CRISPR/Cas9-based localization and functional analysis of Toxoplasma gondii putative protein TGGT1_310420

doi:10.12140/j.issn.1000-7423.2019.02.006

Abstract

Abstract:

Objective To characterize the function and localization of a putative protein TGGT1_310420 expressed on tachyzoite of Toxoplasma gondii. Methods The single guide RNA (sgRNA) targeting TGGT1_310420 was designed online. CRISPR/Cas9 construct targeting GGT1_310420 was obtained by mutating sgRNA of TgUPRT on pSAG1::CAS9-GFP-U6::sgUPRT. The knockout of TGGT1_310420 gene was performed through tagging the mCherry-Ty_HXGPRT sequence after the first 60 nt coding sequences of the endogenous copy using CRISPR/Cas9 genome-editing strategy. The correct insert was confirmed by PCR and DNA sequencing. The expressed fusion protein was detected by SDS-PAGE and Western blotting analysis, and its subcellular localization was determined by immunofluorescence microscopy. The phenotype of the knockout parasites was examined by a plaque assay. Results The CRISPR/Cas9 construct targeting TGGT1_310420 was successfully constructed and confirmed by sequencing. PCR analysis confirmed the integration of the mCherry-Ty_HXGPRT sequence into the correct locus and Western blotting assay detected a single protein band with an apparent relative molecular weight of 36 000. Immunofluorescent assay demonstrated that the first 20 amino acids of TGGT1_310420 fused to mCherry-Ty_HXGPRT was co-localized with T. gondii gliding associated protein 45(TgGAP45)to the parasite’s pellicle. Knockout of TGGT1_310420 gene revealed no measurable alteration in tachyzoites. Conclusion The first 20 amino acids of TGGT1_310420 are sufficient for pellicle targeting and the knockout of TGGT1_310420 did not change the phenotype of T. gomdii tachyzoite.

Key words: Toxoplasma godii, TGGT1_310420, Calcium-dependent protein kinases, CRISPR/Cas9

CLC Number:

R382.5

Yong-gen JIA, Ai-xia YAN, Min-jun HUANG, Yang ZOU, Jun-chao GU. CRISPR/Cas9-based localization and functional analysis of Toxoplasma gondii putative protein TGGT1_310420[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2019, 37(2): 150-155.

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References 18

[1]	Montaya JG, Liesenfeld O.Toxoplasmosis[J]. Lancet, 2004, 363(9425): 1965-1976.
[2]	Tenter AM, Heckeroth AR, Weiss LM.Toxoplasma gondii: from animals to humans[J]. Int J Parasitol, 2000, 30(12/13): 1217-1258.
[3]	Fox BA, Rommereim LM, Guevara RB, et al. The Toxoplasma gondii rhoptry kinome is essential for chronic infection[J]. MBio, 2016, 7(3): e00193-16.
[4]	Blader IJ, Coleman BI, Chen CT, et al. Lytic cycle of Toxoplasma gondii: 15 years later[J]. Annu Rev Microbiol, 2015, 69: 463-485.
[5]	Dvorin JD, Martyn DC, Patel SD, et al. A plant-like kinase in Plasmodium falciparum regulates parasite egress from erythrocytes[J]. Science, 2010, 328(5980): 910-912.
[6]	Lourido S, Shuman J, Zhang C, et al. Calcium-dependent protein kinase 1 is an essential regulator of exocytosis in Toxoplasma[J]. Nature, 2010, 465(7296): 359-362.
[7]	赵旭, 张婷, 张义伟, 等. 弓形虫钙离子结合蛋白的研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(5): 525-528.
[8]	McCoy JM, Whitehead L, Van Dooren GG, et al. TgCDPK3 regulates calcium-dependent egress of Toxoplasma gondii from host cells[J]. PLoS Pathog, 2012, 8(12): e1003066.
[9]	Wallbank BA, Dominicus CS, Broncel M, et al. Characterisation of the Toxoplasma gondii tyrosine transporter and its phosphorylation by the calcium-dependent protein kinase 3[J]. Mol Microbiol, 2018. doi: 10.1111/mmi.14156.
[10]	Gaji RY, Johnson DE, Treeck M, et al. Phosphorylation of a myosin motor by TgCDPK3 facilitates rapid initiation of motility during Toxoplasma gondii egress[J]. PLoS Pathog, 2015, 11(11): e1005268.
[11]	贾永根, 闫爱霞, 黄敏君. 刚地弓形虫 TgPH1 蛋白的重组表达及其与磷脂酰肌醇结合性的鉴定[J]. 中国热带医学, 2019, 19(3): 201-204.
[12]	Lourido S, Tang K, Sibley LD.Distinct signalling pathways control Toxoplasma egress and host-cell invasion[J]. EMBO J, 2012, 31(24): 4524-4534.
[13]	Martin DD, Beauchamp E, Berthiaume LG.Post-translational myristoylation: fat matters in cellular life and death[J]. Biochimie, 2011, 93(1): 18-31.
[14]	Foe IT, Child MA, Majmudar JD, et al. Global analysis of palmitoylated proteins in Toxoplasma gondii[J]. Cell Host Microbe, 2015, 18(4): 501-511.
[15]	Aicart-Ramos C, Valero RA, Rodtiguez-Crespo I.Protein palmitoylation and subcellular trafficking[J]. Biochim Biophys Acta, 2011, 1808(12): 2981-2994.
[16]	Bullen HE, Jia Y, Yamaryo-Botté Y, et al. Phosphatidic acid-mediated signaling regulates microneme secretion in Toxoplasma[J]. Cell Host Microbe, 2016, 19(3): 349-360.
[17]	Jia Y, Marq JB, Bisio H, et al. Crosstalk between PKA and PKG controls pH-dependent host cell egress of Toxoplasma gondii[J]. EMBO J, 2017, 36(21): 3250-3267.
[18]	Ubodi AD, Wilde ML, McRae E A, et al. Protein kinase A negatively regulates Ca2+ signalling in Toxoplasma gondii[J]. PLoS Biol, 2018, 16(9): e2005642.

引物 Primer	序列（5′→3′） Sequence（5′→3′）
P1	5′-ATGCGTGCACTTCCTACCTGGTTTTAGAGCT- AGAAATAGC-3′
P2	5′-AACTTGACATCCCCATTTAC-3′
P3	5′-CCCAGCCTGCCCGTGAGAGCGGGTCTGCGG- CCAGCATGGTGAGCAAGGGCGAGG-3′
P4	5′-TCGCGGCTAGGCGCCTCGGAGGCTCCGCTG- CATAGCAAAAGCTGGAGCTCCACC-3′
P5	5′-TCTGCAGCGAGATTTGCTTA-3′
P6	5′-CAGTTTCTTTATAATGGGGC-3′
P7	5′-GAACAAAGCACGGAGGAGAG-3′
P8	5′-AACGTAGCCAGCGATGTAGG-3′