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


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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  1. Arellano-Aguilar G, Marín-Santillán E, Castilla-Barajas JA, Bribiesca-Juárez MC, Domínguez-Carrillo LG (2017) A brief history of amoebic liver abscess with an illustrative case. Rev Gastroenterol Mex 82(4):344–348. https://doi.org/10.1016/j.rgmx.2016.05.007

  2. Arisue N, Sánchez LB, Weiss LM, Müller M, Hashimoto T (2002) Mitochondrial-type hsp70 genes of the amitochondriate protists, Giardia intestinalis, Entamoeba histolytica and two microsporidians. Parasitol Int 51(1):9–16. https://doi.org/10.1016/S1383-5769(01)00093-9

  3. Bimston D, Song J, Winchester D, Takayama S, Reed JC, Morimoto RI (1998) BAG-1, a negative regulator of Hsp70 chaperone activity, uncouples nucleotide hydrolisis from substrate release. EMBO J 17(23):6871–6878. https://doi.org/10.1093/emboj/17.23.6871

  4. Borges JC, Ramos CH (2006) Spectroscopic and thermodynamic measurements of nucleotide-induced changes in the human 70-kDa heat shock cognate protein. Arch Biochem Biophys 452(1):46–54. https://doi.org/10.1016/j.abb.2006.05.006

  5. Bukau B, Weissman J, Horwich A (2006) Molecular chaperones and protein quality control. Cell 125(3):443–451. https://doi.org/10.1016/j.cell.2006.04.014

  6. Chen VB, Arendall WB, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, Murray LW, Richardson JS, Richardson DC (2010) MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr 66(Pt 1):12–21. https://doi.org/10.1107/S0907444909042073

  7. Chojnacki S, Cowley A, Lee J, Foix A, Lopez R (2017) Programmatic access to bioinformatics tools from EMBL-EBI update:2017. Nucleic Acids Res 45(W1):W550–W553. https://doi.org/10.1093/nar/gkx273

  8. Daugaard M, Rohde M, Jäättelä M (2007) The heat shock protein 70 family: highly homologous proteins with overlapping and distinct functions. FEBS Lett 581(19):3702–3710. https://doi.org/10.1016/j.febslet.2007.05.039

  9. De Beer TA, Berka K, Thornton JM, Laskowski RA (2014) PDBsum additions. Nucleic Acids Res 42:D292–D296. https://doi.org/10.1093/nar/gkt940

  10. Debnath A, Akbar MA, Mazumder A, Kumar S, Das P (2005) Entamoeba histolytica: characterization of human collagen type I and Ca2+ activated differentially expressed genes. Exp Parasitol 110(3):214–219. https://doi.org/10.1016/j.exppara.2005.03.007

  11. DeLano WL (2002) The PyMOL molecular graphics system. DeLano Scientific, Palo Alto http://www.pymol.org.

  12. Diamond LS, Harlow DR, Cunnick CC (1978) A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba. Trans R Soc Trop Med Hyg 72(4):431–432. https://doi.org/10.1016/0035-9203(78)90144-X

  13. Dores-Silva PR, Barbosa LR, Ramos CH, Borges JC (2015) Human mitochondrial Hsp70 (mortalin): shedding light on ATPase activity, interaction with adenosine nucleotides, solution structure and domain organization. PLoS One 10(1):e0117170. https://doi.org/10.1371/journal.pone.0117170

  14. Enríquez-Flores S, Rodríguez-Romero A, Hernández-Alcántara G, Oria-Hernández J, Gutiérrez-Castrellón P, Pérez-Hernández G, de la Mora-de la Mora I, Castillo-Villanueva A, García-Torres I, Méndez ST, Gómez-Manzo S, Torres-Arroyo A, López-Velázquez G, Reyes-Vivas H (2011) Determining the molecular mechanism of inactivation by chemical modification of triosephosphate isomerase from the human parasite Giardia lamblia: a study for antiparasitic drug design. Proteins 79(9):2711–2724. https://doi.org/10.1002/prot.23100

  15. Ghazaei C (2017) Role and mechanism of the Hsp70 molecular chaperone machines in bacterial pathogens. J Med Microbiol 66(3):259–265. https://doi.org/10.1099/jmm.0.000429

  16. Gilchrist CA, Houpt E, Trapaidze N, Fei Z, Crasta O, Asgharpour A, Evans C, Martino-Catt S, Baba DJ, Stroup S, Hamano S, Ehrenkaufer G, Okada M, Singh U, Nozaki T, Mann BJ, Petri WA Jr (2006) Impact of intestinal colonization and invasion on the Entamoeba histolytica transcriptome. Mol Biochem Parasitol 147(2):163–176. https://doi.org/10.1016/j.molbiopara.2006.02.007

