bims-toxgon 2023-11-12 papers

bims-toxgon

Biomed News

on Toxoplasma gondii metabolism

Issue of 2023–11–12
seven papers selected by
Lakesh Kumar, BITS Pilani

Analysis of CDPK1 targets identifies a trafficking adaptor complex that regulates microneme exocytosis in Toxoplasma.
Transcriptomic and metabolomic analyses reveal the essential nature of Rab1B in Toxoplasma gondii.
Establishment and application of an iELISA detection method for measuring apical membrane antigen 1 (AMA1) antibodies of Toxoplasma gondii in cats.
Transcriptome profiling of Madin-Darby bovine kidney cells uncover differences in the susceptibility of cattle to Toxoplasma gondii and Neospora caninum.
Urtica dioica Extract Leads to Cyst Reduction, Enhanced Cell-Mediated Immune Response, and Antioxidant Activity in Experimental Toxoplasmosis.
Toxoplasma gondii IgG Serointensity Is Positively Associated With Frailty.
[Bioinformatics analysis of the RNA binding protein DDX39 of Toxoplasma gondii].

Elife. 2023 11 07. pii: RP85654. [Epub ahead of print]12

Analysis of CDPK1 targets identifies a trafficking adaptor complex that regulates microneme exocytosis in Toxoplasma.

Alex W Chan, Malgorzata Broncel, Eden Yifrach, Nicole R Haseley, Sundeep Chakladar, Elena Andree, Alice L Herneisen, Emily Shortt, Moritz Treeck, Sebastian Lourido.

  Apicomplexan parasites use Ca2+-regulated exocytosis to secrete essential virulence factors from specialized organelles called micronemes. Ca2+-dependent protein kinases (CDPKs) are required for microneme exocytosis; however, the molecular events that regulate trafficking and fusion of micronemes with the plasma membrane remain unresolved. Here, we combine sub-minute resolution phosphoproteomics and bio-orthogonal labeling of kinase substrates in Toxoplasma gondii to identify 163 proteins phosphorylated in a CDPK1-dependent manner. In addition to known regulators of secretion, we identify uncharacterized targets with predicted functions across signaling, gene expression, trafficking, metabolism, and ion homeostasis. One of the CDPK1 targets is a putative HOOK activating adaptor. In other eukaryotes, HOOK homologs form the FHF complex with FTS and FHIP to activate dynein-mediated trafficking of endosomes along microtubules. We show the FHF complex is partially conserved in T. gondii, consisting of HOOK, an FTS homolog, and two parasite-specific proteins (TGGT1_306920 and TGGT1_316650). CDPK1 kinase activity and HOOK are required for the rapid apical trafficking of micronemes as parasites initiate motility. Moreover, parasites lacking HOOK or FTS display impaired microneme protein secretion, leading to a block in the invasion of host cells. Taken together, our work provides a comprehensive catalog of CDPK1 targets and reveals how vesicular trafficking has been tuned to support a parasitic lifestyle.

Keywords:  CDPK; Toxoplasma gondii; cell biology; host–pathogen interactions; infectious disease; microbiology; signaling; vesicular trafficking

DOI:  https://doi.org/10.7554/eLife.85654
Parasit Vectors. 2023 Nov 08. 16(1): 409

Transcriptomic and metabolomic analyses reveal the essential nature of Rab1B in Toxoplasma gondii.

Kai He, Qiangqiang Wang, Xuwen Gao, Tao Tang, Huiyong Ding, Shaojun Long.

