Sarah E. Fryer

Phagocytosis of yeast by Biomphalaria glabrata: Carbohydrate specificity of hemocyte receptors and a plasma opsonin

In vitro phagocytosis of yeast cells by hemocytes of the mollusc Biomphalaria glabrata can occur in the absence of plasma, but is enhanced by opsonization in plasma from certain snail strains. We have investigated the carbohydrate specificity of the hemocyte-bound receptor for phagocytosis and the free plasma opsonin using two dominant carbohydrate components of the yeast cell wall (laminarin and mannan). Phagocytic uptake in the absence of plasma of both untreated and opsonized yeast is strongly inhibited by laminarin, but is unaffected by mannan. In contrast, laminarin does not interfere with opsonization whereas mannan blocks the process, blocking the binding of a 57 kD plasma component as detected by Western immunoblot. These results suggest that the opsonic lectin in plasma is not simply a free form of the hemocyte-bound receptor for yeast phagocytosis.

Opsonization of yeast by the plasma of Biomphalaria glabrata (Gastropoda): a strain-specific, time-dependent process

Summary Phagocytosis of yeast by haemocytes of Biomphalaria glabrata, intermediate host of Schistosoma mansoni, is influenced strongly by host plasma components, although it can occur without involvement of such factors. Plasma from two strains of B. glabrata which are resistant to S. mansoni differs in its opsonic properties from the plasma of a susceptible strain. This may reflect the principles which determine compatibility or incompatibility in this host‐parasite system. Opsonization is a time‐dependent process: short periods of incubation in plasma from all strains reduces subsequent phagocytosis in the absence of plasma factors, whereas longer incubation in resistant strain plasma is markedly opsonic. Haemocytes from all strains examined are equally competent in their recognition of either native or opsonized yeast.

Schistosoma mansoni modulation of phagocytosis in Biomphalaria glabrata

Both short-term (3 hr) exposure of Biomphalaria glabrata snails (M-line and 13-16-R1) to Schistosoma mansoni (PR1) miracidia and in vitro incubation of parasite sporocysts with host hemolymph components altered host phagocytic ability. Hemocytes obtained from susceptible (M-line) snails that had been exposed to parasite miracidia for 3 hr showed reduced levels of phagocytosis of yeast cells in vitro compared to hemocytes from unexposed individuals. Incubation of whole hemolymph with sporocysts in vitro also reduced yeast phagocytosis in this susceptible strain. In contrast, resistant (13-16-R1) hemocytes showed increased levels of yeast phagocytosis after in vitro incubation with the parasite, and the opsonic properties of 13-16-R1 plasma were greater after exposure of snails to miracidia. These strain-specific effects of S. mansoni on host hemocyte phagocytosis and plasma opsonization were seen only when both plasma and hemocytes were present at the time of exposure to the parasite.

Proteinase inhibitory activity in the plasma of a mollusc: Evidence for the presence of α-macroglobulin in Biomphalaria glabrata

1. A methylamine-sensitive inhibitor was present in the plasma of B. glabrata. 2. This inhibitor decreased trypsin activity against a protein substrate, however trypsin retained activity against a low molecular weight substrate in the presence of the inhibitor. 3. Snail plasma protected trypsin from inhibition by soybean trypsin inhibitor. 4. The results give evidence for an alpha-macroglobulin proteinase inhibitor in the plasma of this gastropod mollusc.

The effect of Schistosoma Haematobium infection on the growth and fecundity of three sympatric species of bulinid snails

Three species of bulinid snails were monitored to determine the effect of infection with 2 sympatric strains of Schistosoma haematobium on longevity, growth, and reproductive output, from the onset of cercarial production until death. Bulinus senegalensis was least affected by infection, with total fecundity reduced by less than 34%. Infected Bulinus truncatus showed an acceleration in growth accompanied by a 63% reduction in fecundity, although the majority of snails continued to oviposit at a low level. The longest-lived snails in this study, Bulinus globosus, showed decreased growth and survival when infected. In addition a significant proportion of infected individuals of this species failed to oviposit, and those that retained some reproductive capacity produced fewer embryos than controls. Total fecundity of B. globosus was reduced almost 90% by infection with S. haematobium, yet long-term monitoring of individuals showed that reproductive activity recovered when parasite productivity was low. Results from this and similar studies indicate that the time of infection by a trematode in relation to reproductive maturity of the molluscan host is important in determining the subsequent effects on host growth and fecundity.

Phagocytosis and invertebrate opsonins in relation to parasitism.

At the Woods Hole Meeting, the group of papers in which this was the first to be presented was challenged to address the question: Was a cellular defense the first defense? Phagocytosis is a very primitive cell behavior. Our protist ancestors relied on it as a means of obtaining food, and this was retained by the earliest (metazoan) animals-the sponges, cnidarians, and free-living flatworms. If we entertain the possibility that the origins of the eukaryote condition depended, in part, on the acquisition of intracellular mutualists that are retained as mitochondria, plastids, and other “organelles,” we de fact0 admit the likelihood that members of the Monera (bacteria) were entering bodies long before the animals (Metazoa) appeared. Phagocytic cells were relied on to meet such challenges in early metazoans. Before the evolution of circulatory systems, ameboid phagocytes wandered through the body, phagocytosing foreign materials and damaged self. These considerations do not predicate the conclusion that a cellular defense was the first defense. Allelochemicals released by protists and metazoans alike provide effective “humoral” defenses, killing or stopping the growth of other species in the habitat. We proffer that it is unimportant to be able to say which came first-cellular or humoral; we must be content in the knowledge that both forms of defense have been present from the very earliest times.

