Proteomic Analysis of Biomphalaria glabrata Hemocytes During in vitro Encapsulation of Schistosoma mansoni Sporocysts.

Circulating hemocytes of the snail Biomphalaria glabrata, a major intermediate host for the blood fluke Schistosoma mansoni, represent the primary immune effector cells comprising the host's internal defense system. Within hours of miracidial entry into resistant B. glabrata strains, hemocytes infiltrate around developing sporocysts forming multi-layered cellular capsules that results in larval death, typically within 24-48 h post-infection. Using an in vitro model of hemocyte-sporocyst encapsulation that recapitulates in vivo events, we conducted a comparative proteomic analysis on the responses of hemocytes from inbred B. glabrata strains during the encapsulation of S. mansoni primary sporocysts. This was accomplished by a combination of Laser-capture microdissection (LCM) to isolate sections of hemocyte capsules both in the presence and absence of sporocysts, in conjunction with mass spectrometric analyses to establish protein expression profiles. Comparison of susceptible NMRI snail hemocytes in the presence and absence of sporocysts revealed a dramatic downregulation of proteins in during larval encapsulation, especially those involved in protein/CHO metabolism, immune-related, redox and signaling pathways. One of 4 upregulated proteins was arginase, competitor of nitric oxide synthetase and inhibitor of larval-killing NO production. By contrast, when compared to control capsules, sporocyst-encapsulating hemocytes of resistant BS-90 B. glabrata exhibited a more balanced profile with enhanced expression of shared proteins involved in protein synthesis/processing, immunity, and redox, and unique expression of anti-microbial/anti-parasite proteins. A final comparison of NMRI and BS-90 host hemocyte responses to co-cultured sporocysts demonstrated a decrease or downregulation of 77% of shared proteins by NMRI cells during encapsulation compared to those of the BS-90 strain, including lipopolysaccharide-binding protein, thioredoxin reductase 1 and hemoglobins 1 and 2. Overall, using this in vitro model, results of our proteomic analyses demonstrate striking differences in proteins expressed by susceptible NMRI and resistant BS-90 snail hemocytes to S. mansoni sporocysts during active encapsulation, with NMRI hemocytes exhibiting extensive downregulation of protein expression and a lower level of constitutively expressed immune-relevant proteins (e.g., FREP2) compared to BS-90. Our data suggest that snail strain differences in hemocyte protein expression during the encapsulation process account for observed differences in their cytotoxic capacity to interact with and kill sporocysts.

Isoforms of Hsp70-binding human LDL in adult Schistosoma mansoni worms.

Schistosoma mansoni is one of the most common parasites infecting humans. They are well adapted to the host, and this parasite's longevity is a consequence of effective escape from the host immune system. In the blood circulation, lipoproteins not only help to conceal the worm from attack by host antibodies but also act as a source of lipids for S. mansoni. Previous SEM studies showed that the low-density lipoprotein (LDL) particles present on the surface of adult S. mansoni worms decreased in size when the incubation time increased. In this study, immunocytochemical and proteomic analyses were used to locate and identify S. mansoni binding proteins to human plasma LDL. Ultrathin sections of adult worms were cut transversely from the anterior, medial and posterior regions of the parasite. Immunocytochemical experiments revealed particles of gold in the tegument, muscle region and spine in male worms and around vitelline cells in females. Immunoblotting and 2D-electrophoresis using incubations with human serum, anti-LDL antibodies and anti-chicken IgG peroxidase conjugate were performed to identify LDL-binding proteins in S. mansoni. Analysis of the binding proteins using LC-MS identified two isoforms of the Hsp70 chaperone in S. mansoni. Hsp70 is involved in the interaction with apoB in the cytoplasm and its transport to the endoplasmic reticulum. However, further studies are needed to clarify the functional role of Hsp70 in S. mansoni, mainly related to the interaction with human LDL.