# Calcium-dependent protein kinases 2A involved in the growth of both asexual and sexual stages of Cryptosporidium parvum

## TL;DR

This study shows that a specific protein kinase in Cryptosporidium parvum is important for the parasite's growth and could be a target for new treatments.

## Contribution

The study identifies CpCDPK2A as a key factor in both asexual and sexual stages of Cryptosporidium parvum and demonstrates its potential as a drug target.

## Key findings

- CpCDPK2A is expressed in all intracellular developmental stages of C. parvum.
- Deleting CpCDPK2A significantly reduces in vitro growth of late asexual and sexual stages.
- Gene deletion reduces oocyst shedding and increases survival in infected mice.

## Abstract

Cryptosporidium parvum is a protozoan pathogen that causes moderate to severe diarrhea in both humans and animals. Calcium-dependent protein kinases (CDPKs) are attractive drug targets against cryptosporidiosis given their critical role in the life cycle of Cryptosporidium spp. and their absence in human and animal hosts.

We used CRISPR-Cas9 technology to endogenously tag the CpCDPK2A gene in C. parvum IIdA20G1-HLJ strain with the hemagglutinin (HA) epitope and to delete the CpCDPK2A gene. An immunofluorescence assay was performed to localize the CpCDPK2A expression in the tagged strain and a luciferase assay was performed to compare growth rates of the tagged and deletion strains in vitro. Oocyst shedding, parasite load, villus length/crypt height ratio and survival of infected mice were used to evaluate the function of CpCDPK2A in vivo. The results revealed that CpCDPK2A was expressed in all the intracellular developmental stages, especially in the motile stages of sporozoites and merozoites. While CpCDPK2A is dispensable, deletion of the gene significantly reduced the growth of late asexual and sexual stages in vitro. In an interferon-γ knockout mouse model, gene deletion of CpCDPK2A reduced oocyst shedding by 25-fold and increased survival of infected mice.

These observations suggest that CpCDPK2A may contribute to both asexual and sexual replication of C. parvum and may be a potential target to block the transmission of this important zoonotic pathogen.

Cryptosporidiosis is a major cause of diarrhea in children and young animals, particularly in low- and middle-income countries. Despite its impact, effective drugs or vaccines are lacking. Calcium-dependent protein kinases (CDPKs) in Cryptosporidium parvum are promising therapeutic targets due to their unique structures. Whole genome sequencing and comparative genomic analyses have identified at least eight CpCDPKs in C. parvum. Our study focuses on CpCDPK2A, which is expressed in all intracellular stages and plays a crucial role in both asexual and sexual replication. Deletion of CpCDPK2A significantly reduces infection intensity and pathogenicity without affecting host cell invasion. These findings suggest that CpCDPK2A may be a viable target for the development of prevention strategies against cryptosporidiosis. Further research is needed to explore its vaccine potential and action mechanism.