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 TCA 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.

Keywords: energy utilization; glucose phosphate transport; glucose transport; glycogen; glycolysis.

Figure 1.. Cryptosporidium parvum contains two putative glucose transporters CpGT1 and CpGT2 localized to the feeder organelle.

a Phylogenetic relationship of C. parvum (Cp) putative glucose transporters CpGT1 (gene ID: cgd3_4070), CpGT2 (gene ID: cgd4_2870), Plasmodium falciparum hexose transporter PfHT1, and Toxoplasma gondii glucose transporter TgGT1 and sugar transporter TgST1–3. The tree was constructed by a maximum likelihood method and JTT matrix-based model with 1,000 replications for bootstrapping. Scale bar = 0.1 of substitutions per amino acid site. b Schematic representation of the domain structure of CpGT1 and CpGT2 including putative transmembrane domain (blue) and stop codon (*). c Immunofluorescence localization of CpGT1–3HA in trophozoite (top view) and meront stages (side view) grown on HCT-8 cells. See also supplementary Fig. 1a for constructing and identification of CpGT1–3HA transgenic parasites. For trophozoite staining, cells were fixed at 4 hours post infection (hpi) and stained with rat anti-HA (green), mouse 1B5 (red) and Hoechst (blue). For meront staining, cells were fixed at 24 hpi and stained with rat anti-HA (green), mouse 1E12 (red) and Hoechst (blue). The experiment was performed twice with similar results. Scale bars = 2 µm. d Immunofluorescence localization of CpGT2-smHA in trophozoite stage (top view) and meront stage (side view) grown on HCT-8 cells. See also supplementary Fig. 1b for constructing and identification of CpGT2-smHA transgenic parasites. Stained as in c. The experiment was performed twice with similar results. Scale bars = 2 µm. e Ultrastructural localization of CpGT1–3HA. Parasites were grown in HCT-8 cells, fixed at 20 hpi and processed for immuno-EM and stained with rabbit anti-HA followed by 18-nm colloidal gold goat anti-rabbit IgG. Scale bars = 500 nM. f Ultrastructural localization of CpGT2-smHA. HCT-8 cells were infected with CpGT2-smHA parasites. Stained as same as in e. Scale bars = 500 nM. See also supplementary Fig. 3 for immuno-EM of WT parasite with anti-HA staining as a negative control.

Figure 2.. Formation of the C. parvum feeder organelle and proximity labeling of enriched transporters.

a Schematic representation of CpGT1-mNeon-mCherry transgenic Cp strain. CpGT1 fused to mNeon and mCherry driven by Cp actin promoter. Cartoon depicts the labeling of the feeder organelle with CpGT1-mNeon and the cytosol with mCherry. b Time lapse microscopy at intervals during the merogony cycle. Scale bars = 2 µm. See also supplementary Video 1 for the time-lapse series of a full merogony cycle. c Fold change in fluorescent intensity of mNeon at time intervals during merogony normalized to 0 h post invasion. Each bar represents the mean ± SD for a total of 15 parasites from three combined experiments. d Schematic representation of proximity-dependent biotinylation in Cp. HCT-8 cells were infected with CpGT1-mTurbo-3HA parasites, labeled with biotin, lysed and affinity purified using streptavidin beads, and captured proteins identified by LC-MS/MS. See also supplementary Fig. 1d for constructing and identification of CpGT1-mTurb-3HA transgenic parasites. e Immunofluorescence of biotin-labeled parasites grown in HCT-8 cells. Cells were infected with CpGT1-mTurbo-3HA parasites. After 19 h, cells were treated with 500 µM biotin or vehicle (DMSO) for 1 h, then fixed and stained with rat anti-HA (green), streptavidin-Alex 568 (red) and Hoechst (blue). The experiment was performed twice with similar outcomes. Scale bars = 2 µm. f Western blot analysis of biotinylated proteins in Cp cell lysates. Cells were infected with CpGT1-mTurbo-3HA parasites. After 19 h, cells were treated with 500 µM biotin or vehicle (DMSO) for 1 h, lysed in 1% NP-40 lysis buffer, captured on streptavidin beads and detected by Western blot with IRDye 800CW-labeled streptavidin (green). The experiment was performed three times with similar results. g Heat map of interactors of CpGT1 identified by mass spectrometry. Data were from three independent experiments including proteins (≥2 peptides, 95% peptide threshold, 99% protein threshold) with significant and 2-fold enrichment in biotin versus in vehicle (P < 0.05, unpaired Student’s t tests, two-tailed). The numbers of peptides in biotin samples were given as row Z-scores. The list of gene ID is provided in supplementary Table 2.

