Toxoplasma calcium-dependent protein kinases 3 mediates M1 macrophage polarization by targeting host Arginase-1.

BACKGROUND: Toxoplasma gondii, an obligate intracellular parasite, has developed sophisticated ways to manipulate host immunity, resulting in long-lasting infection and causing serious public health problems in humans and animals. T. gondii type II is the type most frequently associated with human diseases, but the mechanism remains unclear. Toxoplasma calcium-dependent protein kinase 3(CDPK3), a protein located on the T. gondii parasite periphery, is highly expressed in type II strains. Although TgCDPK3 regulates parasite egress from host cells, calcium-based infiltration, and development of tissue cysts, the host target proteins that it modulates are still unclear. METHODS: Firstly, mass spectrometry was used to analyze proteins that selectively bind to TgCDPK3. Subsequently, GST (glutathione-s-transferase) pull-down, immunoprecipitation, and immunofluorescence assay were used to confirm the interaction and colocalization between TgCDPK3 and Arginase-1. Western blotting and Argininaseactivity assays were performed to detect the relative levels of endogenous Arginase-1 and inducible nitric oxide synthase (iNOS) in a murine microglial cell line. Fluorescence activated cell sorting (FACS) assays and enzyme-linked immunosorbent assay (ELISA) analysis were performed to confirm the association of interaction between TgCDPK3 and Arginase-1 within an M1/M2-polarized macrophage. Intracellular multiplication assays and plaque assays were performed to test whether the interaction between TgCDPK3 and Arginase-1 affected intercellular parasite growth. RESULTS: The interaction between TgCDPK3 and Arginase-1 is functionally correlated and leads to a reduction in Arginase-1 activity, ultimately, contributing to the M1-biased phenotype of the host macrophages, which is related to restraining the proliferation of parasites. CONCLUSIONS: Our data showed that CDPK3 mediates M1 macrophage polarization by targeting host Arginase-1, which is beneficial to understanding the mechanism for long term latency establishment of less virulent strains of Toxoplasma.