The TatD-like DNase of Plasmodium is a virulence factor and a potential malaria vaccine candidate

Abstract

Neutrophil extracellular traps (NETs), composed primarily of DNA and proteases, are released from activated neutrophils and contribute to the innate immune response by capturing pathogens. Plasmodium falciparum, the causative agent of severe malaria, thrives in its host by counteracting immune elimination. Here, we report the discovery of a novel virulence factor of P. falciparum, a TatD-like DNase (PfTatD) that is expressed primarily in the asexual blood stage and is likely utilized by the parasite to counteract NETs. PfTatD exhibits typical deoxyribonuclease activity, and its expression is higher in virulent parasites than in avirulent parasites. A P. berghei TatD-knockout parasite displays reduced pathogenicity in mice. Mice immunized with recombinant TatD exhibit increased immunity against lethal challenge. Our results suggest that the TatD-like DNase is an essential factor for the survival of malarial parasites in the host and is a potential malaria vaccine candidate.

Figure 1. Sequence analysis and the predicted structure of PfTatD.

(a) The TatD sequence alignment across various strains of Plasmodium and E. coli. The plasmodial TatD sequences contain a 24–26 AA signal peptide, whose cleavage site was between residues 26 and 27 in the P. falciparum or between 24 and 25 in other genera (blue box). A gap appeared in the predicted TatD DNase domain (yellow box) following a potent cell-binding residue (red box). The AAs that constitute the active site are indicated by red stars along the sequence. (b) The PfTatD 3D structure model was built using SWISS-MODEL. A template that contained 9 α-helices (green bands) and 8 β-pleated sheets (purple bands) in the secondary structure constituted a stable region. The yellow band indicates the gap in the TatD DNase domain. The red cylindrical bonds (red stars) indicate the conserved active site presented in a. (c) The schematic structural composition of the PfTatD domains. A 23-AA signal peptide site at the N terminus, followed by a 73-AA sequence gap (yellow rounded rectangle) that interrupted the TatD DNase domain, and AA residues from 224 to 362 indicated by the cerulean rounded rectangle.

Figure 2. PfTatD is expressed at the mature stage of trophozoite-infected erythrocytes.

(a) Transcriptional analysis of the PF3D7_0112000 gene was performed using quantitative PCR. Transcription was increased with parasite growth and reached its maximum at 32 h in the blood stage. The results are shown as the mean ±s.e.m. of 3 independent experiments. (b) An immunoblot was performed with rabbit anti-rPfTatD IgG (also see Supplementary Fig. 13). The top line indicates the expression of PfTatD every 8 h and the increased expression at 32 h in the blood stage. HSP70, presented in the bottom panel, was used as a control. (c) Indirect immunofluorescence of PfTatD was detected. The green fluorescence indicates that PfTatD was localized to the RBC membrane or the parasitophorous vacuole membrane of a mature trophozoite and was distinct from the nucleus in the schizont. The parasite nuclei were stained with DAPI. Scale bar, 5 μm. (d–f) Immune electron microscopy images of PfTatD. The gold particles were localized to the parasitophorous vacuole membrane, and there was a trend for localization to the periphery. Scale bar, (d) 500 nm and (e,f) 200 nm.

Figure 3. TatD-like DNase expression is associated with parasite virulence.

Quantitative PCR and western blot assays were performed to measure mRNA and protein levels, respectively, of the TatD-like DNase in rodent malarial parasites. In the histograms presented in a,b parasite pathogenicity increased from the left to right, which positively correlated with transcription. Error bars represent the mean ±s.e.m. of three independent experiments. The expression patterns presented in c,d also positively correlated with the transcription results. The top panel indicates the TatD expression in different strains, which were collected at the same stage of the parasites. GAPDH is presented in the bottom panel and was used as a control. The full images of the western blot assays are shown in Supplementary Fig. 13.

Figure 4. A comparison of the parasitaemia and survival of mice infected with WT and mutated strains.

(a) A comparison of the mean parasitaemia with standard variations of the mice that were infected with WT P. berghei, ΔPbTatD, PbTatD-D352A and PbTatD-com parasites from day 1–20 after infection. The parasitaemia in mice that were infected with either ΔPbTatD (red line) or PbTatD-D352A (green line) was lower than in mice infected with WT (black line) or PbTatD-com parasites (blue line). The error bars indicate s.d. (b) The curves representing the survival rates of the mice that were infected with WT, ΔPbTatD, PbTatD-D352A and PbTatD-com strains. The mice that were infected with the WT exhibited 100% death 11 days after IP injection with 10⁶ infected RBCs. The PbTatD-com strain exhibited a similar result as WT, whereas the mice that were infected with the ΔPbTatD and PbTatD-D352A strains exhibited a twofold longer survival time.

Figure 5. Formation of ETs after incubation of infected erythrocytes with macrophages.

Representative images of the extracellular network structures after incubation of J774A.1 macrophages with WT P. berghei (a) and ΔPbTatD (b) infected erythrocytes. The ETs and dead cell nuclei were stained with Sytox green. Scale bar, 20 μm. (c) The relative quantification of ETs released by J774A.1 macrophages stimulated with the WT strain (white bar), ΔPbTatD strain (blue bar), PbTatD-com strain (yellow bar), PbTatD-D352A strain (green bar) or RBCs (red bar) are shown. The WT group was regarded as 100%. The results are the averages of three independent experiments (mean ±s.e.m., *P<0.05 in two-tailed Student's t-test). (d,e) Images of ETs after the addition of rPbTatD-GST or rPbTatD-D352A-GST in the cultivation at various time points. rPbTatD-GST showed DNase activity in a time-dependent manner (d), whereas rPbTatD-D352A-GST did not show any activity (e).

Figure 6. Immunization with the TatD-like DNase generates protective immunity against rodent malaria.

(a) rPbTatD-HIS and rPcTatD-HIS were purified by affinity chromatography using His GraviTrap. The SDS–PAGE and western blotting assays revealed that the recombinant proteins were >90% pure. (b) BALB/c mice immunized with Freund's adjuvant alone or without any immunization (Naive) exhibited 3.12-fold higher parasitaemia than the rPbTatD-immunized group on day 11 post infection; the error bars indicate the s.d. (c) Mice in the rPbTatD-immunized group survived 10 days longer than the two control groups. (d,e) BALB/c mice immunized with Freund's adjuvant alone and naive mice exhibited 4.66-fold higher parasitaemia than the rPcTatD-immunized group on day 8 post challenge; the error bars indicate the s.d. All of the mice that received Freund's adjuvant alone and the naive group died on day 9 post infection; however, eight of the mice in the rPbTatD-immunized group were completely protected. (f,g) The group that received serum from a previously infected mouse exhibited 3.97-fold higher parasitaemia than the group that received anti-rPbTatD serum on day 10 post infection. Compared with the other groups, the group that received anti-rPbTatD serum survived for 5 days longer; the error bars indicate the s.d. (h,i) The group that received serum from a previously infected mouse exhibited 1.79-fold higher parasitaemia than the group that received rPcTatD-specific serum on day 10 post infection. The mice that received rPcTatD-specific serum showed a 100% survival rate. The error bars indicate the s.d.