper mitochondrial positioning is crucial for folate and Coenzyme A metabolism as well as egress signaling. IMPORTANCEToxoplasma gondii is the causative agent of Toxoplasmosis, a disease that affects a third of the worlds population. This parasite has a single, highly dynamic mitochondrion. The parasites mitochondrion changes shape depending on environmental conditions (inside or outside the host cell) or on stressors, such as drugs. Our laboratory characterized the proteins involved in regulating mitochondrial dynamics in the parasite, but the functional importance of these mitochondrial changes has not yet been described. Here, we show that the shape of Toxoplasmas mitochondrion is important for the synthesis of key cofactors, such as folates and coenzyme A. We show that mitochondrial shape in this parasite is important for signaling the parasites exit from the host cell, a critical process in its life cycle. These findings review a previously unknown function of a parasite-specific organelle contact site, providing new insights into the importance of mitochondria for these parasites.

microbiology↗

bioRxiv2024

The essential kinase TgGSK regulates centrosome division and endodyogeny in Toxoplasma gondii

Intracellular replication is crucial for the success of apicomplexan parasites, including Toxoplasma gondii. Therefore, essential players in parasite replication present potential targets for drug development. In this study, we have characterized TgGSK, a glycogen synthase kinase homolog that plays an important role in Toxoplasma endodyogeny. We have shown that TgGSK has a dynamic localization that is concurrent with the cell cycle. In non-dividing parasites, this kinase is highly concentrated in the nucleus. However, during division, TgGSK displays a cytosolic localization, with concentration foci at the centrosomes, a key organelle involved in parasite division, and the basal end. Conditional knockdown of TgGSK determined that it is essential for the completion of the lytic cycle and proper parasite division. Parasites lacking endogenous protein levels of TgGSK exhibited defects in division synchronicity and the segregation of the nucleus and apicoplast into forming daughter cells. These phenotypes are associated with defects in centrosome duplication and fission. Global phosphoproteomic analysis determined TgGSK-dependent phosphorylation of RNA-processing, basal end, and centrosome proteins. Consistent with the putative regulation of RNA-processing proteins, global transcriptomic analysis suggests that TgGSK is needed for proper splicing. Finally, we show that TgGSK interacts with GCN5b, a well-characterized acetyltransferase with roles in transcriptional control. Conversely, GCN5b chemical inhibition results in specific degradation of TgGSK. Thus, these studies reveal the involvement of TgGSK in various crucial processes, including endodyogeny and splicing, and identify acetylation as a possible mechanism by which this essential kinase is regulated. AUTHOR SUMMARYToxoplasma gondii infects nearly a third of the worlds human population. While infection is largely asymptomatic in healthy adults, in immunocompromised or immunosuppressed individuals it can lead to brain lesions and even death. Similarly, toxoplasmosis can result in stillbirth, birth defects, 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. In this study, we investigated the protein TgGSK that is essential for parasite survival and proper division. We showed that TgGSK may perform its essential functions through interaction with the centrosome, an organelle that plays a major role in cell division in many organisms. We also show in this study a role for TgGSK in proper processing of messenger RNAs. Taken together, we have performed an in-depth study of the functional role of the essential protein TgGSK in Toxoplasma gondii. Importantly, TgGSK was shown to have more similarity to plant proteins than mammalian proteins which may allow for the possibility of targeting of this protein for therapeutic treatment of toxoplasmosis.

microbiology↗

bioRxiv2024

Myosin A and F-Actin play a critical role in mitochondrial dynamics and inheritance in Toxoplasma gondii

The single mitochondrion of the obligate intracellular parasite Toxoplasma gondii is highly dynamic. Toxoplasmas mitochondrion changes morphology as the parasite moves from the intracellular to the extracellular environment and during division. Toxoplasmas mitochondrial dynamic is dependent on an outer mitochondrion membrane-associated protein LMF1 and its interaction with IMC10, a protein localized at the inner membrane complex (IMC). In the absence of either LMF1 or IMC10, parasites have defective mitochondrial morphology and inheritance defects. As little is known about mitochondrial inheritance in Toxoplasma, we have used the LMF1/IMC10 tethering complex as an entry point to dissect the machinery behind this process. Using a yeast two-hybrid screen, we previously identified Myosin A (MyoA) as a putative interactor of LMF1. Although MyoA is known to be located at the parasites pellicle, we now show through ultrastructure expansion microscopy (U-ExM) that this protein accumulates around the mitochondrion in the late stages of parasite division. Parasites lacking MyoA show defective mitochondrial morphology and a delay in mitochondrion delivery to the daughter parasite buds during division, indicating that this protein is involved in organellar inheritance. Disruption of the parasites actin network also affects mitochondrion morphology. We also show that parasite-extracted mitochondrion vesicles interact with actin filaments. Interestingly, mitochondrion vesicles extracted out of parasites lacking LMF1 pulled down less actin, showing that LMF1 might be important for mitochondrion and actin interaction. Accordingly, we are showing for the first time that actin and Myosin A are important for Toxoplasma mitochondrial morphology and inheritance.

