bims-toxgon 2024-02-18 papers

bims-toxgon

Biomed News

on Toxoplasma gondii metabolism

Issue of 2024–02–18
twelve papers selected by
Lakesh Kumar, BITS Pilani

Characterization and functional analysis of Toxoplasma Golgi-associated proteins identified by proximity labelling.
Toxoplasma-host endoplasmic reticulum interaction: How T. gondii activates unfolded protein response and modulates immune response.
Toxoplasma and Plasmodium associate with host Arfs during infection.
Mechanisms of adaptation and evolution in Toxoplasma gondii.
Anti-Toxoplasma gondii effect of tylosin in vitro and in vivo.
Stable expression of mucin glycoproteins GP40 and GP15 of Cryptosporidium parvum in Toxoplasma gondii.
Small RNAs from mitochondrial genome recombination sites are incorporated into T. gondii mitoribosomes.
Plasmodium RON11 triggers biogenesis of the merozoite rhoptry pair and is essential for erythrocyte invasion.
Sirtuins in kidney diseases: potential mechanism and therapeutic targets.
Regulation of newly identified lysine lactylation in cancer.
Next-Generation Genetically Encoded Fluorescent Biosensors Illuminate Cell Signaling and Metabolism.
PLK1 phosphorylates RhoGDI1 and promotes cancer cell migration and invasion.

bioRxiv. 2024 Feb 03. pii: 2024.02.02.578703. [Epub ahead of print]

Characterization and functional analysis of Toxoplasma Golgi-associated proteins identified by proximity labelling.

Rebecca R Pasquarelli, Justin J Quan, Vivian Yang, Timmie A Britton, Jihui Sha, James A Wohlschlegel, Peter J Bradley.

Toxoplasma gondii possesses a highly polarized secretory pathway that contains both broadly conserved eukaryotic organelles and unique apicomplexan organelles which play essential roles in the parasite's lytic cycle. As in other eukaryotes, the T. gondii Golgi apparatus sorts and modifies proteins prior to their distribution to downstream organelles. Many of the typical trafficking factors found involved in these processes are missing from apicomplexan genomes, suggesting that these parasites have evolved unique proteins to fill these roles. Here we identify a novel Golgi-localizing protein (ULP1) which contains structural homology to the eukaryotic trafficking factor p115/Uso1. We demonstrate that depletion of ULP1 leads to a dramatic reduction in parasite fitness and replicative ability. Using ULP1 as bait for TurboID proximity labelling and immunoprecipitation, we identify eleven more novel Golgi-associated proteins and demonstrate that ULP1 interacts with the T. gondii COG complex. These proteins include both conserved trafficking factors and parasite-specific proteins. Using a conditional knockdown approach, we assess the effect of each of these eleven proteins on parasite fitness. Together, this work reveals a diverse set of novel T. gondii Golgi-associated proteins that play distinct roles in the secretory pathway. As several of these proteins are absent outside of the Apicomplexa, they represent potential targets for the development of novel therapeutics against these parasites.
Importance: Apicomplexan parasites such as Toxoplasma gondii infect a large percentage of the world's population and cause substantial human disease. These widespread pathogens use specialized secretory organelles to infect their host cells, modulate host cell functions, and cause disease. While the functions of the secretory organelles are now better understood, the Golgi apparatus of the parasite remains largely unexplored, particularly regarding parasite-specific innovations that may help direct traffic intracellularly. In this work, we characterize ULP1, a protein that is unique to parasites but shares structural similarity to the eukaryotic trafficking factor p115/Uso1. We show that ULP1 plays an important role in parasite replication and demonstrate that it interacts with the conserved oligomeric Golgi (COG) complex. We then use ULP1 proximity labelling to identify eleven additional Golgi-associated proteins which we functionally analyze via conditional knockdown. This work expands our knowledge of the Toxoplasma Golgi apparatus and identifies potential targets for therapeutic intervention.

DOI: https://doi.org/10.1101/2024.02.02.578703
Curr Res Microb Sci. 2024 ;6 100223

Toxoplasma-host endoplasmic reticulum interaction: How T. gondii activates unfolded protein response and modulates immune response.

Obed Cudjoe, Roger Afful, Tonny Abraham Hagan.

