Identification and characterization of DNA endonucleases in Plasmodium falciparum 3D7 clone

Abstract

Background: Plasmodium falciparum is the most virulent parasite of the five Plasmodium species that cause human malaria, and biological analysis of the parasite is critical for the development of novel strategies for disease control. DNA endonucleases are important for maintaining the biological activity, gene stability of the parasite and interaction with host immune systems. In this study, ten sequences of DNA endonucleases were found in the genome of P. falciparum 3D7 clone, seven of them were predicted to contain an endonuclease/exonuclease/phosphatase (IPR005135) domain which plays an important role in DNA catalytic activity. The seven DNA endonucleases of P. falciparum were systematically investigated.

Methods: Plasmodium falciparum 3D7 clone was cultured in human O⁺ RBCs, RNA was extracted at 8, 16, 24, 32, 40, and 48 h post invasion and real-time quantitative PCR was carried out to analyse the transcription of the seven DNA endonuclease genes in asexual stages. Immunofluorescence assay was carried out to confirm the location of the encoded proteins expressed in the erythrocytic stages. Finally, the catalytic activity of the DNA nucleases were tested.

Results: Of the seven proteins analysed, two proteins were not soluble. Fragments derived from the rest five endonuclease sequences were successfully expressed as soluble proteins, and which were used to generate antisera for protein localization. The proteins were all located in the nucleus at ring and trophozoite stages. While at schizont stage, proteins encoded by PF3D7_1238600, PF3D7_0107200 and PF3D7_0319200 were in the punctuated forms in the parasite most likely around nuclei of the merozoites. But the proteins encoded by PF3D7_0305600 and PF3D7_1363500 were distributed around the infected erythrocyte membrane. The enzymatic activity of the recombinant GST-PF3D7_1238600 was very efficient without divalent iron, while the activity of the rest four enzymes was iron dependent. Further, divalent irons did not show any specific enhancement on the activity of GST-PF3D7_1238600, but the activity of GST-PF3D7_0107200, GST-PF3D7_1363500 and GST-PF3D7_0319200 were Cu²⁺ dependent. The activity of GST-PF3D7_0305600 was dependent on Mg²⁺ and Mn²⁺. Except GST-PF3D7_1363500, four of the GST tagged recombinant proteins hydrolysed the supercoiled circular plasmid DNA with or without divalent metal ions. The GST-PF3D7_1363500 protein only changed the supercoiled circular plasmid DNA into nicked plasmids, even with Cu²⁺.

Conclusions: Fragments derived from five of the endonuclease sequences of P. falciparum 3D7 clone were successfully expressed. The proteins displayed diverse cell distribution, biochemical and enzymatic activities, which indicated that they carried different biological function in the development of the parasite in the erythrocytes. The DNA repair and DNA degradation capacity of the DNA endonucleases in the biology of the parasite remained further studied.

Keywords: Catalysis; DNA endonuclease; Malaria; Plasmodium falciparum.

Fig. 1

Schematic map of EEP domain within seven DNA endonucleases of P. falciparum 3D7 clone. Grey indicates full length of proteins, and red indicates the EEP domain analysed with InterPro

Fig. 2

Transcription of the seven DNA endonucleases genes of the P. falciparum 3D7 clone. The transcriptions of the seven DNA endonuclease genes at 8, 16, 24, 32, 40 and 48 h post invasion are shown. Transcript levels relative to that of gene PF3D7_0305600 at 16 h post invasion were calculated as 2^−∆∆Ct. A log-scale was calculated and used on the y-axis

Fig. 3

Western blot analysis of the native DNA endonucleases in the P. falciparum 3D7 clone. Lane 1 represents infected erythrocytes, and lane 2 represents uninfected erythrocytes. A single band was detected in infected erythrocytes. Protein specific IgG was used as a primary antibody. Alkaline phosphatase conjugated goat anti-rabbit IgG was used as a secondary antibody

Fig. 4

Localization of the five DNA endonucleases in the P. falciparum 3D7 clone by immunofluorescence assays. Immunofluorescence assay (IFA) was performed with protein-specific IgG as the primary antibody. Alexa Fluor 488-conjugated goat anti-rabbit IgG was used as a secondary antibody. Hoechst 33342 stained the nuclei blue. a Rabbit anti-PF3D7_1238600 IgG was used as a primary antibody. b Rabbit anti-PF3D7_0107200 IgG was used as a primary antibody. c Rabbit anti-PF3D7_0305600 IgG was used as a primary antibody. d Rabbit anti-PF3D7_1363500 IgG was used as a primary antibody. e Rabbit anti-PF3D7_0319200 IgG was used as a primary antibody

Fig. 5

DNA hydrolytic test of the five GST-tagged recombinant proteins without divalent ions. Lane 1 is blank with only genomic DNA incubated at 37 °C for 60 min. Lane 2 is the negative control with genomic DNA and GST protein incubated at 37 °C for 60 min. Lanes 3–8 are genomic DNA and GST-tagged recombinant proteins incubating at 37 °C for 5, 10, 15, 30, 45 and 60 min. a GST-PF3D7_1238600 (0.1 mg/ml); b GST-PF3D7_0107200 (0.1 mg/ml). c. GST-PF3D7_1363500 (0.07 mg/ml); d GST-PF3D7_0319200 (0.1 mg/ml); e GST-PF3D7_0305600—(0.2 mg/ml)

Fig. 6

DNA hydrolytic test of the five GST-tagged recombinant proteins with different divalent metals. Divalent metal ions at different concentrations were added into the reaction and incubated for 30 min. The lane with no protein is the blank control with only genomic DNA incubated for 30 min. a No specific effect of divalent metal ions on the activity of GST-PF3D7_1238600 was observed. b The activity of GST-PF3D7_0107200 was dependent on Cu²⁺ with an optimal concentration of 2 mM. c The activity of GST-PF3D7_0305600 was dependent on Mn²⁺ and Mg²⁺. d The activity of GST-PF3D7_1363500 was dependent on Cu²⁺ with an optimal concentration of 2 mM. e The activity of GST-PF3D7_0319200 was dependent on Cu²⁺ with an optimal concentration of 10 mM

Fig. 7

The catalytic effect of the five GST-tagged recombinant proteins on linear DNA and supercoiled circular plasmid DNA. Linear genomic DNA and supercoiled plasmid were used as substrates in a DNA digestion assay. a GST-PF3D7_1238600 digestion of linear genomic DNA and supercoiled circular plasmid with or without Mn²⁺ and Mg²⁺. b GST-PF3D7_0305600 digestion of linear genomic DNA and supercoiled circular plasmid with Mn²⁺ or Mg²⁺. c GST-PF3D7_0107200 digestion of linear genomic DNA and supercoiled circular plasmid with or without Cu²⁺. d GST-PF3D7_1363500 digestion linear genomic DNA with Cu²⁺. e GST-PF3D7_0319200 digestion of linear genomic DNA and supercoiled circular plasmid with Cu²⁺