bims-toxgon 2023-07-23 papers

bims-toxgon

Biomed News

on Toxoplasma gondii metabolism

Issue of 2023–07–23
nine papers selected by
Lakesh Kumar, BITS Pilani

A heterotrimeric complex of Toxoplasma proteins promotes parasite survival in interferon gamma-stimulated human cells.
LILRB4 regulates the function of decidual MDSCs via the SHP-2/STAT6 pathway during Toxoplasma gondii infection.
A screen of drug-like molecules identifies chemically diverse electron transport chain inhibitors in apicomplexan parasites.
Unique asymmetric distribution of phosphatidylserine and phosphatidylethanolamine in Toxoplasma gondii revealed by nanoscale analysis.
Comparative transcriptome profiling of Eimeria tenella in various developmental stages and functional analysis of an ApiAP2 transcription factor exclusively expressed during sporogony.
The role of Plasmodium V-ATPase in vacuolar physiology and antimalarial drug uptake.
Dual localization of receptor-type adenylate cyclases and cAMP response protein 3 unveils the presence of two putative signaling microdomains in Trypanosoma cruzi.
Activation of autophagy depends on Atg1/Ulk1-mediated phosphorylation and inhibition of the Hsp90 chaperone machinery.
PDE4 inhibitors for disease therapy: advances and future perspective.

PLoS Biol. 2023 Jul 17. 21(7): e3002202

A heterotrimeric complex of Toxoplasma proteins promotes parasite survival in interferon gamma-stimulated human cells.

Eloise J Lockyer, Francesca Torelli, Simon Butterworth, Ok-Ryul Song, Steven Howell, Anne Weston, Philip East, Moritz Treeck.

Toxoplasma gondii secretes protein effectors to subvert the human immune system sufficiently to establish a chronic infection. Relative to murine infections, little is known about which parasite effectors disarm human immune responses. Here, we used targeted CRISPR screening to identify secreted protein effectors required for parasite survival in IFNγ-activated human cells. Independent screens were carried out using 2 Toxoplasma strains that differ in virulence in mice, leading to the identification of effectors required for survival in IFNγ-activated human cells. We identify the secreted protein GRA57 and 2 other proteins, GRA70 and GRA71, that together form a complex which enhances the ability of parasites to persist in IFNγ-activated human foreskin fibroblasts (HFFs). Components of the protein machinery required for export of Toxoplasma proteins into the host cell were also found to be important for parasite resistance to IFNγ in human cells, but these export components function independently of the identified protein complex. Host-mediated ubiquitination of the parasite vacuole has previously been associated with increased parasite clearance from human cells, but we find that vacuoles from GRA57, GRA70, and GRA71 knockout strains are surprisingly less ubiquitinated by the host cell. We hypothesise that this is likely a secondary consequence of deletion of the complex, unlinked to the IFNγ resistance mediated by these effectors.

DOI: https://doi.org/10.1371/journal.pbio.3002202
Parasit Vectors. 2023 Jul 17. 16(1): 237

LILRB4 regulates the function of decidual MDSCs via the SHP-2/STAT6 pathway during Toxoplasma gondii infection.

Yuantao Li, Jingjing Guo, Haixia Zhang, Zhidan Li, Yushan Ren, Yuzhu Jiang, Xianbing Liu, Xuemei Hu.

