Poster Presentation 15th Lorne Infection and Immunity 2025

Identification and characterisation of Cryptosporidium effector proteins in host-pathogen interaction (#317)

Lena Chng 1 2 , Simona Seizova 1 2 , Kharizta Wiradiputri 1 2 , Waail Abdalla 1 2 , Abdul Alammar 1 2 , Alex Uboldi 1 2 , Eva Hesping 1 2 , Justin Boddey 1 2 , Chris Tonkin 1 2
  1. Department of Medical Biology , The University of Melbourne, Melbourne, Victoria, Australia
  2. Walter and Eliza Hall Institute, Parkville, VIC, Australia

The apicomplexan parasite Cryptosporidium causes the severe diarrheal disease, cryptosporidiosis, which is a leading cause of childhood mortality globally. Chronic diarrhoea leads to malnutrition and wasting in children under five, affecting physical and cognitive development1. The lack of diagnostic tools, effective treatment and vaccines for cryptosporidiosis exacerbates the global impact of this neglected pathogen on vulnerable populations. Cryptosporidium infects host enterocytes and like its cousins, Plasmodium and Toxoplasma, secretes proteins into its host cell to establish infection and supress immunity2. These so-called ‘effector’ proteins of both T. gondii and P. falciparum are proteolytically processed by aspartic acid proteases which is essential for protein trafficking and function3,4. In recent years, these proteases have been shown to be excellent drug targets, some of which have progressed to human clinical trials5. However, little is known about Cryptosporidium aspartic acid proteases and their involvement in effector trafficking and cleavage.

In this study, we used TurboID and APEX- expressing host cell lines to identify effector proteins of Cryptosporidium. Using newly developed CRISPR-Cas12 technology and mouse models, we have generated several transgenic HA-tagged parasites and show that several of these effectors localise at the parasite interface with the host enterocyte. Further, we implicate an aspartyl protease, CpASP4, in proteolytic maturation of these effectors. With the use of a newly identified CpASP4 inhibitor compound, we investigate how this protease controls processing and maturation of substrates and show that this process is essential for parasite survival in vitro and in vivo. This study greatly expands our understanding of the importance of proteolytic maturation in Cryptosporidium host-pathogen interactions, and highlights a new therapeutic pathway for the development of an efficacious anti-cryptosporidial agent.

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