Cellular Identification and In Silico Characterization of Protein Phosphatase 2C (PP2C) of Cryptosporidium parvum

Abstract

Purpose

Cryptosporidium parvum is an Apicomplexa parasite that causes watery diarrhea (cryptosporidiosis), especially in children and immunocompromised adults (the latter in a very severe form). No effective treatment exists against infection by this parasite. Phosphatases participate in the regulation of various cellular functions and are thus considered potential therapeutic targets in many diseases. The aim of the present study was to indirectly identify and in silico characterize a protein phosphatase 2C of C. parvum.

Methods

Western blot and indirect immunofluorescence microscopy were performed with a polyclonal antibody against Leishmania major PP2C. Possible cross-reactivity with LmPP2C was assessed by in silico sequence homology to analyze phylogenetic relationships between distinct C. parvum PP2Cs. In addition, another bioinformatics approach was used to predict the possible relationship and function of C. parvum PP2C in the regulation of several cellular processes.

Results

Western blotting showed a protein of approximately 72 kDa. With immunofluorescence, PP2C was localized in the nucleus of oocysts (with some additional labeling in the cytoplasm) and at the apical region of sporozoites. By aligning C. parvum PP2C with known ortholog sequences and carrying out PPI analysis, a determination could be made of the degree of conservation of these enzymes, their possible relationship, and their function in the regulation of several cellular processes associated with a likely nuclear location.

Conclusion

Microscopic localization by immunofluorescence identified CpPP2C at the nucleus in oocysts and at the apical end of the sporozoite body. Hence, this enzyme could be associated with proteins that have an important role in the regulation of transcription and other processes orchestrated by MAPK kinases, according to in silico analysis.

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Acknowledgements

This work is one of the requirements for Jenny Nancy Gómez Sandoval to obtain a PhD degree in Posgrado en Ciencias Biológicas (UNAM). She deeply appreciates the doctoral fellowship from CONACyT, Mexico (fellowship 203485, CVU 205974). The authors gratefully acknowledge technical assistance provided by Marco Gudiño Zayas from the Unidad de Investigación en Medicina Experimental, Facultad de Medicina, UNAM. We are thankful for technical support given by Mónica Mondragón from the Biochemistry Department and Electron Microscopy Facility-LANSE at CINVESTAV, Mexico.

Funding

This project was partially supported by Grants 152433 and 284018 from SEP-Consejo Nacional de Ciencia y Tecnología (SEP-CONACyT, Mexico) and Grant IN218619 from DGAPA-PAPIIT given to MMAG.

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Contributions

MMA-G designed the study and revised the manuscript. PO reviewed the final manuscript. RM-F performed the microscopic analysis. ARE-M carried out Western blot assay. JNG-S performed the in-silico analysis and wrote the first draft of the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to M. Magdalena Aguirre-García.

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Gómez-Sandoval, J.N., Okhuysen, P., Mondragón-Flores, R. et al. Cellular Identification and In Silico Characterization of Protein Phosphatase 2C (PP2C) of Cryptosporidium parvum. Acta Parasit. 65, 704–715 (2020). https://doi.org/10.2478/s11686-020-00209-y

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Keywords

  • Cryptosporidiosis
  • Cryptosporidium parvum
  • Protein phosphatase 2C
  • Diarrhea
  • Sequence alignment
  • PPI analysis