Silvia Moreno Archives - Center for Tropical and Emerging Global Diseases

Interorganellar Communication Through Membrane Contact Sites in Toxoplasma gondii

Donna Huber on August 6, 2023

Figure 1. Reported and potential MCSs between organelles of Toxoplasma gondii. Schematic representation showing proteins recently reported to be involved in MCSs, along with putative MCS candidates (indicated with “?”). For clarity purposes, only the central part of the parasite is shown. Abbreviations: AP, apicoplast; ER, endoplasmic reticulum; PLVAC, plant-like vacuolar compartment; IMC, inner membrane complex; TgTPC, T. gondii two pore channel; VDAC, voltage-dependent anion channel; LMF1, lasso maintenance factor 1.; MCS, membrane contact site.

Apicomplexan parasites are a group of protists that cause disease in humans and include pathogens like Plasmodium spp., the causative agent of malaria, and Toxoplasma gondii, the etiological agent of toxoplasmosis and one of the most ubiquitous human parasites in the world. Membrane contact sites (MCSs) are widespread structures within eukaryotic cells but their characterization in apicomplexan parasites is only in its very beginnings. Basic biological features of the T. gondii parasitic cycle support numerous organellar interactions, including the transfer of Ca²⁺ and metabolites between different compartments. In T. gondii, Ca²⁺ signals precede a series of interrelated molecular processes occurring in a coordinated manner that culminate in the stimulation of key steps of the parasite life cycle. Calcium transfer from the endoplasmic reticulum to other organelles via MCSs would explain the precision, speed, and efficiency that is needed during the lytic cycle of T. gondii. In this short review, we discuss the implications of these structures in cellular signaling, with an emphasis on their potential role in Ca²⁺ signaling.

Diego Huet, Silvia N J Moreno. Contact (Thousand Oaks). 2023 Aug 6;6:25152564231189064. doi: 10.1177/25152564231189064. eCollection 2023 Jan-Dec.

Analysis of the Interactome of the Toxoplasma gondii Tgj1 HSP40 Chaperone

Donna Huber on March 1, 2023

Toxoplasma gondii is an obligate intracellular apicomplexan that causes toxoplasmosis in humans and animals. Central to its dissemination and pathogenicity is the ability to rapidly divide in the tachyzoite stage and infect any type of nucleated cell. Adaptation to different cell contexts requires high plasticity in which heat shock proteins (Hsps) could play a fundamental role. Tgj1 is a type I Hsp40 of T. gondii, an ortholog of the DNAJA1 group, which is essential during the tachyzoite lytic cycle. Tgj1 consists of a J-domain, ZFD, and DNAJ_C domains with a CRQQ C-terminal motif, which is usually prone to lipidation. Tgj1 presented a mostly cytosolic subcellular localization overlapping partially with endoplasmic reticulum. Protein-protein Interaction (PPI) analysis showed that Tgj1 could be implicated in various biological pathways, mainly translation, protein folding, energy metabolism, membrane transport and protein translocation, invasion/pathogenesis, cell signaling, chromatin and transcription regulation, and cell redox homeostasis among others. The combination of Tgj1 and Hsp90 PPIs retrieved only 70 interactors linked to the Tgj1-Hsp90 axis, suggesting that Tgj1 would present specific functions in addition to those of the Hsp70/Hsp90 cycle, standing out invasion/pathogenesis, cell shape motility, and energy pathway. Within the Hsp70/Hsp90 cycle, translation-associated pathways, cell redox homeostasis, and protein folding were highly enriched in the Tgj1-Hsp90 axis. In conclusion, Tgj1 would interact with a wide range of proteins from different biological pathways, which could suggest a relevant role in them.

Jonathan Munera López, Andrés Mariano Alonso, Maria Julia Figueras, Ana María Saldarriaga Cartagena, Miryam A Hortua Triana, Luis Diambra, Laura Vanagas, Bin Deng, Silvia N J Moreno, Sergio Oscar Angel. Proteomes. 2023 Mar 1;11(1):9. doi: 10.3390/proteomes11010009.

