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

Abstract

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.

CRISPR/Cas9 Ribonucleoprotein-Based Genome Editing Methodology in the Marine Protozoan Parasite Perkinsus marinus

“…In Toxoplasma gondii, CRISPR/Cas9 is widely used in high-throughput and genome screening studies to identify essential genes involved in parasite invasion and antiparasitic drug candidates (Di Cristina et al, 2017;Di Cristina and Carruthers, 2018). CRISPR/Cas9based knock-out studies in Cryptosporidium parvum are used to understand the mechanism of the parasite's resistance to antifolate drugs and nutrient acquisition pathways (Vinayak et al, 2015;Pawlowic et al, 2017Pawlowic et al, , 2019.…”

A Conditional Protein Degradation System To Study Essential Gene Function in Cryptosporidium parvum

“…In P. falciparum , toxicity of TMP was overcome by engineering parasites containing a human DHFR (hDHFR) marker integrated into a non-essential gene and using the resistant parasites to generate a transgenic line for tagging an essential gene with DDD ( 40 ). In contrast, C. parvum is naturally resistant to antifolates, since in addition to DHFR, it has another alternative mechanism for nucleotide synthesis through thymidine kinase (TK), thus rendering DHFR dispensable ( 29 , 49 ). Thus, the inherent resistance of C. parvum to the antifolates worked to our advantage for application of TMP in vivo to create a conditional transgenic parasite.…”

Faculty Opinions recommendation of A Conditional Protein Degradation System To Study Essential Gene Function in Cryptosporidium parvum.

“…In P. falciparum, toxicity of TMP was overcome by engineering parasites containing a human DHFR (hDHFR) marker integrated into a non-essential gene and using the resistant parasites to generate a transgenic line for tagging an essential gene with DDD (40). In contrast, C. parvum is naturally resistant to antifolates, since in addition to DHFR, it has another alternative mechanism for nucleotide synthesis through thymidine kinase (TK), thus rendering DHFR dispensable (29,49). Thus, the inherent resistance of C. parvum to the antifolates worked to our advantage for application of TMP in vivo to create a conditional transgenic parasite.…”