   BACKGROUND: Toxoplasma gondii infection can cause adverse pregnancy outcomes, such as recurrent abortion, fetal growth restriction and infants with malformations, among others. Decidual myeloid-derived suppressor cells (dMDSCs) are a novel immunosuppressive cell type at the fetal-maternal interface which play an important role in sustaining normal pregnancy that is related to their high expression of the inhibitory molecule leukocyte immunoglobulin-like receptor B4 (LILRB4). It has been reported that the expression of LILRB4 is downregulated on decidual macrophages after T. gondii infection, but it remains unknown whether T. gondii infection can induce dMDSC dysfunction resulting from the change in LILRB4 expression.
METHODS: LILRB4-deficient (LILRB4-/-) pregnant mice infected with T. gondii with associated adverse pregnancy outcomes, and anti-LILRB4 neutralized antibodies-treated infected human dMDSCs were used in vivo and in vitro experiments, respectively. The aim was to investigate the effect of LILRB4 expression on dMDSC dysfunction induced by T. gondii infection.
RESULTS: Toxoplasma gondii infection was observed to reduce STAT3 phosphorylation, resulting in decreased LILRB4 expression on dMDSCs. The levels of the main functional molecules (arginase-1 [Arg-1], interleukin-10 [IL-10]) and main signaling molecules (phosphorylated Src-homology 2 domain-containing protein tyrosine phosphatase [p-SHP2], phosphorylated signal transducer and activator of transcription 6 [p-STAT6]) in dMDSCs were all significantly reduced in human and mouse dMDSCs due to the decrease of LILRB4 expression induced by T. gondii infection. SHP-2 was found to directly bind to STAT6 and STAT6 to bind to the promoter of the Arg-1 and IL-10 genes during T. gondii infection.
CONCLUSIONS: The downregulation of LILRB4 expression on dMDSCs induced by T. gondii infection could regulate the expression of Arg-1 and IL-10 via the SHP-2/STAT6 pathway, resulting in the dysfunction of dMDSCs, which might contribute to adverse outcomes during pregnancy by T. gondii infection.

Keywords:  Decidual MDSCs; Dysfunction; Fetal; LILRB4; Maternal tolerance; Toxoplasma gondii

DOI:  https://doi.org/10.1186/s13071-023-05856-4
PLoS Pathog. 2023 Jul 20. 19(7): e1011517

A screen of drug-like molecules identifies chemically diverse electron transport chain inhibitors in apicomplexan parasites.

Jenni A Hayward, F Victor Makota, Daniela Cihalova, Rachel A Leonard, Esther Rajendran, Soraya M Zwahlen, Laura Shuttleworth, Ursula Wiedemann, Christina Spry, Kevin J Saliba, Alexander G Maier, Giel G van Dooren.

Apicomplexans are widespread parasites of humans and other animals, and include the causative agents of malaria (Plasmodium species) and toxoplasmosis (Toxoplasma gondii). Existing anti-apicomplexan therapies are beset with issues around drug resistance and toxicity, and new treatment options are needed. The mitochondrial electron transport chain (ETC) is one of the few processes that has been validated as a drug target in apicomplexans. To identify new inhibitors of the apicomplexan ETC, we developed a Seahorse XFe96 flux analyzer approach to screen the 400 compounds contained within the Medicines for Malaria Venture 'Pathogen Box' for ETC inhibition. We identified six chemically diverse, on-target inhibitors of the ETC in T. gondii, at least four of which also target the ETC of Plasmodium falciparum. Two of the identified compounds (MMV024937 and MMV688853) represent novel ETC inhibitor chemotypes. MMV688853 belongs to a compound class, the aminopyrazole carboxamides, that were shown previously to target a kinase with a key role in parasite invasion of host cells. Our data therefore reveal that MMV688853 has dual targets in apicomplexans. We further developed our approach to pinpoint the molecular targets of these inhibitors, demonstrating that all target Complex III of the ETC, with MMV688853 targeting the ubiquinone reduction (Qi) site of the complex. Most of the compounds we identified remain effective inhibitors of parasites that are resistant to Complex III inhibitors that are in clinical use or development, indicating that they could be used in treating drug resistant parasites. In sum, we have developed a versatile, scalable approach to screen for compounds that target the ETC in apicomplexan parasites, and used this to identify and characterize novel inhibitors.

DOI: https://doi.org/10.1371/journal.ppat.1011517
Histochem Cell Biol. 2023 Jul 21.

