Figure 1.
Immunolocalization of SmAQP in adult parasites.
A. Cross section through a male/female couple showing strong staining with anti-SmAQP antibodies in the tegument. B. Higher magnification image of the peripheral tissue of an adult male showing diminished staining in the tegumental tubercles (arrowheads). C. Electron micrograph of the adult tegument showing immunogold labeling of SmAQP. Arrows indicate gold particles at the host/parasite interface. Numbers above scale bars represent microns.
Figure 2.
A. Relative SmAQP expression (mean ± SE) in adult schistosomes 7 days after treatment with none, or control, or SmAQP siRNA. B, Detection by western analysis of SmAQP protein (top panel), and a control protein (SPRM1hc, bottom panel) in extracts prepared from parasites 28 days after treatment with none (lane 1), control (lane 2), or SmAQP (lane 3) siRNA. The arrowhead indicates the diminished level of SmAQP protein seen in lane 3. C. Localization of SmAQP in whole, fixed adult parasites stained with anti-SmAQP antiserum (a–h) 14 days after no (a,b), control (c,d), or SmAQP (e,f) siRNA treatment. An additional control group (g,h) was untreated with siRNA and stained with secondary antibody alone. Panels a,c,e,g are phase contrast images while panels b,d,f,h illustrate antibody binding. Non suppressed parasites (panels a–d) stain brightly for SmAQP while the SmAQP-suppressed parasites (e,f) and parasites stained with secondary antibody alone (g,h) stain very weakly. The bar represents 100 µm.
Figure 3.
Changes in the medium of SmAQP suppressed and control adult schistosomes.
A. pH (mean ± SE) of 1 ml complete RPMI medium containing 10 adult parasites that were treated with none or control or SmAQP siRNA 96 hours earlier. B. Lactate concentration (µM) in medium containing 10 adult parasites that were treated with none or control or SmAQP siRNA 96 hours earlier.
Figure 4.
A. SmAQP, when expressed in X. laevis oocytes, can transport several solutes. SmAQP transports lactate, in addition to mannitol, fructose and L-alanine but not glucose. The substrates were added to a final concentration of 1 mM and accumulation of radiolabeled substance traces on the cells was measured after 1.5 minute (n = 16–18 oocytes). B. The transport of lactate is pH dependent. Solute diffusion coefficient permeability for lactate increased in a pH dependent manner (6.4±0.9×10−6 cm/s pH 7.4 and 20.1±3.5×10−6 cm/s pH 6.3), whereas for mannitol it was unchanged (1.5±0.2×10−6 cm/s pH/.4 and 1.7±0.2×10−6 cm/s pH 6.3) (n = 15–17). C. The transport of lactate is saturable. SmAQP has low affinity for lactate and follows Michaelis-Menten kinetics (n = 14–24). D. Lactate moves through a pore. The energy of activation (Ea) was estimated by plotting the temperature dependent transport (at 4, 15 and 25°C) as an Arrhenius plot. The slope of the linear regression was −1.57±0.15 K (n = 9–15). In all cases, values obtained from control oocytes, not-expressing SmAQP have been subtracted to generate the data shown. Data represent the mean ± SE generated in at least 2 independent experiments.
Figure 5.
A–B. Increase in water osmotic permeability in oocytes expressing SmAQP. A. Volume increase in control, non- injected (NI) oocytes versus oocytes expressing SmAQP when transferred from normal salt solution (206 mOsm/Kg) to a hypotonic solution (72 mOsm/Kg) in the presence or absence of phloretin (0.5 mM). B. The osmotic permeability coefficient Pf of oocytes expressing SmAQP or control, non-injected (NI) oocytes in the presence (+) or absence (−) of phloretin (0.5 mM) (n = 3–6). C–D. Phloretin inhibition of water permeability and solute diffusion. C. The transport of lactate is inhibited by phloretin (0.5 mM, grey bars), but not by HgCl2 (0.3 mM, black bars). “+” signifies oocytes expressing SmAQP, “−” indicates control, non-injected oocytes. D. Kinetics of phloretin inhibition of lactate transport. Phloretin inhibits lactate movement with high affinity (Ki50 = 22 µM, n = 15–16). Values obtained from control oocytes, not-expressing SmAQP have been subtracted to generate the data shown. Data represent the mean ± SE generated in at least 2 independent experiments.