Progress 07/01/04 to 12/31/06

Outputs
While adult schistosomes possess a functional mouth and gut, most nutrients are imported across the parasite's body surface (or tegument). One protein complex that is important in the uptake of amino acids across the tegument by intra-vascular schistosomes is designated SPRM1 (Schistosome permease 1). SPRM1 is a heterodimer and consists of a light chain (SPRM1lc) and a heavy chain (SPRM1hc). The SPRM1 light chain has been cloned and characterized; it is a 55 kDa, multiple membrane-spanning protein that is found in the parasite tegument (and other tissues). When SPRM1lc is expressed with a heterologous (human) heavy chain in Xenopus oocytes, it facilitates the transport of the basic amino acids arginine, lysine, histidine as well as leucine, phenylalanine, methionine and glutamine. The heavy chain acts as a chaperone to permit a functional surface localization of the light chain amino acid transporter. Biochemical characterization demonstrates that, in schistosome extracts, SPRM1lc is associated into a high molecular weight complex with SPRM1hc that can be disrupted by reducing agents. A search of the S. mansoni transcriptome reveals just one sequence with significant homology to mammalian amino acid transporter heavy chains and we have termed this SPRM1hc. SPRM1hc has been cloned and is being characterized. One set of experiments undertaken in this final year of funding was to attempt silencing of the SPRM1hc gene using RNA interference protocols we have developed in the laboratory.

Impacts
We have achieved moderate gene suppression in different developmental stages and are now examining the longer term consequences of suppressing this gene. We propose that surface exposed, host interactive, nutrient transporting proteins like SPRM1 are viable vaccine candidates.

Publications

• No publications reported this period

Progress 01/01/05 to 12/31/05

Outputs
The chronic, debilitating disease schistosomiasis is caused by extracellular blood worms that remain for many years in their definitive host. There is a widely acknowledged need for a protective vaccine for this condition and the current proposal aims at isolating, characterizing and testing one essential, host-interactive membrane protein (designated SPRM1hc) as a vaccine target. SPRM1hc is a heavy chain protein that complexes with a light chain partner (SPRM1lc) that we have shown is important for the uptake of amino acids by schistosomes across their body surfaces. At the start of this work a partial SPRM1hc sequence was available from schistosome expressed sequence tag (EST) data. In order to uncover the entire coding sequence, we compared the EST information with a recently released draft of the Schistosoma mansoni genome sequence. This allowed us identify the complete coding sequence of SPRM1hc as well as to characterize the SPRM1hc gene. We cloned the entire coding sequence into the Xenopus expression vector pSD-EASY and this was used for functional characterization of the protein. Oocytes injected with RNA encoding SPRM1hc together with RNA encoding the light chain partner (SPRM1lc) import selected amino acids. These results involving SPRM1hc/SPRM1lc are very similar to those reported when SPRM1lc is expressed with a heterologous partner confirming that the heavy chain does not contribute to the kinetics or specificity of amino acid import. Affinity purified anti-SPRM1hc antibody were generated from serum of rabbits injected with a SPRM1hc synthetic peptide and these were used to determine the developmental expression of SPRM1hc in several schistosome life-cycle stages. SPRM1hc is a housekeeping protein and is widely expressed. This is not surprising given its role in importing nutrients. The mixed sex adult membrane preparation were resolved in the presence or absence of reducing agent, DTT, and in the absence of this reagent, the protein runs with a slower mobility which we believe represents SPRM1hc complexed with SPRM1lc. In adult sections, SPRM1hc is widely expressed in several tissues including the tegument, as determined using the anti-SPRM1hc antibodies and immunofluorescence microscopy. Immuno-electron microscopy has demonstrated strong staining in the tegumental membranes. This is a very important finding for our purposes since we originally hypothesized that SPRM1hc would localize to the outer tegument to fulfill its role in facilitating amino acid uptake from the host's bloodstream. Ongoing experiments are attempting to suppress the expression of the SPRM1hc gene in the parasites using RNA interference, in order to define its importance for the worms.

Impacts
In this work, we have demonstrated that SPRM1hc is a surface protein that represents a viable vaccine target and we aim to test its protective potential. We have successfully expressed the large, predicted extracellular domain of SPRM1hc in E. coli but have found that purifying the protein for vaccine trials has been problematic. We hope to overcome these technical issues in the future and begin vaccine testing in mice. To overcome these problems we could begin testing our candidate, not in protein form, but as a plasmid construct. For this we need to clone the coding DNA into pVAX and generate endotoxin-free plasmid to use as an immunogen.

Publications

• No publications reported this period

Progress 07/01/04 to 12/31/05

Outputs
The chronic, debilitating disease schistosomiasis is caused by extracellular blood worms that remain for many years in their definitive host. There is a widely acknowledged need for a protective vaccine for this condition and the current proposal aims at isolating, characterizing and testing one essential, host-interactive membrane protein (designated SPRM1hc) as a vaccine target. SPRM1hc is a heavy chain protein that complexes with a light chain partner (SPRM1lc) that we have shown is important for the uptake of amino acids by schistosomes across their body surfaces. At the start of this work a partial SPRM1hc sequence was available from schistosome expressed sequence tag (EST) data. In order to uncover the entire coding sequence, we compared the EST information with a recently released draft of the Schistosoma mansoni genome sequence. This allowed us identify the complete coding sequence of SPRM1hc as well as to characterize the SPRM1hc gene. We cloned the entire coding sequence into the Xenopus expression vector pSD-EASY and this was used for functional characterization of the protein. Oocytes injected with RNA encoding SPRM1hc together with RNA encoding the light chain partner (SPRM1lc) import selected amino acids. These results involving SPRM1hc/SPRM1lc are very similar to those reported when SPRM1lc is expressed with a heterologous partner confirming that the heavy chain does not contribute to the kinetics or specificity of amino acid import. Affinity purified anti-SPRM1hc antibody were generated from serum of rabbits injected with a SPRM1hc synthetic peptide and these were used to determine the developmental expression of SPRM1hc in several schistosome life-cycle stages. SPRM1hc is a housekeeping protein and is widely expressed. This is not surprising given its role in importing nutrients. The mixed sex adult membrane preparation were resolved in the presence or absence of reducing agent, DTT, and in the absence of this reagent, the protein runs with a slower mobility which we believe represents SPRM1hc complexed with SPRM1lc. In adult sections, SPRM1hc is widely expressed in several tissues including the tegument, as determined using the anti-SPRM1hc antibodies and immunofluorescence microscopy. Immuno-electron microscopy has demonstrated strong staining in the tegumental membranes. This is a very important finding for our purposes since we originally hypothesized that SPRM1hc would localize to the outer tegument to fulfill its role in facilitating amino acid uptake from the host's bloodstream. Ongoing experiments are attempting to suppress the expression of the SPRM1hc gene in the parasites using RNA interference, in order to define its importance for the worms.

Impacts
In this work, we have demonstrated that SPRM1hc is a surface protein that represents a viable vaccine target and we aim to test its protective potential. We have successfully expressed the large, predicted extracellular domain of SPRM1hc in E. coli but have found that purifying the protein for vaccine trials has been problematic. We hope to overcome these technical issues in the future and begin vaccine testing in mice. To overcome these problems we could begin testing our candidate, not in protein form, but as a plasmid construct. For this we need to clone the coding DNA into pVAX and generate endotoxin-free plasmid to use as an immunogen.

Publications

• No publications reported this period