Functional Characterization of 17 Protein Serine/Threonine Phosphatases in Toxoplasma gondii Using CRISPR-Cas9 System

Protein serine/threonine phosphatases (PSPs), found in various plants and protozoa, are involved in the regulation of various biological processes. However, very little is known about the role of PSPs in the pathogenicity of the apicomplexan protozoan Toxoplasma gondii. Herein, the subcellular localization of 17 PSPs (PP5, PP7, EFPP, SLP, PPM3F, PPM4, PPM5A, PPM5B, PPM6, PPM8, PPM9, PPM12, PPM14, PPM18, CTD1, CTD2, and CTD3) was examined by 6× HA tagging of endogenous genes in C-terminal. The PSPs were detected in the cytoplasm (PP5, EFPP, PPM8, and CTD2), dense granules (SLP), nucleus (PPM4 and PPM9), inner membrane complex (PPM12), basal complex (CTD3), and apical pole (PP7). The remaining PSPs exhibited low or undetectable level of expression. To characterize the contribution of these genes to the infectivity of T. gondii, knock-out (KO) strains of type I RH strain deficient in the 17 psp genes and KO type II Pru strain deficient in pp7 and slp genes were constructed. The pathogenicity of individual RHΔpsp mutants was characterized in vitro using plaque, egress, and intracellular replication assays, and mouse infection, while pathogenicity of PruΔpp7 and PruΔslp mutant strains was evaluated by examining the parasite lytic cycle in vitro and assessment of brain cyst burden in mice. No significant differences were observed between 16 RHΔpsp strains and wild-type (WT) RH strain. However, RHΔpp7 exhibited significantly lower invasion efficiency and parasitophorous vacuole formation in vitro, and less virulence in mice compared with other RHΔpsp and WT strains. In addition, PruΔpp7 exhibited marked attenuation of virulence and significant reduction in the brain cyst burden in mice compared with PruΔslp and WT strains, suggesting the key role of PP7 in the virulence of T. gondii. Comparative transcriptomic profiling of the 17 psp genes showed that they may play different roles in the pathogenesis of different genotypes or life cycle stages of T. gondii. These findings provide new insight into the role of PSPs in the pathogenesis of T. gondii.

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Hydroxylamine and Carboxymethoxylamine Can Inhibit Toxoplasma gondii Growth through an Aspartate Aminotransferase-Independent Pathway

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01889-19 ◽

2020 ◽

Vol 64 (3) ◽

Cited By ~ 1

Author(s):

Jixu Li ◽

Huanping Guo ◽

Eloiza May Galon ◽

Yang Gao ◽

Seung-Hun Lee ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Drug Targets ◽

Protozoan Parasite ◽

Cell Types ◽

Lytic Cycle ◽

Type I ◽

Content Type ◽

Mechanism Of Inhibition

ABSTRACT Toxoplasma gondii is an obligate intracellular protozoan parasite and a successful parasitic pathogen in diverse organisms and host cell types. Hydroxylamine (HYD) and carboxymethoxylamine (CAR) have been reported as inhibitors of aspartate aminotransferases (AATs) and interfere with the proliferation in Plasmodium falciparum. Therefore, AATs are suggested as drug targets against Plasmodium. The T. gondii genome encodes only one predicted AAT in both T. gondii type I strain RH and type II strain PLK. However, the effects of HYD and CAR, as well as their relationship with AAT, on T. gondii remain unclear. In this study, we found that HYD and CAR impaired the lytic cycle of T. gondii in vitro, including the inhibition of invasion or reinvasion, intracellular replication, and egress. Importantly, HYD and CAR could control acute toxoplasmosis in vivo. Further studies showed that HYD and CAR could inhibit the transamination activity of rTgAAT in vitro. However, our results confirmed that deficiency of AAT in both RH and PLK did not reduce the virulence in mice, although the growth ability of the parasites was affected in vitro. HYD and CAR could still inhibit the growth of AAT-deficient parasites. These findings indicated that HYD and CAR inhibition of T. gondii growth and control of toxoplasmosis can occur in an AAT-independent pathway. Overall, further studies focusing on the elucidation of the mechanism of inhibition are warranted. Our study hints at new substrates of HYD and CAR as potential drug targets to inhibit T. gondii growth.