  Toxoplasma gondii is a neurotropic single-celled zoonotic parasite that can infect human beings and animals. Infection with T. gondii is usually asymptomatic in immune-competent individual, however, it can cause symptomatic and life-threatening conditions in immunocompromised individuals and in developing foetuses. Although the mechanisms that allow T. gondii to persist in host cells are poorly understood, studies in animal models have greatly improved our understanding of Toxoplasma-host cell interaction and how this interaction modulates parasite proliferation and development, host immune response and virulence of the parasite. T. gondii is capable of recruiting the host endoplasmic reticulum (ER), suggesting it may influence the host ER function. Herein, we provide an overview of T. gondii infection and the role of host ER during stressed conditions. Furthermore, we highlight studies that explore T. gondii's interaction with the host ER. We delve into how this interaction activates the unfolded protein response (UPR) and ER stress-mediated apoptosis. Additionally, we examine how T. gondii exploits these pathways to its advantage.

Keywords:  Apoptosis; ER–stress; Endoplasmic reticulum; Immune response; Toxoplasma gondii; UPR

DOI:  https://doi.org/10.1016/j.crmicr.2024.100223
mSphere. 2024 Feb 13. e0077023

Toxoplasma and Plasmodium associate with host Arfs during infection.

Erin A Schroeder, Maria Toro-Moreno, Rene Raphemot, Kayla Sylvester, Isabel C Colón, Emily R Derbyshire.

  The apicomplexans Toxoplasma gondii and Plasmodium are intracellular parasites that reside within a host-derived compartment termed the parasitophorous vacuole (PV). During infection, the parasites must acquire critical host resources and transport them across their PV for development. However, the mechanism by which host resources are trafficked to and across the PV remains uncertain. Here, we investigated host ADP ribosylation factors (Arfs), a class of proteins involved in vesicular trafficking that may be exploited by T. gondii and Plasmodium berghei for nutrient acquisition. Using overexpressed Arf proteins coupled with immunofluorescence microscopy, we found that all Arfs were internalized into the T. gondii PV, with most vacuoles containing at least one punctum of Arf protein by the end of the lytic cycle. We further characterized Arf1, the most abundant Arf inside the T. gondii PV, and observed that active recycling between its GDP/GTP-bound state influenced Arf1 internalization independent of host guanine nucleotide exchange factors (GEFs). In addition, Arf1 colocalized with vesicle coat complexes and exogenous sphingolipids, suggesting a role in nutrient acquisition. While Arf1 and Arf4 were not observed inside the PV during P. berghei infection, our gene depletion studies showed that liver stage development and survival depended on the expression of Arf4 and the host GEF, GBF1. Collectively, these observations indicate that apicomplexans use distinct mechanisms to subvert the host vesicular trafficking network and efficiently replicate. The findings also pave the way for future studies to identify parasite proteins critical to host vesicle recruitment and the components of vesicle cargo.IMPORTANCEThe parasites Toxoplasma gondii and Plasmodium live complex intracellular lifestyles where they must acquire essential host nutrients while avoiding recognition. Although previous work has sought to identify the specific nutrients scavenged by apicomplexans, the mechanisms by which host materials are transported to and across the parasite vacuole membrane are largely unknown. Here, we examined members of the host vesicular trafficking network to identify specific pathways subverted by T. gondii and Plasmodium berghei. Our results indicate that T. gondii selectively internalizes host Arfs, a class of proteins involved in intracellular trafficking. For P. berghei, host Arfs were restricted by the parasite's vacuole membrane, but proteins involved in vesicular trafficking were identified as essential for liver stage development. A greater exploration into how and why apicomplexans subvert host vesicular trafficking could help identify targets for host-directed therapeutics.

Keywords:  Arf; Plasmodium; Toxoplasma; host-parasite interactions; vesicular trafficking

DOI:  https://doi.org/10.1128/msphere.00770-23
Mol Biochem Parasitol. 2024 Feb 12. pii: S0166-6851(24)00008-2. [Epub ahead of print] 111615

Mechanisms of adaptation and evolution in Toxoplasma gondii.

Sergio O Angel, Laura Vanagas, Andres M Alonso.

