Functional Analysis Of The TatD-like DNase Of Plasmodium Parasites And Its Correlation With The Pathogenesis

Innate immune responses provide the first lines of defense against microbial infection. However, much remains unknown about how the Plasmodium parasite counteracts the innate immune response after infection.Proteins of the Tat D deoxyribonuclease family participate in many different biological processes, such as protein export and pathogenesis. All plasmodial Tat D sequences contained a conserved motif with the 21 amino acid sequences characteristic of the Tat D family, which indicates an analogous activity as a deoxyribonuclease.The transcription of Pf Tat D began early after parasite invasion into the erythrocytes, reaching maximum transcription level at 32 h. The expression of the Pf Tat D protein also followed the same trend, as shown in a Western blot with a specific anti-Pf Tat D antibody. Indirect immunofluorescence and immunoelectron microscopy assays revealed that Pf Tat D was primarily localized to the periphery of infected erythrocytes in the mature trophozoite stage.To determine whether the expression of the plasmodial Tat D-like DNase is associated with pathogenicity or the ability of parasite proliferation in the host, we compared the expression of homologous proteins in several rodent parasite strains with distinct lethality in mice. The expression of the Tat D-like DNase was positively correlated with parasite virulence.To further confirm that the Tat D-like DNase is an essential factor of parasite virulence, the Tat D-like DNase-encoding gene from P.berghei ANKA was disrupted by double crossover. The capability of the ΔPb Tat D strain to proliferate in vivo was significantly diminished compared with that of the wild-type strain. This result further supports our supposition that the Tat D-like DNase is an essential factor for the proliferation and consequent pathogenicity of the parasite. Interestingly, when co-cultured with J774 A.1 macrophages, the ΔPb Tat D strain-infected RBCs induced the formation of a network of extracellular DNA from the macrophages that was much more than that of the WT parasite/J774 A.1 macrophages co-culture. Thus, it is likely that the Tat D-like DNase facilitates the counteraction of the malarial parasites against host innate immunity, as in bacterial pathogen. We further demonstrated the DNA hydrolase activity of Pf Tat D by incubating the recombinant protein r Pf Tat D-GST with human DNA. r Pf Tat D-GST was able to hydrolyze double-stranded DNA in a concentration-dependent manner.We further investigated the significance of Tat D-specific antibodies in the protection of the host against parasite infection in vivo. As expected, a significant reduction in parasitemia and delayed death was observed in the immunized group after IP injection with i RBCs. Our data indicate that the P. falciparum Tat D-like DNase may be a promising candidate for malarial vaccine development or a therapeutic target.