Figure 3.. Glucose transport and hexokinase are individually dispensable for C. parvum growth in vivo.

(a) Growth of CpGT1 tagging (TAG) or CpGT1 knockout (KO) transgenic strains (b) Growth of CpGT2 tagging CpGT2 TAG or CpGT2 KO transgenic strains. NSG mice were infected with parasites and infection was monitored by measuring luciferase activity from fecal pellets collected at intervals post infection. Each line represents an individual NSG mouse (n = 6 for CpGT1 TAG, CpGT1 KO and CpGT2 TAG, n = 7 for CpGT2 KO, from two combined experiments). Two-way ANOVA corrected for multiple comparisons by Sidak’s method (*, P = 0.0250, ***, P = 0.0002 at 24 hpi, ***, P = 0.0001 at 30 hpi, ****, P < 0.0001). No significant difference in parasite burden was observed between CpGT1 TAG and CpGT1 KO strains. c Schematic representation of the reaction of hexokinase in the glycolytic pathway. d Diagram of the strategy to construct Cp hexokinase (HK) KO transgenic strain. e Growth of the CpHK KO transgenic strain. NSG mice were infected with parasites and infection was monitored by measuring luciferase activity from fecal pellets collected at intervals post infection. Each line represents an individual NSG mouse (n = 3 from one experiment). f PCR analysis of oocysts obtained from NSG mice infected with parasites as shown. Amplification products correspond to regions annotated in d.

Figure 4.. The C. parvum CpGT1 and CpGT2 transporters salvage glucose phosphate.

a Solid media complementation of a mutant E. coli line lacking the glucose phosphate transport (∆uhpT). Cells were grown in M9 minimal media (M9 MM) supplemented with 10 mM glucose or glucose-6-phosphate (G6P). The indicated dilutions were plated and grown at 37 °C for 40 h before imaging. The experiment was performed three times with similar results. b Solid media complementation of the E. coli the glucose transport mutant (∆ptsG, ∆galp, ∆ptsM) grown in M9 MM supplemented with 10 mM glucose or G6P. The indicated dilutions were plated and grown at 37 °C for 40 h before imaging. The experiment was performed three times with similar results. See also supplementary Fig. 7 for the growth assay of WT E. coli complementing with different genes.

Figure 5.. C. parvum utilizes stored glycogen (amylopectin) for glucose phosphate as a carbon source.

a Schematic representation of stage I of glycolysis, glycogen breakdown and glycogen synthesis. b Schematic representation of Cp engineered to co-express the auxin receptor TIR1 from Oryza sativa and Cp glucan phosphorylase (CpGP) fused to mAID from Arabidopsis thaliana. See also supplementary Fig. 1f for constructing and identification of CpGP-mAID-TIR1 transgenic parasites. c Schematic representation of conditional CpGP-mAID-TIR1 depletion. IAA, indole acetic acid; Ub, ubiquitin; SCF, Skp-1, Cullin, F-box (TIR1)-containing complex. d Immunofluorescence localization of CpGP-mAID-3HA expression. HCT-8 cells were infected with CpGP-mAID-TIR1 parasites and treated with 500 µM IAA or the vehicle (EtOH) for 24 h. Cells then were fixed and stained with rabbit anti-HA (green), mouse anti-TY (red), rat PanCp (cyan) and Hoechst (blue). The experiment was performed twice with similar outcomes. Scale bars = 2 µm. e Growth of CpGP-mAID-TIR1 parasites following treatment with IAA. HCT-8 cells were infected with CpGP-mAID-TIR1 parasites, treated with 500 µM IAA or the vehicle (EtOH) for 24 h or 48 h. Cells were fixed, labeled with rabbit PanCp and Hoechst, and the number of Cp in each well was determined by using a Cytation 3 imager. Each bar represents the mean ± SD for nine replicates in total from three experiments. Statistical analysis performed using was performed by two-way ANOVA corrected for multiple comparisons by Sidak’s method. ns, not significant. ****, P < 0.0001. See also supplementary Fig. 8c for the growth assay of host cell.

Figure 6.. Schematic representation of transportation and utilization of glucose phosphate in C. parvum.

C. parvum has multiple pathways to transport and utilize hexoses for generating energy. Glucose from the gut lumen can be transported into epithelial cells through GLUT1 or GLUT2, then phosphorylated to G6P by HK1 or HK2. The glucose in the host cells could be transported into Cp by CpGT1 and G6P could be transported into Cp by CpGT1 and CpGT2 at the feeder organelle. Glucose and glucose phosphate can be utilized through glycolysis and glycogen synthesis. GLUT1/2, human glucose transporter 1 or 2; HK1/2, human hexokinase 1 or 2.