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

Plasmodium falciparum is the most virulent parasite of all five Plasmodium species that cause human malaria, an estimated 3.3 billion people are at risk of malaria, and 1.2 billion are at high risk [1]. The main pathophysiological symptoms of malaria are caused by repeated merozoite invasion into RBCs and exponential parasite proliferation in the blood stage.

DNA endonucleases are a type of enzymes that hydrolyse internal phosphodiester bonds, which exist in DNA strands. DNase I is a DNA-specific enzyme that was discovered in the cells of spleen, liver and digestive tracts of mammalian hosts [2]. Some pathogens successfully survive from the killing of the host cells by the expression of DNases which can degrade the neutrophil extracellular traps (NETs) [3‐5]. While NETs are mainly composed of DNA and proteases which released from neutrophils and contributed to the innate immune response by capturing pathogens [6, 7]. Further, it was reported that hosts infected with Plasmodium malariae, was accompanied by increased DNase and RNase activities in the sera [8]. During the necrocytosis, DNase I and the plasma fibrinolysis system concentrate at the nucleus of the dead cell and degrade chromosomal DNA, which prevents the appearance of anti-nucleus antibodies [9]. DNase II is a type of acid endonuclease that is independent of divalent metal ions. In mouse fetal development, a deficiency of DNase II leads to the accumulation of large DNA-containing bodies that were resulted from engulfed, but undigested cell corpses in tissues, such as thymus, kidney, spleen, and liver, which could result in dyserythropoietic anaemia and death of the fetus [10]. Deficiency of DNase II in adult mice results in chronic polyarthritis [11]. Apoptotic DNA leads to cell cycle arrest of fibroblasts and epithelial cells. Degraded apoptotic DNA by DNase II activated p53 and p21 pathways, which protected normal cells from apoptotic DNA [12].

The function of DNases is mostly determined by endonuclease/exonuclease/phosphatase (EEP) domain [13‐20]. EEP hydrolyses the phosphodiester bond in nucleic acids, proteins and phospholipids. The EEP domain exists in a large number of enzymes, including AP endonuclease, DNase I, inositol-polyphosphate 5-phosphatase and sphingomyelinase, and these enzymes participate in DNA metabolic processes and intracellular signalling [14, 15].

Plasmodium falciparum contains a 23 Mb nuclear genome encoding 5400 genes on 14 linear chromosomes [21], a 35 Kb apicoplast genome [22] and a 6 Kb mitochondrion genome [23]. Over 50% of the genes’ encoded proteins have not been well studied [21, 24, 25]. Here, proteins with EEP domains that may encompass DNA hydrolytic ability of P. falciparum 3D7 clone were identified and characterized. This study combined a bioinformatics assessment, protein localization and DNA catalytic activity tests. The data generated will facilitate a better understanding of the biology of P. falciparum.

The function of a protein is closely related to its captured domains. Proteins with the same function share similar domains. In this study, a common domain, EEP domain with activity of hydrolysis of phosphodiester bonds in nucleic acids, proteins and phospholipids was identified in 7 protein sequences of DNases in P. falciparum. The EEP domain exists in a large number of enzymes, including AP endonuclease, DNase I, inositol-polyphosphate 5-phosphatase and sphingomyelinase, and these enzymes participate in DNA metabolism processes and intracellular signalling [14, 15].

The DNase I-like superfamily is a member of SCOP 1.75, which groups protein structural domains hierarchically into class, fold, superfamily and family. This superfamily contains three families: DNase I-like, inositol polyphosphate 5-phosphatase and sphingomyelin phosphodiesterase-like. Except the protein PF3D7_1238600, which belongs to the sphingomyelin phosphodiesterase-like family, six of the identified proteins belong to the DNase I-like family. Proteins PF3D7_0305600 and PF3D7_1430600 were AP endonuclease 1 family members in InterPro analysis, and they specifically create a nick at the AP site in the DNA base excision repair pathway. In eukaryotes, there is only one AP endonuclease. However, in E. coli, endonuclease IV and exonuclease III are the AP endonucleases [34].

In transcriptional analysis, the lowest transcription level relative to the internal control gene was used for normalization; the fold changes of the gene PF3D7_0305600 relative to the control at 16 h post invasion was set as one. The transcription levels of the genes PF3D7_1238600 and PF3D7_1363500 were respectively a thousand times and a hundred times higher than that of PF3D7_0305600, and the results were consistent with that obtained by microarray assays recorded in PlasmoDB. Peak transcript levels may represent the main stages of activity of the encoded proteins. All seven genes reached their peak transcription at the late trophozoite and early schizont stages, which was further confirmed by Western blot assays (Fig. 3).

The distribution of the proteins inside the infected erythrocytes were mainly in two patterns. 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 cytoplasm around nuclei of the merozoites (Fig. 4a, b, e). But the proteins encoded by PF3D7_0305600 and PF3D7_1363500 were distributed around the infected erythrocyte membrane (Fig. 4c, d). The phylogenetic analysis indicated that the genes were grouped in separated clusters implying that they perform different function in the development of the parasite.

The DNA catalytic activity of five proteins containing the EEP domain was investigated, and all of the proteins displayed DNA hydrolytic activity with different dependency in divalent irons (Figs. 5, 6 and 7). Thus the proteins with EEP domains encoded by the genes identified in the P. falciparum genome indeed could catalyse DNA in a similar manner as observed in other organisms. However, the function of these enzymes in the biology of the parasite remained further investigation.