AbstractCryptosporidiosis is a major cause of deadly diarrhoeal disease worldwide. The disease is caused by the obligate intracellular parasite, Cryptosporidium. In 2016 alone, 48,000 childhood deaths were attributed to Cryptosporidium. Transmission occurs by faecal-oral infection with the environmentally resilient oocyst stage. Despite the importance of the oocysts in transmission, our understanding of oocyst biogenesis is lacking. Therefore, I aimed to identify new markers of key events throughout oocyst biogenesis.
Existing datasets were used to identify candidate female genes and CRISPR/Cas9 was used to generate stable fluorescent reporter strains. The first protein characterised was fibrillin, an inner oocyst wall protein. In females, fibrillin localised in wall forming bodies (WFBs) distinct from those containing Cryptosporidium oocyst wall protein 1 (COWP1). Knockout (KO) of fibrillin confirmed the protein is not essential, but KO oocysts have a mild ‘wrinkly’ phenotype. Paradoxically, fibrillin KO oocysts conferred moderate tolerance to hydrogen peroxide treatment. Fibrillin is hypothesised to be a glycoprotein that can form disulphide cross-links and confer structural integrity to the inner oocyst wall.
The second protein characterised, amine oxidase 2 (AO2), is an ‘early’ female marker. AO2 is expressed before COWP1 and is the earliest marker of Cryptosporidium females based on current published literature. Expression is significantly decreased in oocysts and AO2 is not recommended as a marker of oocysts although it did localise to the inner oocyst wall. AO2 is hypothesised to be a cross-linking enzyme involved in wall formation.
The third protein characterised, control of complement protein 3 (CCP3) is expressed in females and throughout oocyst biogenesis. CCP3 localised to the crystalloid body in the posterior of sporozoites and to the residual body of oocysts. CCP3 is hypothesised to function during sporozoite division. Together these data provide new markers for oocyst biogenesis, giving better resolution of key events, and advance our knowledge of this important process in Cryptosporidium transmission.
|Date of Award||2023|
|Sponsors||Wellcome Trust, Royal Society & Carnegie Trust for the Universities of Scotland|
|Supervisor||Mattie Pawlowic (Supervisor) & Ian Gilbert (Supervisor)|
- Oocyst wall