Multiple pathways for glucose phosphate transport and utilization support growth of Cryptosporidium parvum

Abstract

Cryptosporidium parvum is an obligate intracellular parasite with a highly reduced mitochondrion that lacks the tricarboxylic acid cycle and the ability to generate ATP, making the parasite reliant on glycolysis. Genetic ablation experiments demonstrated that neither of the two putative glucose transporters CpGT1 and CpGT2 were essential for growth. Surprisingly, hexokinase was also dispensable for parasite growth while the downstream enzyme aldolase was required, suggesting the parasite has an alternative way of obtaining phosphorylated hexose. Complementation studies in E. coli support a role for direct transport of glucose-6-phosphate from the host cell by the parasite transporters CpGT1 and CpGT2, thus bypassing a requirement for hexokinase. Additionally, the parasite obtains phosphorylated glucose from amylopectin stores that are released by the action of the essential enzyme glycogen phosphorylase. Collectively, these findings reveal that C. parvum relies on multiple pathways to obtain phosphorylated glucose both for glycolysis and to restore carbohydrate reserves.

Mendelian segregation and high recombination rates facilitate genetic analyses in Cryptosporidium parvum

“…To test this possibility, we generated two additional transgenic lines of parasites that had an alternative tagged locus on chromosome 1 or 5 and then performed intrachromosomal crosses and evaluated the rate of crossover. The ABC Transporter gene ( cgd1_700 ) has been previously tagged by our lab and shown to have a unique staining pattern in the feeder organelle of all intracellular life stages [ 36 ]. It lies on the distal end of chromosome 1, and here we tagged it with CFP using CRISPR/Cas9 (Figs 3A and S3B–S3D ).…”

“…The transfection and amplification of parasites in immunocompromised mice have been previously described by our lab [ 10 , 36 , 50 , 51 ]. Briefly, 1 x 10 8 freshly excysted sporozoites were electroporated with 100 μg of the repair plasmid, and 66 μg of the CRISPR/Cas9 plasmid and resuspended in 300 uL of cold DPBS.…”

“…Timelapse microscopy. Timelapse microscopy of C. parvum infection has been previously described by our lab [48]. HCT-8 cells were cultured in a poly-lysine coated glass-bottomed 24-well plate for 24 hr before the experiment.…”

“…CRISPR/Cas9 plasmid construction. The pABC-3HA-Nluc-P2A-neo homology repair template and the pACT:Cas9, U6:sgABC plasmids were previously generated in our lab [36]. For this study, an additional CFP fluorescent protein was inserted into the repair template via Gibson assembly cloning with the linearized backbone and a gBlock gene fragment (Integrated DNA Technologies).…”

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“…Amplifying transgenic parasites in mice. The transfection and amplification of parasites in immunocompromised mice have been previously described by our lab [10,36,50,51]. Briefly, 1 x 10 8 freshly excysted sporozoites were electroporated with 100 μg of the repair plasmid, and 66 μg of the CRISPR/Cas9 plasmid and resuspended in 300 uL of cold DPBS.…”