Expression and functional analysis of the TatD-like DNase of Plasmodium knowlesi

Yapan Zhou,Na Yang,Qijun Chen,Lubin Jiang,Xiaoyu Sang,Ning Jiang,Ying Feng,Bo Xiao

doi:10.1186/s13071-018-3251-4

Yapan Zhou, Na Yang + Show 6 more

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https://doi.org/10.1186/s13071-018-3251-4

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Abstract

BackgroundIn recent years, human infection by the simian malaria parasite Plasmodium knowlesi has increased in Southeast Asia, leading to growing concerns regarding the cross-species spread of the parasite. Consequently, a deeper understanding of the biology of P. knowlesi is necessary in order to develop tools for control of the emerging disease. TatD-like DNase expressed at the surface of P. falciparum has recently been shown to counteract host innate immunity and is thus a potential malaria vaccine candidate.MethodsThe expression of the TatD DNase of P. knowlesi (PkTatD) was confirmed by both Western-blot and immunofluorescent assay. The DNA catalytic function of the PkTatD was confirmed by digestion of DNA with the recombinant PkTatD protein in the presence of various irons.ResultsIn the present study, we investigated the expression of the homologous DNase in P. knowlesi. The expression of TatD-like DNase in P. knowslesi (PkTatD) was verified by Western blot and indirect immunofluorescence assays. Like that of the P. falciparum parasite, PkTatD was also found to be located on the surface of erythrocytes infected by the parasites. Biochemical analysis indicated that PkTatD can hydrolyze DNA and this activity is magnesium-dependent.ConclusionsWe identified that PkTatD expressed on the surface of P. knowlesi-infected RBCs is a Mg2+-dependent DNase and exhibits a stronger hydrolytic capacity than TatD from P. falciparum. The data support our previous findings that TatD-like DNase is a unanimously expressed virulence factor of Plasmodium parasites.

Highlights

In recent years, human infection by the simian malaria parasite Plasmodium knowlesi has increased in Southeast Asia, leading to growing concerns regarding the cross-species spread of the parasite
The simian malaria parasite Plasmodium knowlesi is the pathogen of a neglected tropical disease
Until 2004, a proportion of natural P. knowlesi infections acquired by human patients in Sarawak, Malaysia, were likely misdiagnosed as being caused by the morphologically similar parasite P. malariae [3, 4]

Summary

Methods

Bioinformatic analysis of P. knowlesi TatD-like DNase The putative TatD sequence alignments of P. knowlesi, P. berghei, P. falciparum, P. ovale, P. vivax, P. cynomolgi, P. inui, P. reichenowi, P. yoelii, P. chabaudi, P. malariae and E. coli were retrieved from the PlasmoDB database (www.plasmodb.org) and signatures of the sequences were bioinformatically analyzed using DNAMAN 7 (Lynnon Corporation, San Ramon, USA). B DNA was completely hydrolyzed at 35 °C and the enzyme remained active at 41 °C. c PkTatD-GST recombinant protein and DNA were incubated with divalent metal ions at concentrations from 0 to 10 mM. D Mg2+ (10 mM) and DNA were incubated with recombinant PkTatD-GST protein at concentrations from 0 to 5 μM by synthesis and cloned into the pGEX4T-1 and pET28a vectors. The DNA was incubated with GST-tagged recombinant PkTatD protein at a concentration of 0.1, 0.5, 1.5, 3, 4 or 5 μM in PBS buffer (pH 7.4) and a total volume of 20 μl at 37 °C. Abbreviations BCIP/NBT: 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium chloride; IPTG: isopropyl-β-D-thiogalactoside; MCM: malaria culture medium; PBS: phosphate-buffered saline; PfTatD: Plasmodium falciparum TatD-like DNase; PkTatD: Plasmodium knowlesi TatD-like DNase; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis

Results

Background

Results and discussion

Conclusions