Unique asymmetric distribution of phosphatidylserine and phosphatidylethanolamine in Toxoplasma gondii revealed by nanoscale analysis.

Rikako Konishi, Kayoko Fukuda, Sayuri Kuriyama, Tatsunori Masatani, Xuenan Xuan, Akikazu Fujita.

  Toxoplasma gondii is a highly prevalent obligate apicomplexan parasite that is important in clinical and veterinary medicine. It is known that glycerophospholipids phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn), especially their expression levels and flip-flops between cytoplasmic and exoplasmic leaflets, in the membrane of T. gondii play important roles in efficient growth in host mammalian cells, but their distributions have still not been determined because of technical difficulties in studying intracellular lipid distribution at the nanometer level. In this study, we developed an electron microscopy method that enabled us to determine the distributions of PtdSer and PtdEtn in individual leaflets of cellular membranes by using quick-freeze freeze-fracture replica labeling. Our findings show that PtdSer and PtdEtn are asymmetrically distributed, with substantial amounts localized at the luminal leaflet of the inner membrane complex (IMC), which comprises flattened vesicles located just underneath the plasma membrane (see Figs. 2B and 7). We also found that PtdSer was absent in the cytoplasmic leaflet of the inner IMC membrane, but was present in considerable amounts in the cytoplasmic leaflet of the middle IMC membrane, suggesting a barrier-like mechanism preventing the diffusion of PtdSer in the cytoplasmic leaflets of the two membranes. In addition, the expression levels of both PtdSer and PtdEtn in the luminal leaflet of the IMC membrane in the highly virulent RH strain were higher than those in the less virulent PLK strain. We also found that the amount of glycolipid GM3, a lipid raft component, was higher in the RH strain than in the PLK strain. These results suggest a correlation between lipid raft maintenance, virulence, and the expression levels of PtdSer and PtdEtn in T. gondii.

Keywords:  Electron microscopy; Freeze-fracture; Lipid; Quick-freezing

DOI:  https://doi.org/10.1007/s00418-023-02218-0
Parasit Vectors. 2023 Jul 19. 16(1): 241

Comparative transcriptome profiling of Eimeria tenella in various developmental stages and functional analysis of an ApiAP2 transcription factor exclusively expressed during sporogony.

Linlin Chen, Xinming Tang, Pei Sun, Dandan Hu, Yuanyuan Zhang, Chaoyue Wang, Junmin Chen, Jie Liu, Yang Gao, Zhenkai Hao, Ning Zhang, Wenxuan Chen, Fujie Xie, Xun Suo, Xianyong Liu.

   BACKGROUND: The apicomplexan parasites Eimeria spp. are the causative agents of coccidiosis, a disease with a significant global impact on the poultry industry. The complex life cycle of Eimeria spp. involves exogenous (sporogony) and endogenous (schizogony and gametogony) stages. Unfortunately, the genetic regulation of these highly dynamic processes, particularly for genes involved in specific developmental phases, is not well understood.
METHODS: In this study, we used RNA sequencing (RNA-Seq) analysis to identify expressed genes and differentially expressed genes (DEGs) at seven time points representing different developmental stages of Eimeria tenella. We then performed K-means clustering along with co-expression analysis to identify functionally enriched gene clusters. Additionally, we predicted apicomplexan AP2 transcription factors in E. tenella using bioinformatics methods. Finally, we generated overexpression and knockout strains of ETH2_0411800 to observe its impact on E. tenella development.
RESULTS: In total, we identified 7329 genes that are expressed during various developmental stages, with 3342 genes exhibiting differential expression during development. Using K-means clustering along with co-expression analysis, we identified clusters functionally enriched for oocyte meiosis, cell cycle, and signaling pathway. Among the 53 predicted ApiAP2 transcription factors, ETH2_0411800 was found to be exclusively expressed during sporogony. The ETH2_0411800 overexpression and knockout strains did not exhibit significant differences in oocyst size or output compared to the parental strain, while the resulting ETH2_0411800 knockout parasite showed a relatively small oocyst output.
CONCLUSIONS: The findings of our research suggest that ETH2_0411800 is not essential for the growth and development of E. tenella. Our study provides insights into the gene expression dynamics and is a valuable resource for exploring the roles of transcription factor genes in regulating the development of Eimeria parasites.