microbiology↗

bioRxiv2023

The protein phosphatase PPKL is a key regulator of daughter parasite development in Toxoplasma gondii

Apicomplexan parasites, including Toxoplasma gondii, encode many plant-like proteins, which play significant roles and present attractive targets for drug development. In this study, we have characterized the plant-like protein phosphatase PPKL, which is unique to the parasite and absent in its mammalian host. We have shown that its localization changes as the parasite divides. In non-dividing parasites, it is present in the cytoplasm, nucleus, and preconoidal region. As the parasite begins division, PPKL is enriched in the preconoidal region and the cortical cytoskeleton of the nascent parasites. Later in the division, PPKL is present in the basal complex ring. Conditional knockdown of PPKL showed that it is essential for parasite propagation. Moreover, parasites lacking PPKL exhibit uncoupling of division, with normal DNA duplication but severe defects in forming daughter parasites. While PPKL depletion does not impair the duplication of centrosomes, it affects the rigidity and arrangement of the cortical microtubules. Both Co-Immunoprecipitation and proximity labeling identified the kinase DYRK1 as a potential functional partner of PPKL. Complete knockout of DYRK1 phenocopies lack of PPKL, strongly suggesting a functional relationship between these two signaling proteins. Global phosphoproteomics analysis revealed a significant increase in phosphorylation of the microtubule-associated proteins SPM1 in PPKL-depleted parasites, suggesting PPKL regulates the cortical microtubules by mediating the phosphorylation state of SPM1. More importantly, the phosphorylation of cell cycle-associated kinase Crk1, a known regulator of daughter cell assembly, is altered in PPKL-depleted parasites. Thus, we propose that PPKL regulates daughter parasite development by influencing the Crk1-dependent signaling pathway. ImportanceToxoplasma gondii can cause severe disease in immunocompromised or immunosuppressed patients and during congenital infections. Treating toxoplasmosis presents enormous challenges since the parasite shares many biological processes with its mammalian hosts, which results in significant side effects with current therapies. Consequently, proteins that are essential and unique to the parasite represent favorable targets for drug development. Interestingly, Toxoplasma, like other members of the phylum Apicomplexa, has numerous plant-like proteins, many of which play crucial roles and do not have equivalents in the mammalian host. In this study, we found that the plant-like protein phosphatase, PPKL, appears to be a key regulator of daughter parasite development. With the depletion of PPKL, the parasite shows severe defects in forming daughter parasites. This study provides novel insights into the understanding of parasite division and offers a new potential target for the development of antiparasitic drugs.

cell biology↗

bioRxiv2022

The dually localized EF-hand domain-containing protein TgEFP1 regulates the lytic cycle of Toxoplasma gondii

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 Toxoplasmas 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 endo-lysosomal 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.

microbiology↗

bioRxiv2022

IMC10 and LMF1 mediate membrane contact between the mitochondrion and the inner membrane complex in Toxoplasma gondii

The single mitochondrion of Toxoplasma gondii is highly dynamic, being predominantly in a peripherally distributed lasso-shape in intracellular parasites and collapsed in extracellular ones. The peripheral positioning of the mitochondrion is associated with apparent contacts between the mitochondrion membrane and the parasite pellicle. The outer mitochondrial membrane-associated protein LMF1 is critical for the correct positioning of the mitochondrion, and in its absence, intracellular parasites fail to form the lasso-shaped mitochondrion. To identify other proteins that participate in tethering the parasites mitochondrion to the pellicle, we performed a yeast two-hybrid screen for LMF1 interactors. We identified 70 putative interactors, six of which are known to localize to the apical end of the parasite, two to the mitochondrial membrane, and three localize to the inner membrane complex (IMC), a component of the parasite pellicle. Using reciprocal immunoprecipitation and proximity ligation assays, we confirmed the interaction of LMF1 with the pellicle protein IMC10, with a hypothetical protein known to be part of the conoid, and with an ATPase-Guanylyl Cyclase. Conditional knockdown of IMC10 does not affect parasite viability but severely affects mitochondrial morphology in intracellular parasites and mitochondrial distribution to the daughter cells during division. In effect, IMC10 knockdown phenocopies disruption of LMF1, suggesting that these two proteins define a novel membrane tether between Toxoplasmas mitochondrion and the inner membrane complex. IMPORTANCE Toxoplasma gondii is an opportunistic parasite that can cause life-threatening disease in immunocompromised patients and those infected congenitally. As current therapies against this parasite can be poorly tolerated and are not effective against the latent stage of the parasite, there is an urgent need to identify new drug targets. The single mitochondrion of this parasite is a validated drug target, but little is known about the machinery that controls its division and structure, information that would be critical for a thorough exploration of the mitochondrion as a drug target. We have identified parasite-specific proteins that are essential to maintain the normal structure of the mitochondrion. We have discovered a complex of two proteins that tether the mitochondrion to the periphery of the parasite. Loss of this connection results in changes in mitochondrial morphology and cell division defects. Our results provide important insight into the molecular mechanisms regulating Toxoplasma mitochondrial morphology.

