# Small granule protein CP2 of Cryptosporidium translocates to the parasite-host interface during invasion and localizes to parasitophorous vacuole membrane in association with other secretory proteins

## TL;DR

This study reveals how a protein called CP2 in the parasite Cryptosporidium is secreted during infection and contributes to the formation of a protective compartment inside host cells.

## Contribution

The study identifies CP2 as a secreted protein involved in the formation of the parasitophorous vacuole and reveals compensatory mechanisms when CP2 is absent.

## Key findings

- CP2 is stored in small granules and translocated to the parasite-host interface during invasion.
- CP2 localizes to the parasitophorous vacuole membrane and is concentrated near the attachment zone in mature stages.
- Deleting CP2 leads to increased expression of SG3, which also localizes to the PVM and forms external structures.

## Abstract

Cryptosporidium is a major cause of diarrhea in humans and farm animals for which there are no effective drugs. The parasite resides in a parasitophorous vacuole on the surface of gastrointestinal epithelial cells. Although the parasitophorous vacuole membrane (PVM) plays a critical role in parasite development and survival, its composition and biogenesis are not well understood. In this study, we used reverse genetic tools to investigate the secretion and function of a potential PVM protein called CP2. Endogenous gene tagging revealed that, following biosynthesis, CP2 is stored in the small granules (SG) within sporozoites. Upon invasion of host cells, CP2 is translocated to the parasite-host interface. During intracellular development, CP2 is distributed across the entire PVM in trophozoites and microgamonts, but it is concentrated in the lower PVM, near the attachment zone, in mature meronts and macrogametes. Interestingly, CP2 is dispensable; deleting the CP2 gene did not significantly affect the growth or pathogenicity of a virulent Cryptosporidium strain. CP2 knockout resulted in increased expression of a neighboring gene encoding SG3, which is also translocated from the SG to the PVM. SG3 was further localized to knob-like and filamentous structures outside the PVM. Together, these findings suggest that CP2 is secreted to the parasite-host cell interface during invasion and intracellular growth, where it potentially contributes to the formation of the nascent parasitophorous vacuole of Cryptosporidium, together with other SG proteins.

The enteric parasite Cryptosporidium survives inside host cells by forming a protective compartment known as the parasitophorous vacuole. This structure is essential for establishing an infection, yet the process by which it forms remains unknown. We discovered that CP2, a well-known Cryptosporidium glycoprotein, is secreted from the small granules (SG) to the parasite-host interface during invasion, where it becomes is part of the nascent parasitophorous vacuole. Interestingly, the parasite can survive withoutCP2, possibly by increasing the production of another SG protein, SG3. This finding reveals an unexpected flexibility in the parasite’s infection strategy. We also identified two SG3-positive structures that may potentially facilitate interaction between parasites and host cells. These findings shed light on the mechanisms of Cryptosporidium infection and highlight novel aspects of its biology.