Cryptosporidium secreted proteins form a complex layered interface with the host cell

Cryptosporidium parvum, one of the leading causes of diarrheal death in children, remodels its infection site through secreted effector proteins. Several of them accumulate at the host-parasite interface, forming a complex structure whose function and importance for infection remain poorly understood. Here, we localized and functionally characterized the putative dense granule protein DG8. We confirmed the protein to be in the dense granules, forming a ring structure once secreted at the host-parasite interface. Deletion of DG8 showed reduced infection in mice in vivo and early defect in vitro, revealing the importance of DG8 for parasite fitness. Additional secreted proteins were identified as potential partners, among which SG4, a small granule protein, was shown to partially co-localize with DG8. Applying ultrastructure expansion microscopy on intestinal sections with newly generated antibodies against DG8 and SG4, we could precisely position the ring structure above the electron-dense band, around the feeder organelle at the base of the parasitophorous vacuole membrane with an unprecedent resolution. Altogether, better visualization and understanding of the host-parasite interface through expanded intestinal tissue and effectors characterization reveal a complex and essential layered host-parasite interface.