microbiology↗

bioRxiv2022

The tyrosine phosphatase PRL regulates attachment of Toxoplasma gondii to host cells and is essential for virulence

The pathogenesis of Toxoplasma gondii is mainly due to tissue damage caused by the repeating lytic cycles of the parasite. Many proteins localized to the pellicle of the parasite, particularly kinases, have been identified as critical regulators of the Toxoplasma lytic cycle. However, little is known about the associated protein phosphatases. Phosphatase of regenerating liver (PRL), a highly conserved tyrosine phosphatase, is an oncogene that plays pivotal roles in mammalian cells and typically associates with membranes via a conserved prenylation site. PRL in Toxoplasma has a predicted prenylation motif in the C-terminus like other homologs. We have determined that TgPRL localizes to the plasma membrane and that disruption of TgPRL results in a defect in the parasites ability to attach to host cells. This function is dependent both on TgPRLs membrane localization and phosphatase activity. Importantly, in vivo experiments have shown that while mice infected with parental strain parasites die within days of infection, those infected with parasites lacking TgPRL not only survive but also develop immunity that confers protection against subsequent infection with wild-type parasites. Immunoprecipitation experiments revealed that the PRL-CNNM (Cyclin M) complex, which regulates intracellular Mg2+ homeostasis in mammalian cells, is also present in Toxoplasma. Consistent with this interaction, parasites lacking TgPRL had higher intracellular Mg2+ levels than the parental or complemented strains, suggesting TgPRL is involved in regulating intracellular Mg2+ homeostasis. Thus, TgPRL is a vital regulator of the Toxoplasma lytic cycle and virulence, showing its potential as a target of therapeutic intervention. IMPORTANCEInfection with Toxoplasma gondii can lead to severe and even life-threatening diseases in people with compromised or suppressed immune systems. Unfortunately, drugs to combat the parasite are limited, highly toxic, and ineffective against the chronic stage of the parasite. Consequently, there is a strong demand for the discovery of new treatments. A comprehensive understanding of how the parasite propagates in the host cells and which proteins contribute to the parasites virulence will facilitate the discovery of new drug targets. Our study meets this objective and adds new insights to understanding the lytic cycle regulation and virulence of Toxoplasma by determining that the protein phosphatase TgPRL plays a vital role in the parasites ability to attach to host cells and that it is essential for parasite virulence.

microbiology↗

bioRxiv2021

A positive feedback loop mediates crosstalk between calcium, cyclic nucleotide and lipid signalling in Toxoplasma gondii

Fundamental processes of obligate intracellular parasites, such as Toxoplasma gondii and Plasmodium falciparum, are controlled by a set of plant-like calcium dependent kinases (CDPKs), the conserved cAMP- and cGMP-dependent protein kinases (PKA and PKG), secondary messengers and lipid signalling. While some major components of the signalling networks have been identified, how these 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 to activate PKG or CDPKs, respectively. We show that both these inducers trigger near identical signalling pathways and provide evidence for a positive feedback loop involving CDPK3. We measure phospho- and lipid signalling in parasites treated with the Ca2+ ionophore A23187 in a sub-minute timecourse and show CDPK3-dependent regulation of diacylglycerol levels and increased phosphorylation of four phosphodiesterases (PDEs), suggesting their function in the feedback loop. Disruption of CDPK3 leads to elevated cAMP levels and inhibition of PKA signalling rescues the egress defect of {Delta}CDPK3 parasites treated with A23187. Biochemical analysis of the four PDEs identifies PDE2 as the only cAMP-specific PDE among these candidates, while the other PDEs are cGMP specific, two of which are inhibited by the predicted PDE inhibitor BIPPO. Conditional deletion of the four PDEs supports an important, but non-essential role for PDE1 and PDE2 in growth, with PDE2 controlling A23187-mediated egress. In summary we uncover a positive feedback loop that enhances signalling during egress and links several signalling pathways together.