Keywords:  ApiAP2 transcription factor; Eimeria tenella; Gene editing; Life cycle; Transcriptome

DOI:  https://doi.org/10.1186/s13071-023-05828-8
Proc Natl Acad Sci U S A. 2023 07 25. 120(30): e2306420120

The role of Plasmodium V-ATPase in vacuolar physiology and antimalarial drug uptake.

Arne Alder, Cecilia P Sanchez, Matthew R G Russell, Lucy M Collinson, Michael Lanzer, Michael J Blackman, Tim-Wolf Gilberger, Joachim M Matz.

  To ensure their survival in the human bloodstream, malaria parasites degrade up to 80% of the host erythrocyte hemoglobin in an acidified digestive vacuole. Here, we combine conditional reverse genetics and quantitative imaging approaches to demonstrate that the human malaria pathogen Plasmodium falciparum employs a heteromultimeric V-ATPase complex to acidify the digestive vacuole matrix, which is essential for intravacuolar hemoglobin release, heme detoxification, and parasite survival. We reveal an additional function of the membrane-embedded V-ATPase subunits in regulating morphogenesis of the digestive vacuole independent of proton translocation. We further show that intravacuolar accumulation of antimalarial chemotherapeutics is surprisingly resilient to severe deacidification of the vacuole and that modulation of V-ATPase activity does not affect parasite sensitivity toward these drugs.

Keywords:  Plasmodium falciparum; V-ATPase; chloroquine; malaria; vacuole

DOI:  https://doi.org/10.1073/pnas.2306420120
mBio. 2023 Jul 21. e0106423

Dual localization of receptor-type adenylate cyclases and cAMP response protein 3 unveils the presence of two putative signaling microdomains in Trypanosoma cruzi.

Miguel A Chiurillo, Joshua Carlson, Mayara S Bertolini, Aqsa Raja, Noelia Lander.

  Trypanosoma cruzi is the etiologic agent of Chagas disease, a leading cause of disability and premature death in the Americas. This parasite spends its life between a triatomine insect and a mammalian host, transitioning between developmental stages in response to microenvironmental changes. Among the second messengers driving differentiation in T. cruzi, cAMP has been shown to mediate metacyclogenesis and response to osmotic stress, but this signaling pathway remains largely unexplored in this parasite. Adenylate cyclases (ACs) catalyze the conversion of ATP to cAMP. They comprise a multigene family encoding putative receptor-type ACs in T. cruzi. Using protein sequence alignment, we classified them into five groups and chose a representative member from each group to study their localization (TcAC1-TcAC5). We expressed an HA-tagged version of each protein in T. cruzi and performed immunofluorescence analysis. A peculiar dual localization of TcAC1 and TcAC2 was observed in the flagellar distal domain and in the contractile vacuole complex (CVC), and their enzymatic activity was confirmed by gene complementation in yeast. Furthermore, TcAC1 overexpressing parasites showed an increased metacyclogenesis, a defect in host cell invasion, and a reduced intracellular replication, highlighting the importance of this protein throughout T. cruzi life cycle. These mutants were more tolerant to hypoosmotic stress and showed a higher adhesion capacity during in vitro metacyclogenesis, whereas the wild-type phenotype was restored after disrupting TcAC1 localization. Finally, TcAC1 was found to interact with cAMP response protein 3 (TcCARP3), co-localizing with this protein in the flagellar tip and CVC. IMPORTANCE We identified three components of the cAMP signaling pathway (TcAC1, TcAC2, and TcCARP3) with dual localization in Trypanosoma cruzi: the flagellar distal domain and the CVC, structures involved in cell adhesion and osmoregulation, respectively. We found evidence on the role of TcAC1 in both cellular processes, as well as in metacyclogenesis. Our data suggest that TcACs act as signal sensors and transducers through cAMP synthesis in membrane microdomains. We propose a model in which TcACs sense the harsh conditions in the triatomine hindgut (nutrient deprivation, acidic pH, osmotic stress, ionic composition, hydrophobic interactions) and become active. Synthesis of cAMP then triggers cell adhesion prior completion of metacyclogenesis, while mediating a response to osmotic stress in the parasite. These results shed light into the mechanisms driving cAMP-mediated cell differentiation in T. cruzi, while raising new questions on the activation of TcACs and the role of downstream components of this pathway.