The Toxoplasma Plant-Like Vacuolar Compartment (PLVAC)

Donna Huber on October 11, 2022

Toxoplasma gondii belongs to the phylum Apicomplexa and is an important cause of congenital disease and infection in immunocompromised patients. T. gondii shares several characteristics with plants including a non-photosynthetic plastid termed apicoplast and a multi-vesicular organelle that was named the plant-like vacuole (PLV) or vacuolar compartment (VAC). The name plant-like vacuole was selected based on its resemblance in composition and function to plant vacuoles. The name VAC represents its general vacuolar characteristics. We will refer to the organelle as PLVAC in this review. New findings in recent years have revealed that the PLVAC represents the lysosomal compartment of T. gondii which has adapted peculiarities to fulfill specific Toxoplasma needs. In this review, we discuss the composition and functions of the PLVAC highlighting its roles in ion storage and homeostasis, endocytosis, exocytosis, and autophagy.

Andrew J Stasic, Silvia N J Moreno, Vern B Carruthers, Zhicheng Dou. J Eukaryot Microbiol. 2022 Oct 11;e12951. doi: 10.1111/jeu.12951.

The Heptaprenyl Diphosphate Synthase (Coq1) Is the Target of a Lipophilic Bisphosphonate That Protects Mice against Toxoplasma gondii Infection

Donna Huber on September 21, 2022

Prenyldiphosphate synthases catalyze the reaction of allylic diphosphates with one or more isopentenyl diphosphate molecules to form compounds such as farnesyl diphosphate, used in, e.g., sterol biosynthesis and protein prenylation, as well as longer “polyprenyl” diphosphates, used in ubiquinone and menaquinone biosynthesis. Quinones play an essential role in electron transport and are associated with the inner mitochondrial membrane due to the presence of the polyprenyl group. In this work, we investigated the synthesis of the polyprenyl diphosphate that alkylates the ubiquinone ring precursor in Toxoplasma gondii, an opportunistic pathogen that causes serious disease in immunocompromised patients and the unborn fetus. The enzyme that catalyzes this early step of the ubiquinone synthesis is Coq1 (TgCoq1), and we show that it produces the C35 species heptaprenyl diphosphate. TgCoq1 localizes to the mitochondrion and is essential for in vitro T. gondii growth. We demonstrate that the growth defect of a T. gondii TgCoq1 mutant is rescued by complementation with a homologous TgCoq1 gene or with a (C45) solanesyl diphosphate synthase from Trypanosoma cruzi (TcSPPS). We find that a lipophilic bisphosphonate (BPH-1218) inhibits T. gondii growth at low-nanomolar concentrations, while overexpression of the TgCoq1 enzyme dramatically reduced growth inhibition by the bisphosphonate. Both the severe growth defect of the mutant and the inhibition by BPH-1218 were rescued by supplementation with a long-chain (C30) ubiquinone (UQ₆). Importantly, BPH-1218 also protected mice against a lethal T. gondii infection. TgCoq1 thus represents a potential drug target that could be exploited for improved chemotherapy of toxoplasmosis.

IMPORTANCE Millions of people are infected with Toxoplasma gondii, and the available treatment for toxoplasmosis is not ideal. Most of the drugs currently used are only effective for the acute infection, and treatment can trigger serious side effects requiring changes in the therapeutic approach. There is, therefore, a compelling need for safe and effective treatments for toxoplasmosis. In this work, we characterize an enzyme of the mitochondrion of T. gondii that can be inhibited by an isoprenoid pathway inhibitor. We present evidence that demonstrates that inhibition of the enzyme is linked to parasite death. In addition, the inhibitor can protect mice against a lethal dose of T. gondii. Our results thus reveal a promising chemotherapeutic target for the development of new medicines for toxoplasmosis.

Melissa A Sleda, Zhu-Hong Li, Ranjan Behera, Baihetiya Baierna, Catherine Li, Jomkwan Jumpathong, Satish R Malwal, Makoto Kawamukai, Eric Oldfield, Silvia N J Moreno. mBio. 2022 Sep 21;e0196622. doi: 10.1128/mbio.01966-22.

Temporal and thermal profiling of the Toxoplasma proteome implicates parasite Protein Phosphatase 1 in the regulation of Ca 2+-responsive pathways

