Genetic ablation of purine salvage in Cryptosporidium parvum reveals nucleotide uptake from the host cell

Significance Nucleotides are the activated building blocks for DNA and RNA as well as the major form of energy in all living organisms. Cells need to synthesize nucleotides to grow. Interference with this synthesis is thus widely used to treat infections and cancers. Here we discover through genetic experimentation that the parasite Cryptosporidium surprisingly does not require purine nucleotide synthesis. This may reflect the presence of a second novel purine salvage pathway. Alternatively, we propose that the parasite has evolved to import purine nucleotides, making purine salvage dispensable. Nucleotide import may also allow Cryptosporidium to steal energy from host cells. This finding has far-reaching consequences for the development of treatments for this important cause of diarrheal disease.
The apicomplexan parasite Cryptosporidium is a leading global cause of severe diarrheal disease and an important contributor to early-childhood mortality. Waterborne outbreaks occur frequently, even in countries with advanced water treatment capabilities, and there is currently no fully effective treatment. Nucleotide pathways are attractive targets for antimicrobial development, and several laboratories are designing inhibitors of these enzymes as potential treatment for Cryptosporidium infections. Here we take advantage of newly available molecular genetics for Cryptosporidium parvum to investigate nucleotide biosynthesis by directed gene ablation. Surprisingly, we found that the parasite tolerates the loss of classical targets including dihydrofolate reductase-thymidylate synthase (DHFR-TS) and inosine monophosphate dehydrogenase (IMPDH). We show that thymidine kinase provides a route to thymidine monophosphate in the absence of DHFR-TS. In contrast, only a single pathway has been identified for C. parvum purine nucleotide salvage. Nonetheless, multiple enzymes in the purine pathway, as well as the adenosine transporter, can be ablated. The resulting mutants are viable under normal conditions but are hypersensitive to inhibition of purine nucleotide synthesis in their host cell. Cryptosporidium might use as-yet undiscovered purine transporters and salvage enzymes; however, genetic and pharmacological experiments led us to conclude that Cryptosporidium imports purine nucleotides from the host cell. The potential for ATP uptake from the host has significant impact on our understanding of parasite energy metabolism given that Cryptosporidium lacks oxidative phosphorylation and glycolytic enzymes are not constitutively expressed throughout the parasite life cycle.