Keywords:  CARP3; adenylate cyclase; cell adhesion; contractile vacuole complex; cyclic AMP; flagellar distal domain; metacyclogenesis; osmoregulation; trypanosomes

DOI:  https://doi.org/10.1128/mbio.01064-23
Cell Rep. 2023 Jul 13. pii: S2211-1247(23)00818-5. [Epub ahead of print]42(7): 112807

Activation of autophagy depends on Atg1/Ulk1-mediated phosphorylation and inhibition of the Hsp90 chaperone machinery.

Sarah J Backe, Rebecca A Sager, Jennifer A Heritz, Laura A Wengert, Katherine A Meluni, Xavier Aran-Guiu, Barry Panaretou, Mark R Woodford, Chrisostomos Prodromou, Dimitra Bourboulia, Mehdi Mollapour.

  Cellular homeostasis relies on both the chaperoning of proteins and the intracellular degradation system that delivers cytoplasmic constituents to the lysosome, a process known as autophagy. The crosstalk between these processes and their underlying regulatory mechanisms is poorly understood. Here, we show that the molecular chaperone heat shock protein 90 (Hsp90) forms a complex with the autophagy-initiating kinase Atg1 (yeast)/Ulk1 (mammalian), which suppresses its kinase activity. Conversely, environmental cues lead to Atg1/Ulk1-mediated phosphorylation of a conserved serine in the amino domain of Hsp90, inhibiting its ATPase activity and altering the chaperone dynamics. These events impact a conformotypic peptide adjacent to the activation and catalytic loop of Atg1/Ulk1. Finally, Atg1/Ulk1-mediated phosphorylation of Hsp90 leads to dissociation of the Hsp90:Atg1/Ulk1 complex and activation of Atg1/Ulk1, which is essential for initiation of autophagy. Our work indicates a reciprocal regulatory mechanism between the chaperone Hsp90 and the autophagy kinase Atg1/Ulk1 and consequent maintenance of cellular proteostasis.

Keywords:  CP: Molecular biology; Hsp90; Ulk1; atg1; autophagy; chaperone; chaperone code; co-chaperone; phosphorylation

DOI:  https://doi.org/10.1016/j.celrep.2023.112807
Future Med Chem. 2023 Jul 20.

PDE4 inhibitors for disease therapy: advances and future perspective.

Baochan Du, Min Luo, Changyu Ren, Jifa Zhang.

  The PDE4 enzyme family is specifically responsible for hydrolyzing cAMP and plays a vital role in regulating the balance of second messengers. As a crucial regulator in signal transduction, PDE4 has displayed promising pharmacological targets in a variety of diseases, for which its inhibitors have been used as a therapeutic strategy. This review provides a comprehensive summary of the development of PDE4 inhibitors in the past few years, along with the structure, clinical and research progress of multiple inhibitors of PDE4, focusing on the research and development strategies of PDE4 inhibitors. We hope our analysis will provide a significant reference for the future development of new PDE4 inhibitors.

Keywords:  PDE4; cancer; drug discovery; inhibitor; structure–activity relationship

DOI:  https://doi.org/10.4155/fmc-2023-0101