Mitochondrial damage and IL-1β production in monocytes caused by Neospora caninum infection are mediated by dense granule protein 7 and prohibitins

Yu Chen¹Naomi Shimoda¹Coh-ichi NIHEI²Ryuichi Sawa²Yuko Takahashi²Mitsuhiro Nishigori³Shu Nakamura¹Takumi Koshiba³Nanako Ushio-Watanabe¹Yoshifumi Nishikawa^1*

• ¹National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan
• ²Institute of Microbial Chemistry (IMC), Tokyo, Japan
• ³Fukuoka University, Fukuoka, Fukuoka, Japan

The intracellular proliferation of Neospora caninum tachyzoites and the host immune response against infection are key steps in the pathogenesis of neosporosis. In this study, we first demonstrated that the NLRP3 inflammasome in monocytes can be partially activated by N. caninum dense granule protein 7 (NcGRA7) and can mediate IL-1β release. In the infection of human monocytic cell line THP-1 cells with the NcGRA7 knock-out (NcGRA7KO) parasite, decreased IL-1β release was observed compared with Nc1 infection. Transfection with plasmids for reconstitution of the NLRP3 inflammasome enhanced IL-1 production via additional transfection with NcGRA7 cDNA. However, NLRP3 inhibitor did not decrease efficiently IL-1β production from THP-1-infected with wild-type parasites compared with NcGRA7KO-infected cells. Additionally, TNF-α production of NcGRA7KO-infected THP-1 cells was significantly lower than that of the parental strain Nc1-infected cells. Western blot analysis of phosphorylated NF-κB p65 demonstrated that N. caninum infection induced NF-κB p65 phosphorylation; however, no significant difference was observed between Nc1-infected and NcGRA7KO-infected cells. These findings indicate that NcGRA7 does not directly modulate NF-κB activation but likely enhances the production of IL-1β and TNF-α through post-transcriptional mechanisms, thereby contributing to the upregulation of NLRP3. Infection of the THP-1 cells with NcGRA7KO parasites decreased the host mitochondrial damage and apoptosis in THP-1 cells compared to infection with Nc1, suggesting that NcGRA7 plays a crucial role in the pathogenesis of N. caninum. Furthermore, LC‒MS/MS analysis of NcGRA7 immunoprecipitates identified NcGRA7-binding proteins in host cells that localize to host mitochondria. Additionally, mitochondrial fractionation and proteolysis assays using proteinase K showed the distribution of NcGRA7 from the inner mitochondrial membrane to the matrix of host mitochondria. Interestingly, NcGRA7 formed a complex with the prohibitins PHB1 and PHB2. Using inhibitors of PHB1 or transfecting N. caninum-infected THP-1 cells with PHB1 siRNA significantly decreased IL-1β production, but not TNF-α. Our results suggest that NcGRA7 contributes to the NF-κB and inflammasome pathways through its interaction with host cell prohibitins. Understanding the role of PHB1 and NcGRA7 will provide new insights into the host-parasite interaction in pathogenesis.