Donna Huber on August 17, 2022

Apicomplexan parasites cause persistent mortality and morbidity worldwide through diseases including malaria, toxoplasmosis, and cryptosporidiosis. Ca²⁺ signaling pathways have been repurposed in these eukaryotic pathogens to regulate parasite-specific cellular processes governing the replicative and lytic phases of the infectious cycle, as well as the transition between them. Despite the presence of conserved Ca²⁺-responsive proteins, little is known about how specific signaling elements interact to impact pathogenesis. We mapped the Ca²⁺-responsive proteome of the model apicomplexan T. gondii via time-resolved phosphoproteomics and thermal proteome profiling. The waves of phosphoregulation following PKG activation and stimulated Ca²⁺ release corroborate known physiological changes but identify specific proteins operating in these pathways. Thermal profiling of parasite extracts identified many expected Ca²⁺-responsive proteins, such as parasite Ca²⁺-dependent protein kinases. Our approach also identified numerous Ca²⁺-responsive proteins that are not predicted to bind Ca²⁺, yet are critical components of the parasite signaling network. We characterized protein phosphatase 1 (PP1) as a Ca²⁺-responsive enzyme that relocalized to the parasite apex upon Ca²⁺ store release. Conditional depletion of PP1 revealed that the phosphatase regulates Ca²⁺ uptake to promote parasite motility. PP1 may thus be partly responsible for Ca²⁺-regulated serine/threonine phosphatase activity in apicomplexan parasites.

Alice L Herneisen, Zhu-Hong Li, Alex W Chan, Silvia N J Moreno, Sebastian Lourido. Elife. 2022 Aug 17;11:e80336. doi: 10.7554/eLife.80336.

In Vivo Efficacy of SQ109 against Leishmania donovani, Trypanosoma spp. and Toxoplasma gondii and In Vitro Activity of SQ109 Metabolites

Donna Huber on March 14, 2022

SQ109 is an anti-tubercular drug candidate that has completed Phase IIb/III clinical trials for tuberculosis and has also been shown to exhibit potent in vitro efficacy against protozoan parasites including Leishmania and Trypanosoma cruzi spp. However, its in vivo efficacy against protozoa has not been reported. Here, we evaluated the activity of SQ109 in mouse models of Leishmania, Trypanosoma spp. as well as Toxoplasma infection. In the T. cruzi mouse model, 80% of SQ109-treated mice survived at 40 days post-infection. Even though SQ109 did not cure all mice, these results are of interest since they provide a basis for future testing of combination therapies with the azole posaconazole, which acts synergistically with SQ109 in vitro. We also found that SQ109 inhibited the growth of Toxoplasma gondii in vitro with an IC₅₀ of 1.82 µM and there was an 80% survival in mice treated with SQ109, whereas all untreated animals died 10 days post-infection. Results with Trypanosoma brucei and Leishmania donovani infected mice were not promising with only moderate efficacy. Since SQ109 is known to be extensively metabolized in animals, we investigated the activity in vitro of SQ109 metabolites. Among 16 metabolites, six mono-oxygenated forms were found active across the tested protozoan parasites, and there was a ~6× average decrease in activity of the metabolites as compared to SQ109 which is smaller than the ~25× found with mycobacteria.

Kyung-Hwa Baek, Trong-Nhat Phan, Satish R Malwal, Hyeryon Lee, Zhu-Hong Li, Silvia N J Moreno, Eric Oldfield, Joo Hwan No. Biomedicines. 2022 Mar 14;10(3):670. doi: 10.3390/biomedicines10030670.

Toxoplasma bradyzoites exhibit physiological plasticity of calcium and energy stores controlling motility and egress

Donna Huber on December 3, 2021

Toxoplasma gondii has evolved different developmental stages for disseminating during acute infection (i.e. tachyzoites) and for establishing chronic infection (i.e. bradyzoites). Calcium ion (Ca²⁺) signaling tightly regulates the lytic cycle of tachyzoites by controlling microneme secretion and motility to drive egress and cell invasion. However, the roles of Ca²⁺ signaling pathways in bradyzoites remain largely unexplored. Here we show that Ca²⁺ responses are highly restricted in bradyzoites and that they fail to egress in response to agonists. Development of dual-reporter parasites revealed dampened Ca²⁺ responses and minimal microneme secretion by bradyzoites induced in vitro or harvested from infected mice and tested ex vivo. Ratiometric Ca²⁺ imaging demonstrated lower Ca²⁺ basal levels, reduced magnitude, and slower Ca²⁺ kinetics in bradyzoites compared with tachyzoites stimulated with agonists. Diminished responses in bradyzoites were associated with down-regulation of Ca²⁺-ATPases involved in intracellular Ca²⁺ storage in the endoplasmic reticulum (ER) and acidocalcisomes. Once liberated from cysts by trypsin digestion, bradyzoites incubated in glucose plus Ca²⁺ rapidly restored their intracellular Ca²⁺ and ATP stores leading to enhanced gliding. Collectively, our findings indicate that intracellular bradyzoites exhibit dampened Ca²⁺ signaling and lower energy levels that restrict egress, and yet upon release they rapidly respond to changes in the environment to regain motility.