  Toxoplasma has high host flexibility, infecting all nucleated cells of mammals and birds. This implies that during its infective process the parasite must constantly adapt to different environmental situations, which in turn leads to modifications in its metabolism, regulation of gene transcription, translation of mRNAs and stage specific factors. There are conserved pathways that support these adaptations, which we aim to elucidate in this review. We begin by exploring the widespread epigenetic mechanisms and transcription regulators, continue with the supportive role of Heat Shock Proteins (Hsp), the translation regulation, stress granules, and finish with the emergence of contingency genes in highly variable genomic domains, such as subtelomeres. Within epigenetics, the discovery of a new histone variant of the H2B family (H2B.Z), contributing to T. gondii virulence and differentiation, but also gene expression regulation and its association with the metabolic state of the parasite, is highlighted. Associated with the regulation of gene expression are transcription factors (TFs). An overview of the main findings on TF and development is presented. We also emphasize the role of Hsp90 and Tgj1 in T. gondii metabolic fitness and the regulation of protein translation. Translation regulation is also highlighted as a mechanism for adaptation to conditions encountered by the parasite as well as stress granules containing mRNA and proteins generated in the extracellular tachyzoite. Another important aspect in evolution and adaptability are the subtelomeres because of their high variability and gene duplication rate. Toxoplasma possess multigene families of membrane proteins and contingency genes that are associated with different metabolic stresses. Among them parasite differentiation and environmental stresses stand out, including those that lead tachyzoite to bradyzoite conversion. Finally, we are interested in positioning protozoa as valuable evolution models, focusing on research related to the Extended Evolutionary Synthesis, based on models recently generated, such as extracellular adaptation and ex vivo cyst recrudescence.

Keywords:  Extended evolutionary synthesis; Toxoplasma; epigenetics; heat shock; subtelomeres

DOI:  https://doi.org/10.1016/j.molbiopara.2024.111615
Parasit Vectors. 2024 Feb 10. 17(1): 59

Anti-Toxoplasma gondii effect of tylosin in vitro and in vivo.

Ru-Xia Han, Pi-Cheng Jiang, Bing Han, Huai-Yu Zhou, Yong-Liang Wang, Jing-Yu Guan, Zhi-Rong Liu, Shen-Yi He, Chun-Xue Zhou.

   BACKGROUND: Toxoplasma gondii is an important protozoan pathogen with medical and veterinary importance worldwide. Drugs currently used for treatment of toxoplasmosis are less effective and sometimes cause serious side effects. There is an urgent need for the development of more effective drugs with relatively low toxicity.
METHODS: The effect of tylosin on the viability of host cells was measured using CCK8 assays. To assess the inhibition of tylosin on T. gondii proliferation, a real-time PCR targeting the B1 gene was developed for T. gondii detection and quantification. Total RNA was extracted from parasites treated with tylosin and then subjected to transcriptome analysis by RNA sequencing (RNA-seq). Finally, murine infection models of toxoplasmosis were used to evaluate the protective efficacy of tylosin against T. gondii virulent RH strain or avirulent ME49 strain.
RESULTS: We found that tylosin displayed low host toxicity, and its 50% inhibitory concentration was 175.3 μM. Tylsoin also inhibited intracellular T. gondii tachyzoite proliferation, with a 50% effective concentration of 9.759 μM. Transcriptome analysis showed that tylosin remarkably perturbed the gene expression of T. gondii, and genes involved in "ribosome biogenesis (GO:0042254)" and "ribosome (GO:0005840)" were significantly dys-regulated. In a murine model, tylosin treatment alone (100 mg/kg, i.p.) or in combination with sulfadiazine sodium (200 mg/kg, i.g.) significantly prolonged the survival time and raised the survival rate of animals infected with T. gondii virulent RH or avirulent ME49 strain. Meanwhile, treatment with tylosin significantly decreased the parasite burdens in multiple organs and decreased the spleen index of mice with acute toxoplasmosis.
CONCLUSIONS: Our findings suggest that tylosin exhibited potency against T. gondii both in vitro and in vivo, which offers promise for treatment of human toxoplasmosis.