   BACKGROUND: The protozoan parasite Toxoplasma gondii encodes a dozen Rab proteins, which are parts of the small GTPase superfamily and regulate intracellular membrane trafficking. Our previous study showed that depletion of Rab1B caused severe defects regarding parasite growth and morphological structure, yet early defects of endocytic trafficking and vesicle sorting to the rhoptry in T. gondii are not expected to have a strong effect. To understand this discrepancy, we performed an integrated analysis at the level of transcriptomics and metabolomics.
METHODS: In the study, tetracycline-inducible TATi/Ty-Rab1B parasite line treated with ATc at three different time points (0, 18 and 24 h) was used. We first observed the morphological changes caused by Rab1B depletion via transmission electron technology. Then, high-throughput transcriptome along with non-targeted metabolomics were performed to analyze the RNA expression and metabolite changes in the Rab1B-depleted parasite. The essential nature of Rab1B in the parasite was revealed by the integrated omics approach.
RESULTS: Transmission electron micrographs showed a strong disorganization of endo-membranes in the Rab1B-depleted parasites. Our deep analysis of transcriptome and metabolome identified 2181 and 2374 differentially expressed genes (DEGs) and 30 and 83 differentially expressed metabolites (DEMs) at 18 and 24 h of induction in the tetracycline-inducible parasite line, respectively. These DEGs included key genes associated with crucial organelles that contain the rhoptry, microneme, endoplasmic reticulum and Golgi apparatus. The analysis of qRT-PCR verified some of the key DEGs identified by RNA-Seq, supporting that the key vesicular regulator Rab1B was involved in biogenesis of multiple parasite organelles. Functional enrichment analyses revealed pathways related to central carbon metabolisms and lipid metabolisms, such as the TCA cycle, glycerophospholipid metabolism and fatty acid biosynthesis and elongation. Further correlation analysis of the major DEMs and DEGs supported the role of Rab1B in biogenesis of fatty acids (e.g. myrisoleic acid and oleic acid) (R > 0.95 and P < 0.05), which was consistent with the scavenging role in biotin via the endocytic process.
CONCLUSIONS: Rab1B played an important role in parasite growth and morphology, which was supported by the replication assay and transmission electron microscopy observation. Our multi-omics analyses provided detailed insights into the overall impact on the parasite upon depletion of the protein. These analyses reinforced the role of Rab1B in the endocytic process, which has an impact on fatty acid biogenesis and the TCA cycle. Taken together, these findings contribute to our understanding of a key vesicular regulator, Rab1B, on parasite metabolism and morphological formation in T. gondii.

Keywords:  Metabolome; Rab family proteins; Toxoplasma gondii; Transcriptome

DOI:  https://doi.org/10.1186/s13071-023-06030-6
BMC Vet Res. 2023 Nov 03. 19(1): 229

Establishment and application of an iELISA detection method for measuring apical membrane antigen 1 (AMA1) antibodies of Toxoplasma gondii in cats.

Yafan Gao, Yu Shen, Jiyuan Fan, Haojie Ding, Bin Zheng, Haijie Yu, Siyang Huang, Qingming Kong, Hangjun Lv, Xunhui Zhuo, Shaohong Lu.

   BACKGROUND: Diseases caused by Toxoplasma gondii (T. gondii) have introduced serious threats to public health. There is an urgent need to develop a rapid detection method for T. gondii infection in cats, which are definitive hosts. Recombinant apical membrane antigen 1 (rAMA1) was produced in a prokaryotic expression system and used as the detection antigen. The aim of this study was to evaluate and optimize a reliable indirect enzyme-linked immunosorbent assay (iELISA) method based on rAMA1 for the detection of antibodies against T. gondii in cats.
RESULTS: The rAMA1-iELISA method was developed and optimized by the chessboard titration method. There were no cross-reactions between T. gondii-positive cat serum and positive serum for other pathogens, indicating that rAMA1-iELISA could only detect T. gondii in most cases. The lowest detection limit of rAMA1-iELISA was 1:3200 (dilution of positive serum), and the CV of repeated tests within batches and between batches were confirmed to be less than 10%. The results of 247 cat serum samples detected by rAMA1-iELISA (kappa value = 0.622, p < 0.001) were in substantial agreement with commercial ELISA. The ROC curve analysis revealed the higher overall check accuracy of rAMA1-iELISA (sensitivity = 91.7%, specificity = 93.6%, AUC = 0.956, 95% CI 0.905 to 1.000) than GRA7-based iELISA (sensitivity = 91.7%, specificity = 85.5%, AUC = 0.936, 95% CI 0.892 to 0.980). Moreover, the positive rate of rAMA1-iELISA (6.5%, 16/247) was higher than that of GRA7-based iELISA (3.6%, 9/247) and that of commercial ELISA kit (4.9%, 12/247).
CONCLUSION: The iELISA method with good specificity, sensitivity, and reproducibility was established and can be used for large-scale detection of T. gondii infection in clinical cat samples.

Keywords:  Apical membrane antigen 1; Cats; Detection; ELISA; Toxoplasma gondii

DOI:  https://doi.org/10.1186/s12917-023-03775-1
Vet Parasitol. 2023 Nov 04. pii: S0304-4017(23)00203-0. [Epub ahead of print]324 110072

Transcriptome profiling of Madin-Darby bovine kidney cells uncover differences in the susceptibility of cattle to Toxoplasma gondii and Neospora caninum.

Xiaojin Li, Kai He, Weifeng Qian, Chen Li, Suhui Hu, Min Zhang, Tianqi Wang, Wenchao Yan, Meng Qi.

