{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,14]],"date-time":"2026-07-14T04:21:42Z","timestamp":1784002902593,"version":"3.55.0"},"reference-count":40,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2024,6,4]],"date-time":"2024-06-04T00:00:00Z","timestamp":1717459200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Natural Science Foundation of China","award":["32172879"],"award-info":[{"award-number":["32172879"]}]},{"name":"National Natural Science Foundation of China","award":["2022YFD1800200"],"award-info":[{"award-number":["2022YFD1800200"]}]},{"name":"National Natural Science Foundation of China","award":["2662020DKPY016"],"award-info":[{"award-number":["2662020DKPY016"]}]},{"name":"National Natural Science Foundation of China","award":["2262022DKYJ001"],"award-info":[{"award-number":["2262022DKYJ001"]}]},{"name":"National Key Research and Development Program of China","award":["32172879"],"award-info":[{"award-number":["32172879"]}]},{"name":"National Key Research and Development Program of China","award":["2022YFD1800200"],"award-info":[{"award-number":["2022YFD1800200"]}]},{"name":"National Key Research and Development Program of China","award":["2662020DKPY016"],"award-info":[{"award-number":["2662020DKPY016"]}]},{"name":"National Key Research and Development Program of China","award":["2262022DKYJ001"],"award-info":[{"award-number":["2262022DKYJ001"]}]},{"name":"Fundamental Research Funds for the Central Universities in China","award":["32172879"],"award-info":[{"award-number":["32172879"]}]},{"name":"Fundamental Research Funds for the Central Universities in China","award":["2022YFD1800200"],"award-info":[{"award-number":["2022YFD1800200"]}]},{"name":"Fundamental Research Funds for the Central Universities in China","award":["2662020DKPY016"],"award-info":[{"award-number":["2662020DKPY016"]}]},{"name":"Fundamental Research Funds for the Central Universities in China","award":["2262022DKYJ001"],"award-info":[{"award-number":["2262022DKYJ001"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>Babesia duncani (B. duncani), a protozoan parasite prevalent in North America, is a significant threat for human health. Given the regulatory role of pyruvate kinase I (PyK I) in glycolytic metabolism flux and ATP generation, PyK I has been considered the target for drug intervention for a long time. In this study, B. duncani PyK I (BdPyK I) was successfully cloned, expressed, and purified. Polyclonal antibodies were confirmed to recognize the native BdPyK I protein (56 kDa) using Western blotting. AlphaFold software predicted the three-dimensional structure of BdPyK I, and molecular docking with small molecules was conducted to identify potential binding sites of inhibitor on BdPyK I. Moreover, inhibitory effects of six inhibitors (tannic acid, apigenin, shikonin, PKM2 inhibitor, rosiglitazone, and pioglitazone) on BdPyK I were examined under the optimal enzymatic conditions of 3 mM PEP and 3 mM ADP, and significant activity reduction was found. Enzyme kinetics and growth inhibition assays further confirmed the reliability of these inhibitors, with PKM2 inhibitor, tannic acid, and apigenin exhibiting the highest selectivity index as specific inhibitors for B. duncani. Subsequently, key amino acid residues were mutated in both BdPyK I and Homo sapiens pyruvate kinase I (HPyK I), and two differential amino acid residues (isoleucine and phenylalanine) were identified between HPyK I and BdPyK I through PyK activity detection experiments. These findings lay foundation for understanding the role of PyK I in the growth and development of B. duncani, providing insights for babesiosis prevention and drug development.<\/jats:p>","DOI":"10.3390\/microorganisms12061141","type":"journal-article","created":{"date-parts":[[2024,6,5]],"date-time":"2024-06-05T08:43:54Z","timestamp":1717577034000},"page":"1141","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Babesia duncani Pyruvate Kinase Inhibitor Screening and Identification of Key Active Amino Acid