microbiology↗

bioRxiv2019

The secreted acid phosphatase domain-containing GRA44 from Toxoplasma gondii is required for C-myc induction in infected cells

During host cell invasion, the eukaryotic pathogen Toxoplasma gondii forms a parsitophorous vacuole to safely reside within, while partitioned from host cell defense mechanisms. From within this safe niche parasites sabotage multiple host cell systems including gene expression, apoptosis and intracellular immune recognition by secreting a large arsenal of effector proteins. Many parasite proteins studied for active host cell manipulative interactions have been kinases. Translocation of effectors from the parasitophorous vacuole into the host cell is mediated by a putative translocon complex, which includes proteins MYR1, MYR2, and MYR3. Whether other proteins are involved in the structure or regulation of this putative translocon is not known. We have discovered that the secreted protein GRA44, which contains a putative acid phosphatase domain, interacts with members of this complex and is required for host cell effects downstream of effector secretion. We have determined GRA44 is processed in a region with homology to sequences targeted by protozoan proteases of the secretory pathway and that both major cleavage fragments are secreted into the parasitophorous vacuole. Immunoprecipitation experiments showed that GRA44 interacts with a large number of secreted proteins included MYR1. Importantly, conditional knockdown of GRA44 resulted in a lack of host cell cMyc upregulation, which mimics the phenotype seen when members of the translocon complex are genetically disrupted. Thus, the putative acid phosphatase GRA44 is crucial for host cell alterations during Toxoplasma infection and is associated with the translocon complex which Toxoplasma relies upon for success as an intracellular pathogen. IMPORTANCEApproximately one third of humans are infected with the parasite Toxoplasma gondii. Toxoplasma infections can lead to severe disease in those with a compromised or suppressed immune system. Additionally, infections during pregnancy present a significant health risk to the developing fetus. Drugs that target this parasite are limited, have significant side effects, and do not target all disease stages. Thus, a thorough understanding of how the parasite propagates within a host is critical in the discovery of novel therapeutic targets. To replicate Toxoplasma requires to enter the cells of the infected organism. In order to survive the environment inside a cell, Toxoplasma secretes a large repertoire of proteins, which hijack a number of important cellular functions. How these Toxoplasma proteins move from the parasite into the host cell is not well understood. Our work shows that the putative phosphatase GRA44 is part of a protein complex responsible for this process.

microbiology↗

bioRxiv2019

Identification of Fis1 interactors in Toxoplasma gondii reveals a novel protein required for peripheral distribution of the mitochondrion.

Toxoplasma gondiis singular mitochondrion is very dynamic and undergoes morphological changes throughout the parasites life cycle. During parasite division, the mitochondrion elongates, enters the daughter cells just prior to cytokinesis and undergoes fission. Extensive morphological changes also occur as the parasite transitions from the intracellular to the extracellular environment. We show that treatment with the ionophore monensin causes reversible constriction of the mitochondrial outer membrane, and that this effect depends on the function of the fission related protein Fis1. We also observed that mislocalization of the endogenous Fis1 causes a dominant negative effect that affects the morphology of the mitochondrion. As this suggests Fis1 interacts with proteins critical for maintenance of mitochondrial structure, we performed various protein interaction trap screens. In this manner we identified a novel outer mitochondrial membrane protein, LMF1, which is essential for positioning of the mitochondrion in intracellular parasites. Normally, while inside a host cell, the parasite mitochondrion is maintained in a lasso shape that stretches around the parasite periphery where it has regions of coupling with the parasite pellicle, suggesting the presence of membrane contact sites. In intracellular parasites lacking LMF1 the mitochondrion is retracted away from the pellicle and instead is collapsed, as only normally seen in extracellular parasites. We show that this phenotype is associated with defects in parasite fitness and mitochondrial segregation. Thus, LMF1 is necessary for mitochondrial association with the parasite pellicle during intracellular growth and proper mitochondrial morphology is a prerequisite for mitochondrial division.\n\nIMPORTANCEToxoplasma gondii is an opportunistic pathogen that can cause devastating tissue damage in the immunocompromised and the congenitally infected. Current therapies are not effective against all life stages of the parasite and many cause toxic effects. The single mitochondrion of this parasite is a validated drug target and it changes its shape throughout its life cycle. When the parasite is inside of a cell, the mitochondrion adopts a lasso shape that lies in close proximity to the pellicle. The functional significance of this morphology is not understood nor are the proteins involved currently known. We have identified a protein that is required for proper mitochondrial positioning at the periphery and that likely plays a role in tethering this organelle. Loss of this protein results in dramatic changes to the mitochondrial morphology and significant parasite division and propagation defects. Our results give important insight into the molecular mechanisms regulating mitochondrial morphology.

microbiology↗