Yong Fu, Kevin M Brown, Nathaniel G Jones, Silvia Nj Moreno, L David Sibley. Elife. 2021 Dec 3;10:e73011. doi: 10.7554/eLife.73011.

A plastid two-pore channel essential for inter-organelle communication and growth of Toxoplasma gondii

Donna Huber on October 4, 2021

Two-pore channels (TPCs) are a ubiquitous family of cation channels that localize to acidic organelles in animals and plants to regulate numerous Ca²⁺-dependent events. Little is known about TPCs in unicellular organisms despite their ancient origins. Here, we characterize a TPC from Toxoplasma gondii, the causative agent of toxoplasmosis. TgTPC is a member of a novel clad of TPCs in Apicomplexa, distinct from previously identified TPCs and only present in coccidians. We show that TgTPC localizes not to acidic organelles but to the apicoplast, a non-photosynthetic plastid found in most apicomplexan parasites. Conditional silencing of TgTPC resulted in progressive loss of apicoplast integrity, severely affecting growth and the lytic cycle. Isolation of TPC null mutants revealed a selective role for TPCs in replication independent of apicoplast loss that required conserved residues within the pore-lining region. Using a genetically-encoded Ca²⁺ indicator targeted to the apicoplast, we show that Ca²⁺ signals deriving from the ER but not from the extracellular space are selectively transmitted to the lumen. Deletion of the TgTPC gene caused reduced apicoplast Ca²⁺ uptake and membrane contact site formation between the apicoplast and the ER. Fundamental roles for TPCs in maintaining organelle integrity, inter-organelle communication and growth emerge.

Zhu-Hong Li, Thayer P King, Lawrence Ayong, Beejan Asady, Xinjiang Cai, Taufiq Rahman, Stephen A Vella, Isabelle Coppens, Sandip Patel, Silvia N J Moreno. Nat Commun. 2021 Oct 4;12(1):5802. doi: 10.1038/s41467-021-25987-5

Calcium signaling in intracellular protist parasites

Donna Huber on September 24, 2021

Calcium ion (Ca²⁺) signaling is one of the most frequently employed mechanisms of signal transduction by eukaryotic cells, and starts with either Ca²⁺ release from intracellular stores or Ca²⁺ entry through the plasma membrane. In intracellular protist parasites Ca²⁺ signaling initiates a sequence of events that may facilitate their invasion of host cells, respond to environmental changes within the host, or regulate the function of their intracellular organelles. In this review we examine recent findings in Ca²⁺ signaling in two groups of intracellular protist parasites that have been studied in more detail, the apicomplexan and the trypanosomatid parasites.

Roberto Docampo, Silvia Nj Moreno. Current Opinion in Microbiology 2021, 64:33–40. https://doi.org/10.1016/j.mib.2021.09.002

Calcium signaling through a Transient Receptor Channel is important for Toxoplasma gondii growth

Donna Huber on June 9, 2021

Transient Receptor Potential (TRP) channels participate in calcium ion (Ca²⁺) influx and intracellular Ca²⁺ release. TRP channels have not been studied in Toxoplasma gondii or any other apicomplexan parasite. In this work we characterize TgGT1_310560, a protein predicted to possess a TRP domain (TgTRPPL-2) and determined its role in Ca²⁺ signaling in T. gondii, the causative agent of toxoplasmosis. TgTRPPL-2 localizes to the plasma membrane and the endoplasmic reticulum (ER) of T. gondii. The ΔTgTRPPL-2 mutant was defective in growth and cytosolic Ca²⁺ influx from both extracellular and intracellular sources. Heterologous expression of TgTRPPL-2 in HEK-3KO cells allowed its functional characterization. Patching of ER-nuclear membranes demonstrates that TgTRPPL-2 is a non-selective cation channel that conducts Ca²⁺. Pharmacological blockers of TgTRPPL-2 inhibit Ca²⁺ influx and parasite growth. This is the first report of an apicomplexan ion channel that conducts Ca²⁺ and may initiate a Ca²⁺ signaling cascade that leads to the stimulation of motility, invasion and egress. TgTRPPL-2 is a potential target for combating Toxoplasmosis.

Karla Marie Marquez-Nogueras, Myriam Andrea Hortua Triana, Nathan M Chasen, Ivana Y Kuo, Silvia NJ Moreno. Elife. 2021 Jun 9;10:e63417. doi: 10.7554/eLife.63417.

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