The role of tegumental aquaporin from the human parasitic worm, Schistosoma mansoni, in osmoregulation and drug uptake

Abstract

Schistosomes are parasitic platyhelminths that constitute an important public health problem globally. Infection is characterized by the presence of adult worms within the vasculature of their hosts, where they can reside for many years. The worms are covered by an unusual dual lipid bilayer through which they import nutrients. How the parasites import other vital molecules, such as water, is not known. Recent proteomic analysis of the schistosome tegumental membranes revealed the presence of an aquaporin homologue at the host-interactive surface whose cDNA we have cloned and characterized. The cDNA encodes a predicted 304-aa protein (SmAQP) that is found largely in the parasite tegument by immunolocalization and is most highly expressed in the intravascular life stages. Treatment of parasites with short interfering RNAs targeting the SmAQP gene results in potent (>90%) suppression. These suppressed parasites resist swelling when placed in hypotonic medium, unlike their control counterparts, which rapidly double in volume. In addition, SmAQP-suppressed parasites, unlike controls, resist shrinkage when incubated in hyperosmotic solution. While suppressed parasites exhibit lower viability in culture relative to controls and exhibit a stunted appearance following prolonged suppression, they are nonetheless more resistant to killing by the drug potassium antimonyl tartrate (PAT). This is likely because SmAQP acts as a conduit for this drug, as is the case for aquaporins in other systems. These experiments reveal a heretofore unrecognized role of the schistosome tegument in controlling water and drug movement into the parasites and highlight the importance of the tegument in parasite osmoregulation and drug uptake.

Figure 1.

A) Alignment of SmAQP with other members of the AQP protein family. Sm, S. mansoni (GenBank accession no. EU780065); Sj, S. japonicum (AAW24850); Hs, Homo sapiens isoform AQP9 (NP066190); Ce, Caenorhabditis elegans (NP495972); Lb, Leishmania braziliensis (XP001562217). Predicted transmembrane domains are indicated (TM1–6); conserved NPA motifs are boxed; asterisks represent potential N-linked glycosylation sites (at positions 150 and 159). B) Phylogenetic tree of selected AQPs, generated by multiple sequence alignment with hierarchical clustering. Designations (and accession numbers) of AQP homologs compared are as in A. Additional AQPs: HS1–HS10, human AQP isoforms AQP1–AQP10 (NP932766, NP000477, CAG46822, NP001641, NP001642, NP001643, CAI13307, NP001160, NP066190, NP536354). Protozoans: LB, L. braziliensis (XP001562217); TB, Trypanosoma brucei brucei (CAG27022). Nematodes: CE, C. elegans (NP495972); BM, Brugia malayi (XP001892122); TC, Toxocara canis (AAC32826). C) SmAQP gene. White boxes indicate exons 1 and 2. K, kilobase pair.

Figure 2.

Expression of the SmAQP gene in different schistosome developmental stages relative to expression of control triose phosphate isomerase gene. Spor, 24-h cultured sporocyst; Cer, cercaria; Som, 24-h cultured schistosomulum); male and female, 7 wk old.

Figure 3.

A, B) Immunolocalization of SmAQP in 2-d (A) and 7-d (B) cultured schistosomula. Strong tegumental staining is evident. C) Control 7-d schistosomulum probed with secondary antibody alone. Scale bars = 50 μm.

Figure 4.

A) Relative SmAQP expression in cultured schistosomula 2 d after no treatment (none) or treatment with control or SmAQP siRNA. Data represent means ± se. B) Detection by Western blot analysis of SmAQP protein (top panel) and a control protein (SPRM1hc; bottom panel) in extracts prepared from parasites 5 d after no treatment (none; lane 1) or treatment with control (lane 2) or SmAQP siRNA (lane 3). Arrowhead indicates relatively diminished level of SmAQP protein in lane 3 (∼33,000 M_r).

Figure 5.

A) Size of cultured schistosomula 5 d after no treatment (none) or treatment with control or SmAQP siRNA, when incubated either in complete RPMI medium (RPMI) or after 5 min in distilled water (H₂O). Each point represents an individual parasite; line indicates the mean for that group; n = 22/treatment. Mean size of AQP-suppressed group in water differs significantly from both control counterparts; P < 0.05. B, C) Representative group of control untreated schistosomula in RPMI medium (B) and after 5 min in distilled water (C). Scale bars = 50 μm.

Figure 6.

A) Size of cultured schistosomula at different time points after incubation in distilled H₂O. Data represent means ± sd. B) Size of cultured schistosomula in RPMI or after 5 min in H₂O or on return to RPMI following incubation in H₂O for the time periods indicated; n = 15/treatment.

Figure 7.

A) Size of cultured schistosomula 5 d after treatment with control or SmAQP siRNA when incubated either in complete RPMI medium (−) or after 30 min in hyperosmotic solution (+). Each point represents an individual parasite; line indicates mean for that group; n = 15/treatment. Mean size of AQP-suppressed group in water differs significantly from the control group in hyperosmotic solution; P < 0.05. B, C) Images of a representative group of control untreated schistosomula in RPMI medium (B) and after 20 min in hyperosmotic solution (C). Scale bars = 50 μm.

Figure 8.

Number of dead schistosomula following 2 h incubation in RPMI containing 10 μM PAT, 8 d after no treatment (none) or treatment with control or SmAQP siRNA (open bar). Significantly fewer parasites were killed in the SmAQP-suppressed group vs. either control group; P < 0.05. Data represent means ± sd.

Figure 9.

A) Viability of 14- and 21-d cultured schistosomula after no treatment (−) or treatment with control (+) or SmAQP siRNA (AQP) on d 2. Significantly fewer SmAQP-suppressed parasites (open bars) are viable at both time points vs. controls (shaded bars); P < 0.05. Data represent means ± sd. B) Size of 21-d cultured schistosomula after no treatment (none) or treatment with control or SmAQP siRNA on d 2. Mean size of SmAQP-suppressed group differs significantly from both controls; P < 0.05; n = 15/treatment. C, D) Representative images of 21-d-old SmAQP-suppressed parasites (C) vs. their generally more elongated control counterparts (D). Scale bars = 50 μm.