Genetic ablation of purine salvage inCryptosporidium parvumreveals nucleotide uptake from the host cell

Abstract

The apicomplexan parasiteCryptosporidiumis 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 forCryptosporidiuminfections. Here we take advantage of newly available molecular genetics forCryptosporidium parvumto 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 forC. parvumpurine 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.Cryptosporidiummight use as-yet undiscovered purine transporters and salvage enzymes; however, genetic and pharmacological experiments led us to conclude thatCryptosporidiumimports 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 thatCryptosporidiumlacks oxidative phosphorylation and glycolytic enzymes are not constitutively expressed throughout the parasite life cycle.