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Novel Strategy for In Vitro Validation of Babesia orientalis Heat Shock Proteins Chaperone Activity and Thermostability

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Abstract

Background

Babesia orientalis is an intra-erythrocytic protozoan parasite that causes babesiosis in water buffalo. The genome of B. orientalis has been reported and various genes have been accurately annotated, including heat shock proteins (HSP). Three B. orientalis HSPs (HSP90, HSP70 and HSP20) have been previously identified as potential antigenic targets. Here, a new validation strategy for the chaperone activities and cell protection characteristics of the three HSPs was developed in vitro.

Methods

BoHSP20, BoHSP70 and BoHSP90B were amplified from cDNA, followed by cloning them into the pEGFP-N1 vector and transfecting the vector plasmid separately into 293T and Hela mammalian cells. Their expression and localization were determined by fluorescence microscopy. The biological functions and protein stability were testified through an analysis of the fluorescence intensity duration. Their role in the protection of cell viability from heat-shock treatments was examined by MTT assay (cell proliferation assay based on thiazolyl blue tetrazolium bromide).

Results

Fusion proteins pEGFP-N1-BoHSP20, pEGFP-N1-BoHSP70, and pEGFP-N1-BoHSP90B (pBoHSPs: pBoHSP20; pBoHSP70 and pBoHSP90B) were identified as 47 kDa/97 kDa/118 kDa with a 27 kDa GFP tag, respectively. Prolonged fluorescent protein half-time was observed specifically in pBoHSPs under heat shock treatment at 55 °C, and BoHSP20 showed relatively better thermotolerance than BoHSP70 and BoHSP90B. Significant difference was found between pBoHSPs and controls in the cell survival curve after 2 h of 45 °C heat shock.

Conclusion

Significant biological properties of heat stress-associated genes of B. orientalis were identified in eukaryote by a new strategy. Fusion proteins pBoHSP20, pBoHSP70 and pBoHSP90B showed good chaperone activity and thermo-stability in this study, implying that BoHSPs played a key role in protecting B. orientalis against heat-stress environment during parasite life cycle. In conclusion, the in vitro model explored in this study provides a new way to investigate the biological functions of B. orientalis proteins during the host–parasite interaction.

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Data Availability

The datasets generated during the current study are available from the corresponding author upon reasonable request.

Abbreviations

HSPs:

Heat shock proteins

BoHSPs:

Babesia orientalis Heat shock proteins

BoHSP20:

Babesia orientalis Heat shock protein 20

BoHSP70:

Babesia orientalis Heat shock protein 70

BoHSP90B:

Babesia orientalis Heat shock protein 90 B

pBoHSPs:

Babesia orientalis Heat shock proteins eukaryotically expressing on pEGFP-N1 vector

pBoHSP20:

Babesia orientalis Heat shock protein 20 eukaryotically expressing on pEGFP-N1 vector

pBoHSP70:

Babesia orientalis Heat shock protein 70 eukaryotically expressing on pEGFP-N1 vector

pBoHSP90B:

Babesia orientalis Heat shock protein 90B eukaryotically expressing on pEGFP-N1 vector

ACD:

Alpha-crystallin domain

NBD:

Nucleotide-binding domain

MTT:

Thiazolyl blue tetrazolium bromide

s:

Seconds

m:

Minutes

h:

Hours

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Acknowledgements

This study was supported by the National Key Research and Development Program of China (2022YFD1800200), the National Natural Science Foundation of China (31772729), the Natural Science Foundation of Hubei Province (2017CFA020), and the Natural Science Foundation of Guangdong Province (2021A1515010485). This study was supported by the National Key Research and Development Program of China (2022YFD1800200), the National Natural Science Foundation of China (Grant No. 31930108), the Natural Science Foundation of Guangdong Province (2021A1515010485), Top-notch Young Talent Supporting Program (to L. He), and the Fundamental Research Funds for the Central Universities (2262022DKYJ001).

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Authors and Affiliations

  1. State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, Hubei, People’s Republic of China

    Jinfang Hu, Lizhe Fan, Yuan Huang, Pei He, Lan He & Junlong Zhao

  2. Key Laboratory for Development of Veterinary Diagnostic Products, Ministry of Agriculture, Huazhong Agricultural University, Wuhan, 430070, People’s Republic of China

    Lizhe Fan, Yuan Huang, Pei He, Lan He & Junlong Zhao

  3. Guangdong Province Key Laboratory of Laboratory Animals, Guangdong Laboratory Animals Monitoring Institute, Guangzhou, 510633, Guangdong, China

    Jinfang Hu

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Contributions

JH wrote the draft of the manuscript. JZ and LH revised the manuscript. LF, YH, and PH had work in Parasitology lab, Huazhong Agricultural University, China. They contribute the basic need for B. orientalis heat shock protein research. All authors read and approved the final manuscript.

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Correspondence to Junlong Zhao.

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Hu, J., Fan, L., Huang, Y. et al. Novel Strategy for In Vitro Validation of Babesia orientalis Heat Shock Proteins Chaperone Activity and Thermostability. Acta Parasit. 69, 591–598 (2024). https://doi.org/10.1007/s11686-023-00775-x

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