Biological Characterization of Alternative Oxidase in <i>Cryptosporidium</i> Mitosome

Cryptosporidium parvum is an important enteric parasite that causes diarrhea in humans and animals. There are currently no effective drugs to treat cryptosporidiosis. C. parvum undergoes reduced mitochondrial energy metabolism and has a mitochondrial remnant called the mitosome. Comparative genomic analyses have revealed the presence of an alternative respiratory pathway in the C. parvum mitosome and identified the alternative oxidase (CpAOX) as its functional component. Therefore, CpAOX has long been considered a potential drug target for the treatment of cryptosporidiosis. In this study, we used genetic manipulation tools to investigate the localization and biological significance of CpAOX. Endogenous gene tagging revealed that CpAOX is localized in the oval or calabash-shaped mitosome adjacent to the parasite nucleus and is expressed in most life stages of C. parvum except male gamonts. Deletion of the CpAOX gene had no significant effect on parasite growth either in vitro or in vivo, but reduced the pathogenicity of C. parvum in interferon-γ knockout mice, resulting in milder clinical signs, attenuated intestinal damage, and prolonged survival of infected mice. The results of this study suggest that CpAOX is not essential for parasite growth and survival, but is likely to be involved in C. parvum fitness and pathogenesis.