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Functional characterization and subcellular distribution of two recombinant cytosolic HSP70 isoforms from Entamoeba histolytica under normal and stress conditions

Abstract

Amoebiasis is a human intestinal disease caused by the parasite Entamoeba histolytica. It has been previously demonstrated that E. histolytica heat shock protein 70 (EhHSP70) plays an important role in amoebic pathogenicity by protecting the parasite from the dangerous effects of oxidative and nitrosative stresses. Despite its relevance, this protein has not yet been characterized. In this study, the EhHSP70 genes were cloned, and the two recombinant EhHSP70 proteins were expressed, purifying and biochemically characterized. Additionally, after being subjected to some host stressors, the intracellular distribution of the proteins in the parasite was documented. Two amoebic HSP70 isoforms, EhHSP70-A and EhHSP70-B, with 637 and 656 amino acids, respectively, were identified. Kinetic parameters of ATP hydrolysis showed low rates, which were in accordance with those of the HSP70 family members. Circular dichroism analysis showed differences in their secondary structures but similarities in their thermal stability. Immunocytochemistry in trophozoites detected EhHSP70 in the nuclei and cytoplasm as well as a slight overexpression when the parasites were subjected to oxidants and heat. The structural differences of amoebic HSP70s with their human counterparts may be used to design specific inhibitors to treat human amoebiasis.

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Acknowledgements

This work is part of the doctoral dissertation of F.S. This research was supported by the DGAPA grant IN-214617 and the CONACyT-Mexico grant 247430. We acknowledge Mr. Marco Gudiño for image processing and Andrés Saralegui, MSc and Jaime Arturo Pimentel, PhD for acquiring confocal images and performing the densitometry analysis.

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Correspondence to Alfonso Olivos-García.

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This research does not contain any studies with human participants. All experiments involving animals were performed in strict accordance with the Mexican Law for the Production, Care and Use of Laboratory Animals (NOM-062-ZOO-1999). All animal procedures were carried out under protocol number 091–2016, which was approved by the Institutional Animal Care and Use Committees of the Facultad de Medicina, Universidad Nacional Autónoma de México. All efforts were made to minimize animal suffering.

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Santos, F., Marcial-Quino, J., Gómez-Manzo, S. et al. Functional characterization and subcellular distribution of two recombinant cytosolic HSP70 isoforms from Entamoeba histolytica under normal and stress conditions. Parasitol Res (2020). https://doi.org/10.1007/s00436-020-06621-7

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Keywords

  • HSP70
  • Entamoeba histolytica
  • Stress response
  • Recombinant protein