  17. Gómez-Manzo S, Marcial-Quino J, Vanoye-Carlo A, Enríquez-Flores S, de la Mora-de la Mora I, González-Valdez A, García-Torres I, Martínez-Rosas V, Sierra-Palacios E, Lazcano-Pérez F, Rodríguez-Bustamante E, Arreguin-Espinosa R (2015) Mutations of glucose-6-phosphate dehydrogenase Durham, Santa-Maria and A+ variants are associated with loss functional and structural stability of the protein. Int J Mol Sci 16(12):28657–28668. https://doi.org/10.3390/ijms161226124

  18. Katz S, Trebicz-Geffen M, Ankri S (2014) Stress granule formation in Entamoeba histolytica: cross-talk between EhMLBP, EhRLE3 reverse transcriptase and polyubiquitinated proteins. Cell Microbiol 16(8):1211–1223. https://doi.org/10.1111/cmi.12273

  19. Krieger E, Joo K, Lee J, Lee J, Raman S, Thompson J, Tyka M, Baker D, Karplus K (2009) Improving physical realism, stereochemistry, and side-chain accuracy in homology modeling: four approaches that performed well in CASP8. Proteins 77:114–122. https://doi.org/10.1002/prot.22570

  20. Liu FH, Wu SJ, Hu SM, Hsiao CD, Wang C (1999) Specific interaction of the 70-kDa heat shock cognate protein with the tetratricopeptide repeats. J Biol Chem 274(48):34425–34432. https://doi.org/10.1074/jbc.274.48.34425

  21. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193(1):265–275

  22. Maralikova B, Ali V, Nakada-Tsukui K, Nozaki T, van der Giezen M, Henze K, Tovar J (2010) Bacterial-type oxygen detoxification and iron-sulfur cluster assembly in amoebal relict mitochondria. Cell Microbiol 12(3):331–342. https://doi.org/10.1111/j.1462-5822.2009.01397.x

  23. Marie C, Petri WA Jr (2014) Regulation of virulence of Entamoeba histolytica. Annu Rev Microbiol 68:493–520. https://doi.org/10.1146/annurev-micro-091313-103550

  24. Matambo TS, Odunuga OO, Boshoff A, Blatch GL (2004) Overproduction, purification, and characterization of the Plasmodium falciparum heat shock protein 70. Protein Expr Purif 33(2):214–222. https://doi.org/10.1016/j.pep.2003.09.010

  25. Mayer MP, Bukau B (2005) Hsp70 chaperones: cellular functions and molecular mechanism. Cell Mol Life Sci 62(6):670–684. https://doi.org/10.1007/s00018-004-4464-6

  26. Mirelman D, Ankri S, Katz U, Padilla-Vaca F, Bracha R (2000) Pathogenesis of Entamoeba histolytica depends on the concerted action of numerous virulence factors. Arch Med Res 31(4 Suppl):S214–S215. https://doi.org/10.1016/S0188-4409(00)00234-4

  27. Nauck M, Wölfle D, Katz N, Jungermann K (1981) Modulation of the glucagon-dependent induction of phosphoenolpyruvate carboxykinase and tyrosine aminotransferase by arterial and venous oxygen concentrations in hepatocyte cultures. Eur J Biochem 119(3):657–661. https://doi.org/10.1111/j.1432-1033.1981.tb05658.x

  28. O’Brien MC, McKay DB (1998) Threonine 204 of the chaperone protein Hsc70 influences the structure of the active site, but is not essential for ATP hydrolysis. J Biol Chem 268(32):24323–24329

  29. Olivos-García A, Saavedra E, Nequiz M, Santos F, Luis-García ER, Gudiño M, Pérez-Tamayo R (2016) The oxygen reduction pathway and heat shock stress response are both required for Entamoeba histolytica pathogenicity. Curr Genet 62(2):295–300. https://doi.org/10.1007/s00294-015-0543-5

  30. Olson CL, Nadeau KC, Sullivan MA, Winquist AG, Donelson JE, Walsh CT, Engman DM (1994) Molecular and biochemical comparison of the 70-KDa heat shock proteins of Trypanosoma cruzi. J Biol Chem 269(5):3868–3874

  31. Ortner S, Plaimauer B, Binder M, Wiedermann G, Scheiner O, Duchêne M (1992) Humoral immune response against a 70-kilodalton heat shock protein of Entamoeba histolytica in a group of patients with invasive amoebiasis. Mol Biochem Parasitol 54(2):175–183. https://doi.org/10.1016/0166-6851(92)90110-6

  32. Roy A, Kucukural A, Zhang Y (2010) I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc 5(4):725–738. https://doi.org/10.1038/nprot.2010.5

