Plasmodium falciparum specific helicase 2 is a dual, bipolar helicase and is crucial for parasite growth

Abstract

Human malaria infection is a major challenge across the globe and is responsible for millions of deaths annually. Rapidly emerging drug resistant strains against the new class of anti-malarial drugs are major threat to control the disease burden worldwide. Helicases are present in every organism and have important role in various nucleic acid metabolic processes. Previously we have reported the presence of three parasite specific helicases (PSH) in Plasmodium falciparum 3D7 strain. Here we present the detailed biochemical characterization of PfPSH2. PfPSH2 is DNA and RNA stimulated ATPase and is able to unwind partially duplex DNA and RNA substrates. It can translocate in both 3' to 5' and 5' to 3' directions. PfPSH2 is expressed in all the stages of intraerythrocytic development and it is localized in cytoplasm in P. falciparum 3D7 strain. The dsRNA mediated inhibition study suggests that PfPSH2 is important for the growth and survival of the parasite. This study presents the detailed characterization of PfPSH2 and lays the foundation for future development of PfPSH2 as drug target.

Figure 1

Multiple Sequence Alignment of the amino acid sequence of the PfPSH2 with orthologs present in apicomplexans Toxoplasma gondii and Neospora caninum. The alignment was done using clustal omega program (www.ebi.ac.uk/Tools/msa/clustalo/). For motif representation, all signature motifs of PfPSH2 are highlighted and boxed in purple color and the name of each motif (from I to VI) is written in roman numerals.

Figure 2

(A) Schematic representation of signature motifs of DEAD box family of PfPSH2. All the domains are presented with their respective amino acid positions; (B) SDS PAGE analysis. Commassie blue stained gel. Lane M is molecular weight marker and lanes 1 and 2 are purified PfPSH2 and PfPSH2M proteins (~105 kDa); (C) Western Blot analysis. Lane numbers are similar to B.

Figure 3

ATPase activity analysis of PfPSH2 and PfPSH2M. (A) ATPase activity in the presence of DNA with increasing concentration (25 nM to 300 nM) of PfPSH2 protein (lanes 1–5); the experiment was repeated at least three times; (B) ATPase activity in the presence of RNA with increasing concentration (25 nM to 300 nM) of PfPSH2 protein (lanes 1–5), the experiment was repeated at least three times; (C) Graphical representation of quantitative data of Fig. 3A,B, the triangle and circle data points depict % ATP hydrolysis in the presence of DNA or RNA, respectively. (D) Time dependent ATPase activity of PfPSH2 (lanes 1–6) in the presence of DNA; the experiment was repeated at least three times (E) Time dependent ATPase activity of PfPSH2 (lanes 1–6) in the presence of RNA; experiment was repeated at least three times; (F) Graphical representation of quantitative data of Fig. 3D,E, data points with triangle and circle depict % ATP hydrolysis in the presence of DNA or RNA at different time intervals; (G) ATPase assay with increasing protein concentration (25 nM to 400 nM) of PfPSH2 and PfPSH2M in the presence of DNA (H) ATPase assay with increasing protein concentration (25 nM to 400 nM) of PfPSH2 and PfPSH2M in the presence of RNA. Lane C in panels A, B, D, E, G and H represents control reaction without protein.

Figure 4

DNA Helicase activity assay of PfPSH2. (A) Lanes 1–7, reactions with increasing concentration (5 nM to 200 nM) of PfPSH2 protein; the experiment was repeated at least three times; (B) Graphical representation of quantitative data of Fig. 4A. (C) Lanes 1–5, reactions with increasing concentration (25 nM to 300 nM) of PfPSH2M protein; the experiment was repeated at least two times; (D) Lanes 1–5, show time dependent helicase activity with fixed concentration (80 nM) of PfPSH2 protein; the experiment was repeated at least two times; (E) Graphical representation of quantitative data of Fig. 4D. (F) Lanes 1–7, helicase activity of PfPSH2 at different concentrations of ATP (0.25 M to 5 M); the experiment was repeated at least two times. (G) Graphical representation of quantitative data of Fig. 4F. (H) Helicase activity of PfPSH2 in the presence of different nucleotides/deoxynucleotides triphosphates such as dGTP, GTP, dCTP, CTP, dATP, ATP, dTTP and UTP (lanes 1–8); the experiment was repeated at least two times; (I) Graphical representation of quantitative data of Fig. 4H. In A, C, D, F and H, lane C is control reaction without protein and lane B is boiled substrate.