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iTRAQ-Based Phosphoproteomic Analysis of Toxoplasma gondii Tachyzoites Provides Insight Into the Role of Phosphorylation for its Invasion and Egress

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2020.586466 ◽

2020 ◽

Vol 10 ◽

Author(s):

Cheng He ◽

Mei-zhen Xu ◽

Shuai Pan ◽

Hui Wang ◽

Hong-juan Peng ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Protein Function ◽

Parasitophorous Vacuole ◽

Interaction Network ◽

Bioinformatic Analysis ◽

Lytic Cycle ◽

Transcriptional Level ◽

Cellular Processes ◽

Phosphorylation Level

The invasion and egress are two key steps in lytic cycle vital to the propagation of Toxoplasma gondii infection, and phosphorylation is believed to play important roles in these processes. However, the phosphoproteome of T. gondii at these two stages has not been characterized. In this study, we profiled the phosphoproteome of tachyzoites at the stages of “just invading” (JI) and “prior to egress” (PE) based on iTRAQ quantitative analysis, in which a total of 46 phosphopeptides, 42 phosphorylation sites, and 38 phosphoproteins were detected. In the comparison of PE vs. JI, 10 phosphoproteins were detected with their phosphorylation level significantly changed, and four of them were demonstrated to be significantly down-regulated at the transcriptional level. Bioinformatic analysis of these identified phosphoproteins suggested that phosphorylation-mediated modulation of protein function was employed to regulate the pathway of toxoplasmosis and metabolism and cellular processes correlated with tachyzoite’s binding, location, and metabolism, and thus play vital roles in the parasite lytic cycle. Moreover, cytoskeletal network (CN)-associated Inner Membrane Complex (IMC1, IMC4, IMC6 and IMC12), Intravascular Network (IVN)-related GRAs (GRA2, GRA3, GRA7 and GRA12), and Parasitophorous Vacuole Membrane (PVM)-localized ROP5 were shown to be enriched at the central nodes in the protein interaction network generated by bioinformatic analysis, in which the phosphorylation level of IMC4, GRA2, GRA3, and GRA12 were found to be significantly regulated. This study revealed the main cellular processes and key phosphoproteins crucial for the invasion and egress of T. gondii, which will provide new insights into the developmental biology of T. gondii in vitro and contribute to the understanding of pathogen-host interaction from the parasite perspective.

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Functional Characterization of Two Thioredoxin Proteins of Toxoplasma gondii Using the CRISPR-Cas9 System

Frontiers in Veterinary Science ◽

10.3389/fvets.2020.614759 ◽

2021 ◽

Vol 7 ◽

Author(s):

Zhi-Wei Zhang ◽

Ting-Ting Li ◽

Jin-Lei Wang ◽

Qin-Li Liang ◽

Hai-Sheng Zhang ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Expression Patterns ◽

Functional Characterization ◽

Plaque Assay ◽

Future Research ◽

Type I ◽

Worldwide Distribution ◽

Total Antioxidant

Toxoplasmosis caused by infection with Toxoplasma gondii is an important parasitic zoonosis with a worldwide distribution. In this study, we examined the functions of two thioredoxins (namely CTrp26 and CTrx1) of T. gondii tachyzoites by generation of HA tag strains or gene deficient parasites in Type I RH strain (ToxoDB#10). Immunofluorescence analysis (IFA) was used to investigate the subcellular localization of the thioredoxins (Trxs). Results of IFA showed that both CTrp26 and CTrx1 were located in the cytoplasm of T. gondii. Functional characterizations of CTrp26 and CTrx1-deficient parasites were performed by plaque assay, intracellular replication, egress, H2O2 resistance, detection of reactive oxygen species (ROS) level and total antioxidant capacity (T-AOC) assays in vitro, as well as mouse infection in vivo. Our results showed that deletion of CTrp26 or CTrx1 did not influence the ability of T. gondii RH strain to replicate, egress, form plaque, resist H2O2 exposure, maintain the ROS level, and T-AOC, and also did not serve as virulence factors in Kunming mice. Taken together, these results provide new properties of the two Trxs. Although they are not essential for RH strain, they may have roles in other strains of this parasite due to their different expression patterns, which warrants future research.

