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.

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.

SUMMARYRNA interference (RNAi) is a potent gene silencing process that is playing an increasingly important role in investigations of gene function in schistosomes. Here we review what is known about the process in these parasites and provide an update on the methodology and machinery of RNAi. Data are presented to demonstrate that: (1) not all schistosome genes can be suppressed to the same extent, using the methods employed here; (2) while there is variation in the level of suppression achieved for one target gene (SmAP) in adult parasites, all individuals exhibit robust (>80%) suppression; (3) short interfering RNAs (siRNAs) can effect suppression when delivered by soaking (and not just via electroporation, as reported previously); (4) Male/female adult pairs need not be separated prior to siRNA delivery by electroporation for effective gene suppression in both genders and (5) electroporation of siRNAs in medium is as efficient as in commercial electroporation buffer. Regarding the machinery of RNAi in schistosomes, a homologue of the C. elegans multi-membrane spanning, RNA importing protein SID-1 is identified in silico. The gene encoding this protein contains 21 exons and spans over 50 kb to potentially encode a 115,556 Mr protein (SmSID-1). These analyses, and a review of the literature, permit us to derive and present here a draft of potential RNAi pathways in schistosomes.

The initiation and progression of several forms of retinal degenerations involve excessive, repetitive, and/or sustained oxidative stress that, in turn, mediate photoreceptor cell damage and death. Since phosphatidylinositol 3-kinase (PI3K)/Akt and mTOR/p70S6-kinase pathways are part of survival signaling in cells confronted with oxidative stress, we asked whether or not docosahexaenoic acidderived neuroprotectin D1 (NPD1) mediates survival upon single-dose and/or repetitive oxidative stress through this pathway. For this purpose, we used human retinal pigment epithelial (ARPE-19) cells challenged by exposure to hydrogen peroxide (H 2 O 2 ) plus tumor necrosis factor alpha (TNF-α). We found that in single-dose oxidative stress-induced apoptosis, phosphorylation of Akt, mTOR, and p70S6K was both time-and dose-dependent. Inhibition of PI3K or mTOR/p70S6K by wortmannin and rapamycin, respectively, increased apoptosis and inhibited phosphorylation of Akt and p70S6K induced by single-dose oxidative stress. While two exposures of a low-dose, nondamaging oxidation induced apoptosis and upregulation of Akt, mTOR, and p70S6K, longer treatment of the cells with three exposures of low dose to low-dose stress showed no changes in the levels of Akt, mTOR, or p70S6K, and resulted in enhanced apoptosis compared to higher doses. Removing the oxidative stress-inducing agents following the single-dose or short term repetitive oxidative stress at the peak of Akt, mTOR, and p70S6K phosphorylation (i.e, 30 minutes after induction) led to recovery, with no apoptosis after 16 hours of incubation. Cells that were induced with three low doses of stress did not show recovery when oxidative stress was removed 30 minutes after the last exposure. NPD1 protected the RPE cells against both single-dose and repetitive oxidative stress-induced apoptosis and promoted higher levels of phosphorylated Akt, mTOR, and p70S6K. Together, our results show that a) repetitive oxidative stress is dose dependent and may not be recovered by removing the oxidative stress-inducing agents, b) PI3K/Akt and mTOR/p70S6K pathways play a major role in the protection against oxidative stress-induced apoptosis in ARPE-19 cells, and c) NPD1 exerts protection under these conditions by inducing PI3K/Akt and mTOR/ p70S6K pathways.

Adult schistosomes are intravascular parasites that metabolize imported glucose largely via glycolysis. How the parasites get rid of the large amounts of lactic acid this generates is unknown at the molecular level. Here, we report that worms whose aquaporin gene (SmAQP) has been suppressed using RNAi fail to rapidly acidify their culture medium and excrete less lactate compared to controls. Functional expression of SmAQP in Xenopus oocytes demonstrates that this protein can transport lactate following Michaelis-Menten kinetics with low apparent affinity (Km = 41±5. 8 mM) and with a low energy of activation (Ea = 7.18±0.7 kcal/mol). Phloretin, a known inhibitor of lactate release from schistosomes, also inhibits lactate movement in SmAQP-expressing oocytes. In keeping with the substrate promiscuity of other aquaporins, SmAQP is shown here to be also capable of transporting water, mannitol, fructose and alanine but not glucose. Using immunofluorescent and immuno-EM, we confirm that SmAQP is localized in the tegument of adult worms. These findings extend the proposed functions of the schistosome tegument beyond its known capacity as an organ of nutrient uptake to include a role in metabolic waste excretion.