Keywords:  Sulfadiazine sodium; Tachyzoite; Toxoplasma gondii; Tylosin

DOI:  https://doi.org/10.1186/s13071-024-06157-0
Parasit Vectors. 2024 Feb 15. 17(1): 65

Stable expression of mucin glycoproteins GP40 and GP15 of Cryptosporidium parvum in Toxoplasma gondii.

Muxiao Li, Xiaohua Sun, Haoyu Chen, Na Li, Yaoyu Feng, Lihua Xiao, Yaqiong Guo.

   BACKGROUND: Cryptosporidium spp. are common protozoa causing diarrhea in humans and animals. There are currently only one FDA-approved drug and no vaccines for cryptosporidiosis, largely due to the limited knowledge of the molecular mechanisms involved in the invasion of the pathogens. Previous studies have shown that GP60, which is cleaved into GP40 and GP15 after expression, is an immunodominant mucin protein involved in the invasion of Cryptosporidium. The protein is highly O-glycosylated, and recombinant proteins expressed in prokaryotic systems are non-functional. Therefore, few studies have investigated the function of GP40 and GP15.
METHODS: To obtain recombinant GP40 with correct post-translational modifications, we used CRISPR/Cas9 technology to insert GP40 and GP15 into the UPRT locus of Toxoplasma gondii, allowing heterologous expression of Cryptosporidium proteins. In addition, the Twin-Strep tag was inserted after GP40 for efficient purification of GP40.
RESULTS: Western blotting and immunofluorescent microscopic analyses both indicated that GP40 and GP15 were stably expressed in T. gondii mutants. GP40 localized not only in the cytoplasm of tachyzoites but also in the parasitophorous vacuoles, while GP15 without the GPI anchor was expressed only in the cytoplasm. In addition, a large amount of recTgGP40 was purified using Strep-TactinXT supported by a visible band of ~ 50 kDa in SDS-PAGE.
CONCLUSIONS: The establishment of a robust and efficient heterologous expression system of GP40 in T. gondii represents a novel approach and concept for investigating Cryptosporidium mucins, overcoming the limitations of previous studies that relied on unstable transient transfection, which involved complex steps and high costs.

Keywords:  Cryptosporidium parvum; Expression; GP40; Glycoprotein; Toxoplasma gondii

DOI:  https://doi.org/10.1186/s13071-024-06159-y
Elife. 2024 Feb 16. pii: e95407. [Epub ahead of print]13

Small RNAs from mitochondrial genome recombination sites are incorporated into T. gondii mitoribosomes.

Sabrina Tetzlaff, Arne Hillebrand, Nikiforos Drakoulis, Zala Gluhic, Sascha Maschmann, Peter Lyko, Susann Wicke, Christian Schmitz-Linneweber.

  The mitochondrial genomes of apicomplexans comprise merely three protein-coding genes, alongside a set of thirty to forty genes encoding small RNAs (sRNAs), many of which exhibit homologies to rRNA from E. coli. The expression status and integration of these short RNAs into ribosomes remains unclear and direct evidence for active ribosomes within apicomplexan mitochondria is still lacking. In this study, we conducted small RNA sequencing on the apicomplexan Toxoplasma gondii to investigate the occurrence and function of mitochondrial sRNAs. To enhance the analysis of sRNA sequencing outcomes, we also re-sequenced the T. gondii mitochondrial genome using an improved organelle enrichment protocol and Nanopore sequencing. It has been established previously that the T. gondii genome comprises 21 sequence blocks that undergo recombination among themselves but that their order is not entirely random. The enhanced coverage of the mitochondrial genome allowed us to characterize block combinations at increased resolution. Employing this refined genome for sRNA mapping, we find that many small RNAs originated from the junction sites between protein-coding blocks and rRNA sequence blocks. Surprisingly, such block border sRNAs were incorporated into polysomes together with canonical rRNA fragments and mRNAs. In conclusion, apicomplexan ribosomes are active within polysomes and are indeed assembled through the integration of sRNAs, including previously undetected sRNAs with merged mRNA-rRNA sequences. Our findings lead to the hypothesis that T. gondii's block-based genome organization enables the dual utilization of mitochondrial sequences as both messenger RNAs and ribosomal RNAs, potentially establishing a link between the regulation of rRNA and mRNA expression.