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Toxoplasma gondii excretion of glycolytic products is associated with acidification of the parasitophorous vacuole during parasite egress

10.1101/2021.11.25.469974 ◽

2021 ◽

Author(s):

My-Hang Huynh ◽

Vern B. Carruthers

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Parasitophorous Vacuole ◽

Indirect Evidence ◽

Lytic Cycle ◽

Vacuolar Acidification ◽

Lactate And Pyruvate ◽

Acidic Conditions ◽

Parasite Egress

The Toxoplasma gondii lytic cycle is a repetition of host cell invasion, replication, egress, and re-invasion into the next host cell. While the molecular players involved in egress have been studied in greater detail in recent years, the signals and pathways for triggering egress from the host cell have not been fully elucidated. A perforin-like protein, PLP1, has been shown to be necessary for permeabilizing the parasitophorous vacuole (PV) membrane or exit from the host cell. In vitro studies indicated that PLP1 is most active in acidic conditions, and indirect evidence using superecliptic pHluorin indicated that the PV pH drops prior to parasite egress. Using ratiometric pHluorin, a GFP variant that responds to changes in pH with changes in its bimodal excitation spectrum peaks, allowed us to directly measure the pH in the PV prior to and during egress by live-imaging microscopy. A statistically significant change was observed in PV pH during egress in both wild-type RH and Δplp1 vacuoles compared to DMSO-treated vacuoles. Interestingly, if parasites are chemically paralyzed, a pH drop is still observed in RH but not in Δplp1 tachyzoites. This indicates that the pH drop is dependent on the presence of PLP1 or motility. Efforts to determine transporters, exchangers, or pumps that could contribute to the drop in PV pH identified two formate-nitrite transporters (FNTs). Auxin-induced conditional knockdown and knockouts of FNT1 and FNT2 reduced the levels of lactate and pyruvate released by the parasites and lead an abatement of vacuolar acidification. While additional transporters and molecules are undoubtedly involved, we provide evidence of a definitive reduction in vacuolar pH associated with induced and natural egress and characterize two transporters that contribute to the acidification.

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TGF-β signaling–deficient hematopoietic stem cells have normal self-renewal and regenerative ability in vivo despite increased proliferative capacity in vitro

Blood ◽

10.1182/blood-2003-04-1300 ◽

2003 ◽

Vol 102 (9) ◽

pp. 3129-3135 ◽

Cited By ~ 110

Author(s):

Jonas Larsson ◽

Ulrika Blank ◽

Hildur Helgadottir ◽

Jon Mar Björnsson ◽

Mats Ehinger ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Negative Regulator ◽

Type I ◽

Hematopoietic Stem ◽

Knock Out

Abstract Studies in vitro implicate transforming growth factor β (TGF-β) as a key regulator of hematopoiesis with potent inhibitory effects on progenitor and stem cell proliferation. In vivo studies have been hampered by early lethality of knock-out mice for TGF-β isoforms and the receptors. To directly assess the role of TGF-β signaling for hematopoiesis and hematopoietic stem cell (HSC) function in vivo, we generated a conditional knock-out model in which a disruption of the TGF-β type I receptor (TβRI) gene was induced in adult mice. HSCs from induced mice showed increased proliferation recruitment when cultured as single cells under low stimulatory conditions in vitro, consistent with an inhibitory role of TGF-β in HSC proliferation. However, induced TβRI null mice show normal in vivo hematopoiesis with normal numbers and differentiation ability of hematopoietic progenitor cells. Furthermore HSCs from TβRI null mice exhibit a normal cell cycle distribution and do not differ in their ability long term to repopulate primary and secondary recipient mice following bone marrow transplantation. These findings challenge the classical view that TGF-β is an essential negative regulator of hematopoietic stem cells under physiologic conditions in vivo.

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TgCentrin2 is required for invasion and replication in the human parasite Toxoplasma gondii

10.1101/491316 ◽

2018 ◽

Author(s):

Jacqueline M. Leung ◽

Jun Liu ◽

Laura A. Wetzel ◽

Ke Hu

Keyword(s):

Toxoplasma Gondii ◽

Functional Characterization ◽

Stability Control ◽

Lytic Cycle ◽

Knock Out ◽

Human Parasite ◽

Ef Hand ◽

Parasite Survival ◽

Successive Generations

AbstractCentrins are EF-hand containing proteins ubiquitously found in eukaryotes and are key components of centrioles/basal bodies as well as certain contractile fibers. We previously identified three centrins in the human parasite Toxoplasma gondii, all of which localized to the centrioles. However, one of them, TgCentrin2 (CEN2), is also targeted to ring-shaped structures at the apical and basal ends of the parasite, as well as to multiple annuli at the junction between the apical cap and the rest of the membrane cortex. The role(s) that TgCentrin2 plays in these locations was unknown. Here we report the functional characterization of TgCentrin2 in the parasite lytic cycle. After multiple unsuccessful attempts to knock out or knock down the TgCentrin2 gene with existing tools, we designed a new conditional knockdown method that combines transcriptional and protein stability control to achieve tight regulation of TgCentrin2 levels in the parasite. We discovered that under knockdown conditions, there was an ordered loss of TgCentrin2 from its four compartments, due to differences in incorporation kinetics and structural inheritance over successive generations. This was correlated with the development of major invasion deficiency at early stages of CEN2 knockdown, and replication defects at later stages. These results indicate that TgCentrin2 is incorporated into multiple cytoskeletal structures to serve distinct functions in T. gondii that are required for parasite survival.