Residues"],"prefix":"10.3390","volume":"12","author":[{"given":"Fangjie","family":"Li","sequence":"first","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pengfei","family":"Zhao","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sen","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wanxin","family":"Luo","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yingjun","family":"Xia","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8229-8258","authenticated-orcid":false,"given":"Dongfang","family":"Li","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lan","family":"He","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China"},{"name":"Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture of the People\u2019s Republic of China, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Junlong","family":"Zhao","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China"},{"name":"Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture of the People\u2019s Republic of China, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,6,4]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Wang, S., Li, D., Chen, F., Jiang, W., Luo, W., Zhu, G., Zhao, J., and He, L. (2022). Establishment of a Transient and Stable Transfection System for Babesia duncani Using a Homologous Recombination Strategy. Front. Cell Infect. Microbiol., 12.","DOI":"10.3389\/fcimb.2022.844498"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1663","DOI":"10.3389\/fphar.2019.01663","article-title":"Identifying the Naphthalene-Based Compound 3,5-Dihydroxy 2-Napthoic Acid as a Novel Lead Compound for Designing Lactate Dehydrogenase-Specific Antibabesial Drug","volume":"10","author":"Yu","year":"2019","journal-title":"Front. Pharmacol."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Drews, S.J., Kjemtrup, A.M., Krause, P.J., Lambert, G., Leiby, D.A., Lewin, A., O\u2019Brien, S.F., Renaud, C., Tonnetti, L., and Bloch, E.M. (2023). Transfusion-transmitted Babesia spp.: A changing landscape of epidemiology, regulation, and risk mitigation. J. Clin. Microbiol., 61.","DOI":"10.1128\/jcm.01268-22"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2084","DOI":"10.1128\/JCM.01334-06","article-title":"First case of human babesiosis in Korea: Detection and characterization of a novel type of Babesia sp. (KO1) similar to ovine babesia","volume":"45","author":"Kim","year":"2007","journal-title":"J. Clin. Microbiol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1323","DOI":"10.1016\/S0020-7519(00)00137-5","article-title":"Human babesiosis: An emerging tick-borne disease","volume":"30","author":"Kjemtrup","year":"2000","journal-title":"Int. J. Parasitol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"336","DOI":"10.1186\/s13071-016-1596-0","article-title":"A review of canine babesiosis: The European perspective","volume":"9","author":"Sainz","year":"2016","journal-title":"Parasites Vectors"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"284","DOI":"10.7326\/0003-4819-119-4-199308150-00006","article-title":"Babesiosis in Washington State: A new species of Babesia?","volume":"119","author":"Quick","year":"1993","journal-title":"Ann. Intern. Med."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1016\/j.ijpara.2018.07.002","article-title":"Evidence for transmission of the zoonotic apicomplexan parasite Babesia duncani by the tick Dermacentor albipictus","volume":"49","author":"Swei","year":"2019","journal-title":"Int. J. Parasitol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"450","DOI":"10.1128\/jcm.35.2.450-454.1997","article-title":"Human babesiosis in Taiwan: Asymptomatic infection with a Babesia microti-like organism in a Taiwanese woman","volume":"35","author":"Shih","year":"1997","journal-title":"J. Clin. Microbiol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"109539","DOI":"10.1016\/j.vetpar.2021.109539","article-title":"Animal reservoirs of zoonotic Babesia species: A global systematic review and meta-analysis of their prevalence, distribution and species diversity","volume":"298","author":"Karshima","year":"2021","journal-title":"Vet. Parasitol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"939","DOI":"10.3324\/haematol.12450","article-title":"Pyruvate kinase deficiency protects against malaria in humans","volume":"93","author":"Durand","year":"2008","journal-title":"Haematologica"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1805","DOI":"10.1056\/NEJMoa072464","article-title":"Pyruvate kinase deficiency and malaria","volume":"358","author":"Ayi","year":"2008","journal-title":"N. Engl. J. Med."