  33. Sánchez R, Saralegui A, Olivos-García A, Scapolla C, Damonte G, Sanchez-Lopez R, Alagón A, Stock RP (2005) Entamoeba histolytica: intracellular distribution of the sec61alpha subunit of the secretory pathway and down-regulation by antisense peptide nucleic acids. Exp Parasitol 109(4):241–251. https://doi.org/10.1016/j.exppara.2004.12.011

  34. Santi-Rocca J, Smith S, Weber C, Pineda E, Hon CC, Saavedra E, Olivos-García A, Rousseau S, Dillies MA, Coppée JY, Guillén N (2012) Endoplasmic reticulum stress-sensing mechanism is activated in Entamoeba histolytica upon treatment with nitric oxide. PLoS One 7(2):e31777. https://doi.org/10.1371/journal.pone.0031777

  35. Santos F, Nequiz M, Hernández-Cuevas NA, Hernández K, Pineda E, Encalada R, Guillén N, Luis-García E, Saralegui A, Saavedra E, Pérez Tamayo R, Olivos-García A (2015) Maintenance of intracellular hypoxia and adequate heat shock response are essential requirements for pathogenicity and virulence of Entamoeba histolytica. Cell Microbiol 17(7):1037–1051. https://doi.org/10.1111/cmi.12419

  36. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH image to ImageJ: 25 years of image analysis. Nat Methods 9(7):671–675

  37. Teixeira JE, Huston CD (2008) Evidence of a continuous endoplasmic reticulum in the protozoan parasite Entamoeba histolytica. Eukaryot Cell 7(7):1222–1226. https://doi.org/10.1128/EC.00007-08

  38. Tovar J, Cox SS, van der Giezen M (2007) A mitosome purification protocol based on percoll density gradients and its use in validating the mitosomal nature of Entamoeba histolytica mitochondrial Hsp70. Methods Mol Biol 390:167–177

  39. Tovy A, Hertz R, Siman-Tov R, Syan S, Faust D, Guillen N, Ankri S (2011) Glucose starvation boosts Entamoeba histolytica virulence. PLoS Negl Trop Dis 5(8):e1247. https://doi.org/10.1371/journal.pntd.0001247

  40. Vicente JB, Ehrenkaufer GM, Saraiva LM, Teixeira M, Singh U (2009) Entamoeba histolytica modulates a complex repertoire of novel genes in response to oxidative and nitrosative stresses: implications for amebic pathogenesis. Cell Microbiol 11(1):51–69. https://doi.org/10.1111/j.1462-5822.2008.01236.x

  41. Vickery LE, Cupp-Vickery JR (2007) Molecular chaperones HscA/Ssq1 and HscB/Jac1 and their roles in iron-sulfur protein maturation. Crit Rev Biochem Mol Biol 42(2):95–111. https://doi.org/10.1080/10409230701322298

  42. Weber C, Guigon G, Bouchier C, Frangeul L, Moreira S, Sismeiro O, Gouyette C, Mirelman D, Coppee JY, Guillén N (2006) Stress by heat shock induces massive down regulation of genes and allows differential allelic expression of the Gal/GalNAc lectin in Entamoeba histolytica. Eukaryot Cell 5(5):871–875. https://doi.org/10.1128/EC.5.5.871-875.2006

  43. Weber C, Koutero M, Dillies MA, Varet H, Lopez-Camarillo C, Coppée JY, Hon CC, Guillén N (2016) Extensive transcriptome analysis correlates the plasticity of Entamoeba histolytica pathogenesis to rapid phenotype changes depending on the environment. Sci Rep 6:35852. https://doi.org/10.1038/srep35852

  44. WHO/PAHO/UNESCO report. A consultation with experts on amoebiasis. Mexico City, Mexico 28–29 1997. Epidemiol Bull 18(1): 13–14

  45. Yang J, Zhang Y (2015) I-TASSER server: new development for protein structure and function predictions. Nucleic Acids Res 43(W1):W174–W181. https://doi.org/10.1093/nar/gkv342

  46. Zhai P, Stanworth C, Liu S, Silberg JJ (2008) The human escort protein hep binds to the ATPase domain of mitochondrial hsp70 and regulates ATP hydrolysis. J Biol Chem 283(38):26098–26106. https://doi.org/10.1074/jbc.M803475200

  47. Zhang C, Freddolino PL, Zhang Y (2017) COFACTOR: improved protein function prediction by combining structure, sequence and protein-protein interaction information. Nucleic Acids Res 45(W1):W291–W299. https://doi.org/10.1093/nar/gkx366

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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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  • HSP70
  • Entamoeba histolytica
  • Stress response
  • Recombinant protein