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Two phosphoglucomutase paralogs regulate triggered secretion of the Toxoplasma micronemes

10.1101/157743 ◽

2017 ◽

Author(s):

Sudeshna Saha ◽

Bradley I. Coleman ◽

Tiffany Sansom ◽

Rashmi Dubey ◽

Ira J. Blader ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Cell Invasion ◽

Lytic Cycle ◽

Host Cell Invasion ◽

Plain Language ◽

Functional Interaction ◽

Similar Phenotype

AbstractParafusin is a phosphoglucomutase (PGM) paralog that acts as a signaling scaffold protein in calcium mediated exocytosis across many eukaryotes. In Toxoplasma gondii the parafusin related protein 1 (PRP1) has been associated in indirect and heterologous studies with the regulated exocytosis of the micronemes, which are required for successful host cell invasion and egress. Here we directly assessed the role of PRP1 by deleting the gene from the parasite. We observed a specific defect in microneme secretion in response to high Ca2+ fluxes, but not to phosphatidic acid fluxes controlling microneme release. We observed no defect in constitutive microneme secretion which was sufficient to support completion of the lytic cycle. Furthermore, deletion of the other PGM in Toxoplasma, PGM2, as well as the double PRP1/PGM2 deletion resulted in a similar phenotype. This suggests a functional interaction between these two genes. Strikingly, tachyzoites without both paralogs are completely viable in vitro and during acute mice infections. This indicates that PGM activity is neither required for glycolysis. In conclusion, the PRP1-PGM2 pair is required for a burst in microneme secretion upon high Ca2+ fluxes, but this burst is not essential to complete the lytic cycle of the parasite.Plain Language SummaryCalcium mediated control of microneme secretion is essential for host cell invasion and egress of Toxoplasma gondii. Here it is shown that the two phosphoglucomutases in Toxoplasma both function in the translation of a spike in calcium into a burst in microneme secretion.

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The Extracellular Small Leucine-Rich Proteoglycan Biglycan Is a Key Player in Gastric Cancer Aggressiveness

Cancers ◽

10.3390/cancers13061330 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1330

Author(s):

Filipe Pinto ◽

Liliana Santos-Ferreira ◽

Marta T. Pinto ◽

Catarina Gomes ◽

Celso A. Reis

Keyword(s):

Clinical Value ◽

Knock Out ◽

Angiogenic Potential ◽

In Vivo Experiments ◽

Cancer Aggressiveness ◽

Advanced Stages ◽

Oncogenic Gene

Biglycan (BGN gene), an extracellular proteoglycan, has been described to be associated with cancer aggressiveness. The purpose of this study was to clarify the clinical value of biglycan as a biomarker in multiple independent GC cohorts and determine the in vitro and in vivo role of biglycan in GC malignant features. We found that BGN is commonly over-expressed in all analyzed cohorts, being associated with disease relapse and poor prognosis in patients with advanced stages of disease. In vitro and in vivo experiments demonstrated that biglycan knock-out GC cells display major phenotypic changes with a lower cell survival, migration, and angiogenic potential when compared with biglycan expressing cells. Biglycan KO GC cells present increased levels of PARP1 and caspase-3 cleavage and a decreased expression of mesenchymal markers. Importantly, biglycan deficient GC cells that were supplemented with exogenous biglycan were able to restore biological features, such as survival, clonogenic and migratory capacities. Our in vitro and in vivo findings were validated in human GC samples, where BGN expression was associated with several oncogenic gene signatures that were associated with apoptosis, cell migration, invasion, and angiogenesis. This study provided new insights on biglycan role in GC that should be taken in consideration as a key cellular regulator with major impact in tumor progression and patients’ clinical outcome.