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"775","DOI":"10.1111\/j.1365-2141.2010.08165.x","article-title":"Malaria: Looking for selection signatures in the human PKLR gene region","volume":"149","author":"Machado","year":"2010","journal-title":"Br. J. Haematol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Machado, P., Manco, L., Gomes, C., Mendes, C., Fernandes, N., Salom\u00e9, G., Sitoe, L., Chibute, S., Langa, J., and Ribeiro, L. (2012). Pyruvate kinase deficiency in sub-Saharan Africa: Identification of a highly frequent missense mutation (G829A;Glu277Lys) and association with malaria. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0047071"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Van Bruggen, R., Gualtieri, C., Iliescu, A., Louicharoen Cheepsunthorn, C., Mungkalasut, P., Trape, J.F., Modiano, D., Sirima, B.S., Singhasivanon, P., and Lathrop, M. (2015). Modulation of Malaria Phenotypes by Pyruvate Kinase (PKLR) Variants in a Thai Population. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0144555"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Bakszt, R., Wernimont, A., Allali-Hassani, A., Mok, M.W., Hills, T., Hui, R., and Pizarro, J.C. (2010). The crystal structure of Toxoplasma gondii pyruvate kinase 1. PLoS ONE, 5.","DOI":"10.1371\/journal.pone.0012736"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Xia, N., Ye, S., Liang, X., Chen, P., Zhou, Y., Fang, R., Zhao, J., Gupta, N., Yang, S., and Yuan, J. (2019). Pyruvate Homeostasis as a Determinant of Parasite Growth and Metabolic Plasticity in Toxoplasma gondii. mBio, 10.","DOI":"10.1128\/mBio.00898-19"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1894","DOI":"10.1073\/pnas.0914845107","article-title":"The alternative splicing repressors hnRNP A1\/A2 and PTB influence pyruvate kinase isoform expression and cell metabolism","volume":"107","author":"Clower","year":"2010","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1525","DOI":"10.1016\/j.cellsig.2015.02.032","article-title":"Pyruvate kinase M2 accelerates pro-inflammatory cytokine secretion and cell proliferation induced by lipopolysaccharide in colorectal cancer","volume":"27","author":"Yang","year":"2015","journal-title":"Cell Signal."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"22","DOI":"10.3389\/fonc.2018.00022","article-title":"Posttranslational Modifications of Pyruvate Kinase M2: Tweaks that Benefit Cancer","volume":"8","author":"Prakasam","year":"2018","journal-title":"Front. Oncol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"184","DOI":"10.1016\/j.canlet.2014.01.031","article-title":"PKM2 contributes to cancer metabolism","volume":"356","author":"Wong","year":"2015","journal-title":"Cancer Lett."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"An, X., Yu, L., Wang, S., Ao, Y., Zhan, X., Liu, Q., Zhao, Y., Li, M., Shu, X., and Li, F. (2021). Kinetic Characterization and Inhibitor Screening of Pyruvate Kinase I From Babesia microti. Front. Microbiol., 12.","DOI":"10.3389\/fmicb.2021.710678"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1007\/s11892-019-1270-y","article-title":"Thiazolidinediones: The Forgotten Diabetes Medications","volume":"19","author":"Lebovitz","year":"2019","journal-title":"Curr. Diab. Rep."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"9","DOI":"10.2147\/NDT.S121149","article-title":"Pioglitazone could induce remission in major depression: A meta-analysis","volume":"13","author":"Colle","year":"2017","journal-title":"Neuropsychiatr. Dis. Treat."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Alhaddad, A., Radwan, A., Mohamed, N.A., Mehanna, E.T., Mostafa, Y.M., El-Sayed, N.M., and Fattah, S.A. (2023). Rosiglitazone Mitigates Dexamethasone-Induced Depression in Mice via Modulating Brain Glucose Metabolism and AMPK\/mTOR Signaling Pathway. Biomedicines, 11.","DOI":"10.3390\/biomedicines11030860"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"343","DOI":"10.1016\/j.ejmech.2017.06.064","article-title":"Discovery of novel naphthoquinone derivatives as inhibitors of the tumor cell specific M2 isoform of pyruvate kinase","volume":"138","author":"Ning","year":"2017","journal-title":"Eur. J. Med. Chem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"680068","DOI":"10.3389\/fimmu.2021.680068","article-title":"Pyruvate Kinase M2 Contributes to TLR-Mediated Inflammation and Autoimmunity by Promoting Pyk2 Activation","volume":"12","author":"Zhang","year":"2021","journal-title":"Front. Immunol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"4297","DOI":"10.1038\/onc.2011.137","article-title":"Shikonin and its analogs inhibit cancer cell glycolysis by targeting tumor pyruvate kinase-M2","volume":"30","author":"Chen","year":"2011","journal-title":"Oncogene"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"5547","DOI":"10.1039\/C8FO01161C","article-title":"Tannic acid directly targets pyruvate kinase isoenzyme M2 to attenuate colon cancer cell proliferation","volume":"9","author":"Yang","year":"2018","journal-title":"Food Funct."