CULTURE OF CELLS FROM JUVENILE WORMS OF SCHISTOSOMA MANSONI

Tissue disruption methods were developed and serum-free cell culture media formulated for the maintenance in vitro of cells from juvenile worms (day 18 after infection) of Schistosoma mansoni. Cultures maintained viability for up to 6 mo when plated on a feeder layer of irradiated rat liver cells and survived primarily as clusters of small (2.5-4 microns diameter) cells with a high nuclear-to-cytoplasmic ratio and relatively few organelles identified by electron microscopy. Cultures synthesized a protein profile similar to that of intact worms, and the cell clusters maintained a time- and concentration-dependent contractile response to serotonin. Cells synthesizing DNA were detected by precursor incorporation and flow cytometry in cultures initially and also after several weeks in vitro, although the percentage of cells synthesizing DNA decreased with time. Efforts to identify peptide growth factor-responsive tyrosine phosphorylation were negative, and the overall amount of S. mansoni phosphotyrosine-containing proteins identified by western blot with anti-phosphotyrosine monoclonal antibody was much less than that found in a peptide growth factor-responsive mouse cell line.

Phagocytosis of latex beads by Biomphalaria glabrata hemocytes is modulated in a strain-specific manner by adsorbed plasma components.

Circulating phagocytes of the mollusc Biomphalaria glabrata help determine the fate of invading Schistosoma mansoni larvae. In mediating cytotoxicity against this trematode parasite, phagocytic hemocytes may interact with unidentified plasma components. In this study, positively and negatively charged latex beads were coated with plasma for use in in vitro phagocytosis assays. Hemocytes from S. mansoni-susceptible and -resistant strains of B. glabrata were tested for their ability to internalize beads coated with homologous and heterologous plasma components. Electrophoretic analysis showed that differences in the plasma polypeptides adsorbed to latex beads were charge-related, but no consistent strain-specific differences were detected. Hemocytes from a susceptible strain phagocytosed negatively charged latex pretreated in plasma form a resistant strain of snail at a higher rate than beads treated in plasma from the susceptible strain. Pretreatment of susceptible hemocytes with resistant plasma reduced subsequent phagocytosis of beads coated with resistant plasma, consistent with blocking of receptors on the susceptible hemocytes with resistant strain plasma components. Exogenous lectins detected no differences in the carbohydrates on beads coated with plasma from either snail strain. However, strain differences in endogenous lectins are suggested by differential binding of neoglycoproteins to plasma coated beads. The plasma component(s) responsible for the differential phagocytosis of beads treated in the two plasma strains by hemocytes from the susceptible strain of B. glabrata remain(s) to be identified.

Killing of Schistosoma mansoni Sporocysts by Biomphalaria glabrata Hemolymph in vitro. Alteration of Hemocyte Behavior after Poly-L-Lysine Treatment of Plastic, and the Kinetics of Killing by Different Host Strains

Behavior of hemocytes of the gastropod mollusc Biomphalaria glabrata was markedly changed on plastic by treatment of the substrate with 0.1 mg/ml poly-L-lysine compared to behavior on untreated plastic. On lysine, the cells showed minimal spreading, moved significantly faster, and formed aggregates. Cell-mediated cytotoxicity (CMC) assays were set up on the modified and untreated substrates to compare the killing capacities of B. glabrata hemocytes against Schistosoma mansoni sporocysts. Hemolymph from 1316-R1 (resistant) snails showed higher killing in lysine-treated wells; no significant difference in sporocyst mortality was observed in MO (susceptible) hemolymph between treated and untreated wells. The CMC assays on poly-L-lysine-treated plastic were used to compare the kinetics of parasite killing in hemolymph from 2 susceptible (MO, MRLc) and 2 resistant (1316-R1, 10R2) host strains. Marked differences could be observed between the two resistant snail strains, suggesting different mechanisms of parasite recognition, killing, or both.

Inhibition of cysteine proteinase from Schistosoma mansoni larvae by alpha-macroglobulin from the plasma of Biomphalaria glabrata.

The hemolymph of Biomphalaria glabrata, a molluscan host of Schistosoma mansoni, contains an alpha-macroglobulin proteinase inhibitor (alphaM). In this study we have demonstrated that this host molecule inhibits a cysteine proteinase produced by larval S. mansoni. Inhibition by alphaM involves conformational changes through proteolytic cleavage by the proteinase, thus the enzyme must be active for interactions to occur. A specific cysteine proteinase inhibitor (E64) was used to block the interaction between parasite cysteine proteinase and host alphaM during an in vitro parasite killing assay. Increased sporocyst mortality was not observed in hemolymph from susceptible strains of B. glabrata when E64 was included, nor was there decreased killing in similarly treated hemolymph from a resistant strain. This suggests that the inhibition of this parasite proteinase by host alphaM is not involved in processes determining either resistance or susceptibility to this trematode.