Pore-Forming and Saposin-like Proteins from the Gut of Blood-Feeding Helminths

Field: Immunology and Microbiology (Life Sciences)

Blood-feeding parasites rely on the acquisition of amino acids contained within erythrocytes, namely haemoglobin (Hb), to meet their nutritional requirements for growth, reproduction and survival in the host. While the Hb degradation pathways have been partially elucidated in haematophagous hookworms and schistosomes, the molecular mechanisms by which these parasites lyse erythrocytes to release the Hb are still unknown. Despite earlier publications claiming that hookworms did not possess haemolysins, haemolytic activity in both adult and larval stages of Ancylostoma caninum, the common dog hookworm, has been identified. Activity was detergentsoluble and heat-stable, and was unaffected by the addition of protease inhibitors, metal ions, chelators and reducing agents. Trypsin ablated lysis, indicating that the haemolytic agent is a protein. A closely migrating doublet of A. caninum proteins (60-65 kDa) bound to erythrocyte membranes after lysis of cells, and in addition, chromatographic separation of detergent extracts resulted in the purification of similar sized proteins with trypsin-sensitive haemolytic activity. Scanning electron microscopy was used to observe erythrocytes lysed with particulate, buffer-insoluble worm extracts. Red cell remnants (ghosts) remained, with pores approximately 100 nm in diameter forming in their cell membranes, suggesting that lysis is likely caused by pore-formation followed by osmotic disruption of the cell. Recently, cDNAs encoding proteins with haemolytic capacity were identified from the liver flukes Clonorchis sinensis and Fasciola hepatica. These fluke cytolysins share sequence homology with amoebapores, pore-forming peptides from the protozoan parasite, Entamoeba histolytica, and can be grouped within a family of distantly related saposin-like proteins (SAPLIPs). Two A. caninum expressed sequence tags (ESTs) encoding SAPLIPs were identified and full-length cDNA sequences were obtained from a cDNA library by PCR – these were termed Ac-slp-1 and Ac-slp-2. Both mRNAs were expressed in all life stages studied, with Ac-slp-1 predominantly expressed in third-stage larvae (L3) before and after activation with host serum. Recombinant proteins were expressed in insect cells and used to raise specific antisera in mice. Both proteins were localised to small punctate organelles or vesicles within the intestinal cells of adult worms. There was also weak staining on the microvillar brush border of the intestine. Both proteins also localised to similar vesicles in the intestinal cells of L3 using transmission electron microscopy. Some SAPLIPs dimerise via a head-to-tail mechanism, but the purified recombinant hookworm proteins had C-terminal purification tags which might preclude them from undergoing dimerisation, therefore possibly interfering with the subsequent detection of membrane binding and haemolysis. Similarly, two cDNAs termed Sm-slp-1 and Sm-slp-2 were chosen for further analysis from the human blood fluke Schistosoma mansoni. The latter cDNA encoded a double SAPLIP domain. Although recombinant proteins from the two cDNAs were successfully expressed in insect cells, only Sm-SLP-1 was produced at sufficient levels to warrant further investigation. Specific antiserum was raised in mice against Sm-SLP-1 and used in fluorescence microscopy to localise the anatomical expression site to the gastrodermis of both male and female adult worms. Again, and likely due to the inability to remove C-terminal tags from the recombinant protein, potential dimerisation could not be addressed. In addition, no haemolytic activity was observed, but a role in haemolysis cannot be excluded until Sm-SLP-1 is produced in recombinant form with a flush C-terminus. Sm-SLP-1 was considered a putative vaccine candidate due to its anatomic location and potential role in feeding. Vaccination of mice with recombinant Sm-SLP-1 did not result in a decrease in worm burdens or eggs in vaccinated animals. Although antibodies bound to the native protein in the gastrodermis of the parasite, they neither damaged the parasite nor did they interfere with critical physiological processes. However, mice infected with schistosomes did generate a specific antibody response to SLP-1. Similarly, sera from individuals residing in a schistosomiasis endemic area of Brazil also had serum antibodies that recognised Sm-SLP-1. The highest levels of antibodies were detected in subjects with moderate/heavy infections compared with subjects who were exposed to schistosomiasis but egg-negative. This indicates that Sm-SLP-1 could be developed as a biomarker to determine intensity of S. mansoni infection. These studies have identified novel intestinal molecules which potentially play key roles in host-parasite interactions and in the haemolysis of ingested red cells, and might aid in the immunodiagnosis of infections with blood-feeding helminths.