Characterization of Host Protein Prohibitin-2 Interacting With Cryptosporidium parvum Cyclophilin 23

Cryptosporidium parvum is a prevalent protozoan pathogen responsible for moderate to severe diarrhea in humans and animals, posing a significant threat to global public health. Host-parasite interactions play a crucial role in the infection process of C. parvum. Cyclophilin 23 (CpCyP23), a secreted protein of C. parvum, is implicated in parasite development and pathogenicity. As a secretory protein, its function often relies on interactions with host cell proteins. However, it remains unclear whether CpCyP23 has host interaction partners and what specific roles such interactions play during infection. In this study, using CpCyP23 as bait, we screened for potential interacting proteins in HCT-8 cells through pull-down assays combined with mass spectrometry (MS) analysis. The interaction was subsequently confirmed by co-immunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assays. Immunofluorescence (IF) was employed to determine subcellular localization. Furthermore, after confirming the altered expression of the interacting protein during parasite infection, we analyzed its impact on parasite infection using small interfering RNA (siRNA) knockdown and overexpression techniques, coupled with parasite infection assays. The results identified the host cell protein Prohibitin-2 (PHB2) as an interacting partner of CpCyP23. Co-IP and BiFC assays validated their interaction, and IF revealed their colocalization in the cytoplasm of HCT-8 cells. The mRNA level of PHB2 was significantly upregulated at 3 and 12 h postinfection (hpi) with C. parvum. Functional experiments further demonstrated that the expression level of PHB2 positively correlates with the infection burden of C. parvum. In summary, this study reveals an interaction between the host cell protein PHB2 and CpCyP23 and identifies PHB2 expression level as a key host factor significantly influencing C. parvum infection. These findings provide experimental evidence and a potential target for elucidating the pathogenic mechanisms of C. parvum and developing novel anti-infection strategies.