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Jiang, W., Wang, S., Li, D., Zhang, Y., Luo, W., Zhao, J., and He, L. (2023). Continuous In Vitro Culture of Babesia duncani in a Serum-Free Medium. Cells, 12.","DOI":"10.3390\/cells12030482"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1114","DOI":"10.1038\/s41596-021-00676-1","article-title":"Accurate determination of protein:ligand standard binding free energies from molecular dynamics simulations","volume":"17","author":"Fu","year":"2022","journal-title":"Nat. Protoc."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"935","DOI":"10.1038\/nrd1549","article-title":"Docking and scoring in virtual screening for drug discovery: Methods and applications","volume":"3","author":"Kitchen","year":"2004","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"269","DOI":"10.1016\/0022-2836(82)90153-X","article-title":"A geometric approach to macromolecule-ligand interactions","volume":"161","author":"Kuntz","year":"1982","journal-title":"J. Mol. Biol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"9137","DOI":"10.1021\/bi990690n","article-title":"Role of lysine 240 in the mechanism of yeast pyruvate kinase catalysis","volume":"38","author":"Bollenbach","year":"1999","journal-title":"Biochemistry"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"8051","DOI":"10.1007\/s00432-022-04501-4","article-title":"Small molecule inhibitors for cancer metabolism: Promising prospects to be explored","volume":"149","author":"Liu","year":"2023","journal-title":"J. Cancer Res. Clin. Oncol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"126","DOI":"10.1080\/14756366.2017.1404591","article-title":"Synthesis and antitumor activity of novel 2,3-didithiocarbamate substituted naphthoquinones as inhibitors of pyruvate kinase M2 isoform","volume":"33","author":"Ning","year":"2018","journal-title":"J. Enzym. Inhib. Med. Chem."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"823","DOI":"10.1080\/09205063.2021.2021352","article-title":"Comparative study of poly tannic acid functionalized magnetic particles before and after modification for immobilized penicillin G acylase","volume":"33","author":"Tu","year":"2022","journal-title":"J. Biomater. Sci. Polym. Ed."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Xie, L., Liu, Y., Zhang, W., and Xu, S. (2021). A Dopamine\/Tannic-Acid-Based Co-Deposition Combined with Phytic Acid Modification to Enhance the Anti-Fouling Property of RO Membrane. Membranes, 11.","DOI":"10.3390\/membranes11050342"},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Gr\u00fcning, N.M., Du, D., Keller, M.A., Luisi, B.F., and Ralser, M. (2014). Inhibition of triosephosphate isomerase by phosphoenolpyruvate in the feedback-regulation of glycolysis. Open Biol., 4.","DOI":"10.1098\/rsob.130232"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1718","DOI":"10.1038\/s41467-021-21960-4","article-title":"Citrullination of pyruvate kinase M2 by PADI1 and PADI3 regulates glycolysis and cancer cell proliferation","volume":"12","author":"Coassolo","year":"2021","journal-title":"Nat. Commun."}],"container-title":["Microorganisms"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2607\/12\/6\/1141\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T14:53:19Z","timestamp":1760107999000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2607\/12\/6\/1141"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,6,4]]},"references-count":40,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2024,6]]}},"alternative-id":["microorganisms12061141"],"URL":"https:\/\/doi.org\/10.3390\/microorganisms12061141","relation":{},"ISSN":["2076-2607"],"issn-type":[{"value":"2076-2607","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,6,4]]}}}