Suppressing glucose transporter gene expression in schistosomes impairs parasite feeding and decreases survival in the mammalian host

Abstract

Adult schistosomes live in the host's bloodstream where they import nutrients such as glucose across their body surface (the tegument). The parasite tegument is an unusual structure since it is enclosed not by the typical one but by two closely apposed lipid bilayers. Within the tegument two glucose importing proteins have been identified; these are schistosome glucose transporter (SGTP) 1 and 4. SGTP4 is present in the host interactive, apical tegumental membranes, while SGTP1 is found in the tegumental basal membrane (as well as in internal tissues). The SGTPs act by facilitated diffusion. To examine the importance of these proteins for the parasites, RNAi was employed to knock down expression of both SGTP genes in the schistosomula and adult worm life stages. Both qRT-PCR and western blotting analysis confirmed successful gene suppression. It was found that SGTP1 or SGTP4-suppressed parasites exhibit an impaired ability to import glucose compared to control worms. In addition, parasites with both SGTP1 and SGTP4 simultaneously suppressed showed a further reduction in capacity to import glucose compared to parasites with a single suppressed SGTP gene. Despite this debility, all suppressed parasites exhibited no phenotypic distinction compared to controls when cultured in rich medium. Following prolonged incubation in glucose-depleted medium however, significantly fewer SGTP-suppressed parasites survived. Finally, SGTP-suppressed parasites showed decreased viability in vivo following infection of experimental animals. These findings provide direct evidence for the importance of SGTP1 and SGTP4 for schistosomes in importing exogenous glucose and show that these proteins are important for normal parasite development in the mammalian host.

Figure 1. Schistosome glucose transporter protein (SGTP) gene expression analysis.

A. Relative SGTP1 (grey bars) and SGTP4 (black bars) gene expression (mean ± SE) in schistosomula 7 days after treatment with control or SGTP siRNA. “SGTP1+4” indicates parasites treated with both SGTP1 and SGTP4 siRNAs. B. Relative SGTP1 (grey bars) and SGTP4 (black bars) gene expression (mean ± SE) in adult parasites 14 days after treatment with the indicated siRNA. C. Protein levels in adult worm extracts obtained 14 days following treatment with the indicated siRNA. Western blot results are shown for SGTP1 protein (top panel), SGTP4 (middle panel) and a control protein (SPRM1hc, bottom panel). The arrowheads indicate the diminished level of SGTP proteins seen in the left lanes.

Figure 2. Glucose uptake analysis in schistosomes treated with SGTP siRNA.

Relative uptake of 2-deoxyglucose (mean ± SE) into schistosomula 14 days after treatment with the indicated siRNA. An additional control group was treated with the glucose transport inhibitor, cytochalasin B (control +CytB, right bar).

Figure 3. Relative viability of schistosomes treated with SGTP siRNA.

Schistosomula (mean ± SD) were treated with the indicated siRNA and their viability was established following culture for 14 days in RPMI medium either containing high glucose (10 mM, grey bars) or low glucose (0.05 mM, white bars).

Figure 4. Schistosome survival in vivo in mice following treatment with SGTP siRNA.

Schistosomula were treated with the indicated siRNA and used to infect mice. Worm numbers recovered from individual mice in each group are shown as dots. The lines indicate the mean for each group and P values are given at top.

Figure 5. Expression of SGTP1 and SGTP4 (mean ± SE) in schistosomula at different times after treatment with the indicated siRNA.

Parasites were either maintained in culture for 7 days after treatment (white bars) or for 4 week after treatment (grey bars), or were recovered from infected mice 4 weeks after treatment (black bars). A, SGTP1; B, SGTP4.