  Toxoplasma gondii and Neospora caninum are two major apicomplexan protozoan parasites with heteroxenous life cycles and worldwide distributions. The transplacental transmission of N. caninum causes bovine abortion, which resulting in serious economic losses to the dairy industry. Although T. gondii was also reported to cause abortions in pregnant woman and small ruminants, scarce cases about the symptom to the host cattle and the causality remains unknown. In this study, transcriptome analysis of Madin Darby bovine kidney (MDBK) cells infected with T. gondii and N. caninum was performed to uncover the differences in susceptibility of cattle to the two parasites. The results showed that 256 and 2225 differentially expressed genes (DEGs) were detected in cells infected with N. caninum and T. gondii, respectively. Moreover, significant biological differences were revealed by the functional analysis including GO and KEGG enrichment. One serpin peptidase inhibitor (SEPRINA14), which is associated with immunosuppression during pregnancy, was found to significantly decrease in cells infected with N. caninum and increase in cells infected with T. gondii-infected cells. Pattern recognition receptors TLR3 and NOD2 were also significantly upregulated in N. caninum-infected MDBK cells, but not in T. gondii. They could induce an increased inflammatory response leading to severe tissue damage. In addition, the interleukin 12 receptor subunit beta 2 (IL12β2), which plays an essential role in Th1 and Th2 cell differentiation and inflammatory bowel disease, was also markedly upregulated in the N. caninum infected cells, which led to an imbalance in the Th1 and Th2 cells by promoting the Th1 cellular response. Altogether, our findings recognized a new understanding on the differences between T. gondii and N. caninum infection of MDBK cells, where SEPRINA14, TLR3, NOD2, and IL12β2 may be the key genes that affect the difference in susceptibility of cattle to T. gondii and N. caninum, especially in pregnant animals. This study provides more clues as to why N. caninum is more likely to cause abortions in cattle.

Keywords:  MDBK cells; Neospora caninum; Toxoplasma gondii; Transcriptome

DOI:  https://doi.org/10.1016/j.vetpar.2023.110072
Acta Parasitol. 2023 Nov 04.

Urtica dioica Extract Leads to Cyst Reduction, Enhanced Cell-Mediated Immune Response, and Antioxidant Activity in Experimental Toxoplasmosis.

Ali Peyvandi, Tahmineh Gorgani-Firouzjaee, Hossein Najafzadehvarzi, Jalal Jafarzadeh.

   BACKGROUND: Toxoplasmosis is a cosmopolitan parasitic infection caused by Toxoplasma gondii which is commonly treated by pyrimethamine (PYR) plus sulfadiazine (SDZ) with several adverse side effects. The present study evaluated the therapeutic effects of Urtica dioica L. aqueous extract (UDE) on acute and chronic toxoplasmosis in mice.
METHODS: For this purpose, mice were infected with 20 cysts (acute infection) or 10 cysts (chronic infection) of T. gondii (Me49 strain). The mice were treated with 200 mg/kg of UDE intraperitoneally (IP) and intragastric route (IG). The UDE-treated mice were compared with the PYR + SDZ treatment. The histopathological changes, cyst count, total antioxidant capacity (TAC), malondialdehyde (MDA) assay, and serum INF-γ were also evaluated.
RESULTS: In the acute toxoplasmosis, UDE by IP and IG administration significantly reduced the number of brain cysts by 93.74 and 92.55%, respectively, and increased the survival rate to 80% compared with 60% in untreated controls. In the chronic infection, cyst burden decreased at 88.2 and 83.4%, respectively, for IP and IG treatments. Moreover, UDE significantly increased INF- γ levels in acute and chronic toxoplasmosis. Tissue inflammatory lesions were reduced in the UDE-treated subgroups compared to the untreated group. UDE treatment significantly reduced MDA levels and elevated TAC in both acute and chronic infections.
CONCLUSION: The results show that U. dioica possesses significant immunostimulant and antioxidant activity with a higher cyst reduction in the brain during acute toxoplasmosis. Further studies are required to investigate the fractionations of UDE against T. gondii and its combination with other standard drugs.

Keywords:  Acute; Chronic; Extract; Mice; Toxoplasmosis; Urtica dioica

DOI:  https://doi.org/10.1007/s11686-023-00727-5
J Gerontol A Biol Sci Med Sci. 2023 Nov 06. pii: glad228. [Epub ahead of print]

Toxoplasma gondii IgG Serointensity Is Positively Associated With Frailty.