Keywords:  infectious disease; microbiology; none

DOI:  https://doi.org/10.7554/eLife.95407
bioRxiv. 2024 Jan 31. pii: 2024.01.29.577654. [Epub ahead of print]

Plasmodium RON11 triggers biogenesis of the merozoite rhoptry pair and is essential for erythrocyte invasion.

David Anaguano, Opeoluwa Adewale-Fasoro, Grace S Vick, Sean Yanik, James Blauwkamp, Manuel A Fierro, Sabrina Absalon, Prakash Srinivasan, Vasant Muralidharan.

Malaria is a global and deadly human disease caused by the apicomplexan parasites of the genus Plasmodium . Parasite proliferation within human red blood cells (RBC) is associated with the clinical manifestations of the disease. This asexual expansion within human RBCs, begins with the invasion of RBCs by P. falciparum , which is mediated by the secretion of effectors from two specialized club-shaped secretory organelles in merozoite-stage parasites known as rhoptries. We investigated the function of the Rhoptry Neck Protein 11 (RON11), which contains seven transmembrane domains and calcium-binding EF-hand domains. We generated conditional mutants of the P. falciparum RON11. Knockdown of RON11 inhibits parasite growth by preventing merozoite invasion. The loss of RON11 did not lead to any defects in processing of rhoptry proteins but instead led to a decrease in the amount of rhoptry proteins. We utilized ultrastructure expansion microscopy (U-ExM) to determine the effect of RON11 knockdown on rhoptry biogenesis. Surprisingly, in the absence of RON11, fully developed merozoites had only rhoptry each. The single rhoptry in RON11 deficient merozoites were morphologically typical with a bulb and a neck oriented into the apical polar ring. Moreover, rhoptry proteins are trafficked accurately to the single rhoptry in RON11 deficient parasites. These data show that in the absence of RON11, the first rhoptry is generated during schizogony but upon the start of cytokinesis, the second rhoptry never forms. Interestingly, these single-rhoptry merozoites were able to attach to host RBCs but are unable to invade RBCs. Instead, RON11 deficient merozoites continue to engage with RBC for prolonged periods eventually resulting in echinocytosis, a result of secreting the contents from the single rhoptry into the RBC. Together, our data show that RON11 triggers the de novo biogenesis of the second rhoptry and functions in RBC invasion.

DOI: https://doi.org/10.1101/2024.01.29.577654
Cell Commun Signal. 2024 Feb 12. 22(1): 114

Sirtuins in kidney diseases: potential mechanism and therapeutic targets.

Qi Jin, Fang Ma, Tongtong Liu, Liping Yang, Huimin Mao, Yuyang Wang, Liang Peng, Ping Li, Yongli Zhan.

  Sirtuins, which are NAD+-dependent class III histone deacetylases, are involved in various biological processes, including DNA damage repair, immune inflammation, oxidative stress, mitochondrial homeostasis, autophagy, and apoptosis. Sirtuins are essential regulators of cellular function and organismal health. Increasing evidence suggests that the development of age-related diseases, including kidney diseases, is associated with aberrant expression of sirtuins, and that regulation of sirtuins expression and activity can effectively improve kidney function and delay the progression of kidney disease. In this review, we summarise current studies highlighting the role of sirtuins in renal diseases. First, we discuss sirtuin family members and their main mechanisms of action. We then outline the possible roles of sirtuins in various cell types in kidney diseases. Finally, we summarise the compounds that activate or inhibit sirtuin activity and that consequently ameliorate renal diseases. In conclusion, targeted modulation of sirtuins is a potential therapeutic strategy for kidney diseases. Video Abstract.

Keywords:  Endothelial cells; Kidney diseases; Macrophages; Podocyte; Renal tubular epithelial cells; Sirtuins

DOI:  https://doi.org/10.1186/s12964-023-01442-4
Cancer Lett. 2024 Feb 10. pii: S0304-3835(24)00073-9. [Epub ahead of print]587 216680

Regulation of newly identified lysine lactylation in cancer.

Xin Gao, Chaoyu Pang, Zhiyao Fan, Yunshan Wang, Yangmiao Duan, Hanxiang Zhan.