Figure 5

Direction specific helicase activity assay. (A) Helicase activity of PfPSH2 with the 5′ to 3′ direction- specific substrate. Lanes 1–4 are reactions with 80 nM of PfPSH2 and lanes 5–8 are reactions with 160 nM of PfPSH2, respectively at various time points. The experiment was repeated at least two times; (B) Graphical representation of quantitative data of Fig. 5A, data points with circles and triangles depict % unwinding with 80 nM and 160 nM of PfPSH2, respectively. (C) Helicase activity of PfPSH2 with the 3′ to 5′ direction specific substrate. Lanes 1–4 are reactions with 80 nM of PfPSH2 and lanes 5–8 are reactions with 160 nM of PfPSH2, respectively at various time points. The experiment was repeated at least two times; (D) Graphical representation of quantitative data of Fig. 5C, data points with circles and triangles depict % unwinding of 3′-5′ direction substrate with 80 nM and 160 nM of PfPSH2, respectively. In A and C, lane C is control reaction without protein and lane B is boiled substrate.

Figure 6

RNA helicase activity assay. (A) RNA helicase activity of PfPSH2. Lanes 1–4 are reactions with 80 nM and lanes 5–8 are reactions with 160 nM of PfPSH2, respectively at various time points. The experiment was repeated at least two times; (B) Graphical representation of quantitative data of Fig. 6A, data points with circles and triangles depict % unwinding of RNA substrate with 80 nM and 160 nM of PfPSH2, respectively. (C) RNA helicase activity of PfPSH2M. Lanes 1–4 are reactions with 80 nM and lanes 5–8 are reactions with 160 nM of PfPSH2M, respectively at various time points. The experiment was repeated at least two times. In A and C, lane C represents control reaction without protein and lane B is boiled substrate.

Figure 7

Localization of PSH2 in different intra-erythrocytic stages of P. falciparum. The cells were fixed and stained with pre-immune sera or anti-PfPSH2 antisera and anti-PfPABP antisera followed by Alexa fluor 488 and Alexa fluor 594-conjugated secondary antibodies and then counterstained with DAPI. In each panel, single confocal image of each stage is shown. (A) Staining with pre-immune sera (i) phase contrast (TD) image; (ii) image of cell stained with DAPI (blue); (iii) pre-immune sera PSH2; (iv) PSH2 + DAPI (v) All merged; (B–H) Staining with anti-PSH2 and anti-PABP sera (B) merozoite stage, (C) ring stage, (D) trophozoite stage, (E) schizont stage, (F) schizont stage (i) phase contrast (TD) image; (ii) image of cell stained with DAPI (blue); (iii) immunofluorescent stained cell (PSH2); (iv) immunofluorescent stained cell (PABP); (v) Merged image of panel ii, iii and iv; (vi) Merged image of panel i-v (vii) Pearson’s correlation coefficients of merged images are written. (G) Western blot analysis using lysate of mixed stage P. falciparum 3D7 strain culture. Lane M is prestained protein molecular weight marker and lane 1 is parasite lysate immunoprecipitated with anti-PfPSH2N antiserum and lane 2 is parasite lysate immunoprecipitated with pre-immune serum.

Figure 8

Effect of dsRNA on parasite growth. (A–C) Giemsa-stained parasite infected RBCs at 24 hours (A) before dsRNA treatment (Control); (B) After GFP-dsRNA treatment; (C) After PfPSH2 dsRNA treatment; (D–F) Immunofluorescence microscopy with PfPSH2 anti-sera; (D) untreated parasite (E) GFP dsRNA treated parasite (F) PfPSH2 dsRNA treated parasite; In D-F (i) TD and merge image; (ii) image of cell stained with DAPI (blue); (iii) immunofluorescent stained cell (PSH2); (iv) image of cell stained with DAPI + PSH2; (G) Growth inhibition of parasite calculated by manual microscopic counting of Giemsa stained slides (H) Real-time PCR analysis results of GFP dsRNA treated samples and PfPSH2 dsRNA treated samples. The experiments were repeated at least three times.