Hira Mohyuddin, Blanca Laffon, João P Teixeira, Solange Costa, Armanda Teixeira-Gomes, Eduardo Pásaro, Niel Constantine, Aline Dagdag, Heidi K Ortmeyer, Boris Tizenberg, Liubov Afram, Poyu Yen, Christopher Marano, Christopher A Lowry, Andrew J Hoisington, Jill A RachBeisel, Vanessa Valdiglesias, Carlota Lema-Arranz, Natalia Fernández-Bertólez, Ana Maseda, José C Millán-Calenti, Elizabeth J Kovacs, Johanna M Gostner, Dietmar Fuchs, Lisa A Brenner, Laura Lorenzo-López, Teodor T Postolache.

   BACKGROUND: Persistent inflammation related to aging ("inflammaging") is exacerbated by chronic infections and contributes to frailty in older adults. We hypothesized associations between Toxoplasma gondii (T. gondii), a common parasite causing an oligosymptomatic unremitting infection, and frailty, and secondarily between T. gondii and previously reported markers of immune activation in frailty.
METHODS: We analyzed available demographic, social, and clinical data in Spanish and Portuguese older adults [N = 601; age: mean (SD) 77.3 (8.0); 61% women]. Plasma T. gondii immunoglobulin G (IgG) serointensity was measured with an enzyme-linked immunosorbent assay. The Fried criteria were used to define frailty status. Validated translations of Mini-Mental State Examination, Geriatric Depression Scale, and the Charlson Comorbidity Index were used to evaluate confounders. Previously analyzed biomarkers that were significantly associated with frailty in both prior reports and the current study, and also related to T. gondii serointensity, were further accounted for in multivariable logistic models with frailty as outcome.
RESULTS: In T. gondii-seropositives, there was a significant positive association between T. gondii IgG serointensity and frailty, accounting for age (p = .0002), and resisting adjustment for multiple successive confounders. Among biomarkers linked with frailty, kynurenine/tryptophan and soluble tumor necrosis factor receptor II were positively associated with T. gondii serointensity in seropositives (p < .05). Associations with other biomarkers were not significant.
CONCLUSIONS: This first reported association between T. gondii and frailty is limited by a cross-sectional design and warrants replication. While certain biomarkers of inflammaging were associated with both T. gondii IgG serointensity and frailty, they did not fully mediate the T. gondii-frailty association.

Keywords:  Chronic toxoplasmosis; Inflammaging; Kynurenine to tryptophan ratio; sTNF-RII

DOI:  https://doi.org/10.1093/gerona/glad228
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi. 2023 Oct 11. 35(4): 358-365

[Bioinformatics analysis of the RNA binding protein DDX39 of Toxoplasma gondii].

Z Yang, J Wang, Y Qi, X Tian, X Mei, Z Zhang, S Wang.

   OBJECTIVE: To analyze the RNA binding protein of Toxoplasma gondii (TgDDX39) using bioinformatics technology, and to evaluate the immunogenicity of TgDDX39, so as to provide insights into development of toxoplasmosis vaccines.
METHODS: The amino acid sequences of TgDDX39 were retrieved from the ToxoDB database, and the physicochemical properties, transmembrane structure domain, signal peptide sites, post-translational modification sites, coils, secondary and tertiary structures, hydrophobicity, and antigenic epitopes of the TgDDX39 protein were predicted using online bioinformatics tools, incluiding ProtParam, TMHMM 2.0, SignalP 5.0, NetPhos 3.1, COILS, SOPMA, Phyre2, ProtScale, ABCpred, SYFPEITHI and DNA-STAR.
RESULTS: TgDDX39 protein was predicted to be an unstable hydrophilic protein with the molecular formula of C2173H3458N598O661S18, which contained 434 amino acids and had an estimated molecular weight of 49.1 kDa and a theoretical isoelectric point of 5.55. The protein was predicted to have an extremely low possibility of signal peptides, without transmembrane regions, and contain 27 phosphorylation sites. The β turn and random coils accounted for 39.63% of the secondary structure of the TgDDX39 protein, and a coiled helix tended to produce in one site. In addition, the TgDDX39 protein contained multiple B and T cell antigenic epitopes.
CONCLUSIONS: Bioinformatics analyses predict that TgDDX39 protein has high immunogenicity and contains multiple antigenic epitopes. TgDDX39 protein is a potential candidate antigen for vaccine development.

Keywords:  Bioinformatics; DDX39; Immunogenicity; RNA binding protein; Toxoplasma gondii

DOI:  https://doi.org/10.16250/j.32.1374.2023002