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WNT-5A regulates TGF-β-related activities in liver fibrosis

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00160.2016 ◽

2017 ◽

Vol 312 (3) ◽

pp. G219-G227 ◽

Cited By ~ 21

Author(s):

Leonie Beljaars ◽

Sara Daliri ◽

Christa Dijkhuizen ◽

Klaas Poelstra ◽

Reinoud Gosens

Keyword(s):

Liver Fibrosis ◽

Functional Role ◽

Collagen Type ◽

Collagen Type I ◽

Matrix Proteins ◽

Type I ◽

Human Livers

WNT-5A is a secreted growth factor that belongs to the noncanonical members of the Wingless-related MMTV-integration family. Previous studies pointed to a connection between WNT-5A and the fibrogenic factor TGF-β warranting further studies into the functional role of WNT-5A in liver fibrosis. Therefore, we studied WNT-5A expressions in mouse and human fibrotic livers and examined the relation between WNT-5A and various fibrosis-associated growth factors, cytokines, and extracellular matrix proteins. WNT-5A gene and protein expressions were significantly increased in fibrotic mouse and human livers compared with healthy livers. Regression or therapeutic intervention in mice resulted in decreased hepatic WNT-5A levels paralleled by lower collagen levels. Immunohistochemical analysis showed WNT-5A staining in fibrotic septa colocalizing with desmin staining indicating WNT-5A expression in myofibroblasts. In vitro studies confirmed WNT-5A expression in this cell type and showed that TGF-β significantly enhanced WNT-5A expression in contrast to PDGF-BB and proinflammatory cytokines IL-1β and TNF-α. Additionally, TGF-β induces the expression of the WNT receptors FZD2 and FZD8. After silencing of WNT-5A, reduced levels of collagen type I, vimentin, and fibronectin in TGF-β-stimulated myofibroblasts were measured compared with nonsilencing siRNA-treated controls. Interestingly, the antifibrotic cytokine IFNγ suppressed WNT-5A in vitro and in vivo. IFNγ-treated fibrotic mice showed significantly less WNT-5A expression compared with untreated fibrotic mice. In conclusion, WNT-5A paralleled collagen I levels in fibrotic mouse and human livers. WNT-5A expression in myofibroblasts is induced by the profibrotic factor TGF-β and plays an important role in TGF-β-induced regulation of fibrotic matrix proteins, whereas its expression can be reversed upon treatment, both in vitro and in vivo. NEW & NOTEWORTHY This study describes the localization and functional role of WNT-5A in human and mouse fibrotic livers. Hepatic WNT-5A expression parallels collagen type I expression. In vivo and in vitro, the myofibroblasts were identified as the key hepatic cells producing WNT-5A. WNT-5A is under control of TGF-β and its activities are primarily profibrotic.

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ADAR1 function affects HPV replication and is associated to recurrent human papillomavirus-induced dysplasia in HIV coinfected individuals

Scientific Reports ◽

10.1038/s41598-019-56422-x ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Maria Pujantell ◽

Roger Badia ◽

Iván Galván-Femenía ◽

Edurne Garcia-Vidal ◽

Rafael de Cid ◽

...

Keyword(s):

Human Papillomavirus ◽

Immune Activation ◽

Hpv Infection ◽

Innate Immune ◽

Type I ◽

Innate Immune Activation ◽

Enhanced Expression

AbstractInfection by human papillomavirus (HPV) alters the microenvironment of keratinocytes as a mechanism to evade the immune system. A-to-I editing by ADAR1 has been reported to regulate innate immunity in response to viral infections. Here, we evaluated the role of ADAR1 in HPV infection in vitro and in vivo. Innate immune activation was characterized in human keratinocyte cell lines constitutively infected or not with HPV. ADAR1 knockdown induced an innate immune response through enhanced expression of RIG-I-like receptors (RLR) signaling cascade, over-production of type-I IFNs and pro-inflammatory cytokines. ADAR1 knockdown enhanced expression of HPV proteins, a process dependent on innate immune function as no A-to-I editing could be identified in HPV transcripts. A genetic association study was performed in a cohort of HPV/HIV infected individuals followed for a median of 6 years (range 0.1–24). We identified the low frequency haplotype AACCAT significantly associated with recurrent HPV dysplasia, suggesting a role of ADAR1 in the outcome of HPV infection in HIV+ individuals. In summary, our results suggest that ADAR1-mediated innate immune activation may influence HPV disease outcome, therefore indicating that modification of innate immune effectors regulated by ADAR1 could be a therapeutic strategy against HPV infection.

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