  Metabolic reprogramming is a typical hallmark of cancer. Enhanced glycolysis in tumor cells leads to the accumulation of lactate, which is traditionally considered metabolic waste. With the development of high-resolution liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), the lactate-derived, lysine lactylation(Kla), has been identified. Kla can alter the spatial configuration of chromatin and regulate the expression of corresponding genes. Metabolic reprogramming and epigenetic remodeling have been extensively linked. Accumulating studies have subsequently expanded the framework on the key roles of this protein translational modification (PTM) in tumors and have provided a new concept of cancer-specific regulation by Kla.

Keywords:  Cancer; Glycolysis; Lactate; Lysine lactylation; Metabolic reprogramming

DOI:  https://doi.org/10.1016/j.canlet.2024.216680
Annu Rev Biophys. 2024 Feb 12.

Next-Generation Genetically Encoded Fluorescent Biosensors Illuminate Cell Signaling and Metabolism.

Michelle S Frei, Sohum Mehta, Jin Zhang.

Genetically encoded fluorescent biosensors have revolutionized the study of cell signaling and metabolism, as they allow for live-cell measurements with high spatiotemporal resolution. This success has spurred the development of tailor-made biosensors that enable the study of dynamic phenomena on different timescales and length scales. In this review, we discuss different approaches to enhancing and developing new biosensors. We summarize the technologies used to gain structural insights into biosensor design and comment on useful screening technologies. Furthermore, we give an overview of different applications where biosensors have led to key advances over recent years. Finally, we give our perspective on where future work is bound to make a large impact. Expected final online publication date for the Annual Review of Biophysics, Volume 53 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

DOI: https://doi.org/10.1146/annurev-biophys-030722-021359
Cancer Cell Int. 2024 Feb 14. 24(1): 73

PLK1 phosphorylates RhoGDI1 and promotes cancer cell migration and invasion.

Jeewon Lim, Yo Sep Hwang, Hyang Ran Yoon, Jiyun Yoo, Suk Ran Yoon, Haiyoung Jung, Hee Jun Cho, Hee Gu Lee.

   BACKGROUND: Rho guanine nucleotide dissociation inhibitor 1 (RhoGDI1) plays an important role in diverse cellular processes by regulating Rho guanosine triphosphate (GTP)ases activity. RhoGDI1 phosphorylation regulates the spatiotemporal activation of Rho GTPases during cell migration. In this study, we identified polo-like kinase 1 (PLK1) as a novel kinase of RhoGDI1 and investigated the molecular mechanism by which the interaction between RhoGDI1 and PLK1 regulates cancer cell migration.
METHODS: Immunoprecipitation, GST pull-down assay, and proximity ligation assay (PLA) were performed to analyze the interaction between RhoGDI1 and PLK1. In vitro kinase assay and immunoprecipitation were performed with Phospho-(Ser/Thr) antibody. We evaluated RhoA activation using RhoGTPases activity assay. Cell migration and invasion were analyzed by transwell assays.
RESULTS: GST pull-down assays and PLA showed that PLK1 directly interacted with RhoGDI1 in vitro and in vivo. Truncation mutagenesis revealed that aa 90-111 of RhoGDI1 are critical for interacting with PLK1. We also showed that PLK1 phosphorylated RhoGDI1 at Thr7 and Thr91, which induces cell motility. Overexpression of the GFP-tagged RhoGDI1 truncated mutant (aa 90-111) inhibited the interaction of PLK1 with RhoGDI1 and attenuated RhoA activation by PLK1. Furthermore, the overexpression of the RhoGDI1 truncated mutant reduced cancer cell migration and invasion in vitro and suppressed lung metastasis in vivo.
CONCLUSIONS: Collectively, we demonstrate that the phosphorylation of RhoGDI1 by PLK1 promotes cancer cell migration and invasion through RhoA activation. This study connects the interaction between PLK1 and RhoGDI1 to the promotion of cancer cell behavior associated with malignant progression, thereby providing opportunities for cancer therapeutic interventions.

Keywords:  Cancer; Migration; PLK1; RhoA; RhoGDI1

DOI:  https://doi.org/10.1186/s12935-024-03254-z