Randall C. Bender

Mechanisms of molluscan host resistance and of parasite strategies for survival.

In parallel with massive research efforts in human schistosomiasis over the past 30 years, persistent efforts have been made to understand the basis for compatibility and incompatibility in molluscan schistosomiasis. Snail plasma contains molecules that are toxic to trematodes, but these seem to kill only species that never parasitize the mollusc used as the source of plasma. A sporocyst will be killed actively by haemocytes alone if they are from a snail that is resistant to the trematode. Oxygen-dependent killing mechanisms play a major role. Enzymes such as NADPH oxidase, superoxide dismutase, myeloperoxidase and nitric oxide synthase are critical components of the putative killing pathways. Metabolic intermediates such as hydrogen peroxide and nitric oxide appear to be more important against trematodes than the shorter-lived intermediates that are more important in anti-microbial defences. Products secreted by trematode larvae influence the physiology of snail haemocytes, implying active counter-defences mounted by the parasite, but these remain largely unexplored. A possible molecular basis for the susceptibility/resistance dichotomy in molluscan schistosomiasis is suggested to be deficient forms of enzymes in the respiratory burst pathway, and a selective disadvantage for schistosome resistance is an integral component of this model.

KILLING OF SCHISTOSOMA MANSONI SPOROCYSTS BY HEMOCYTES FROM RESISTANT BIOMPHALARIA GLABRATA: ROLE OF REACTIVE OXYGEN SPECIES

The fate of Schistosoma mansoni (Trematoda) sporocysts in its molluscan host Biomphalaria glabrata (Gastropoda) is determined by circulating phagocytes (hemocytes). When the parasite invades a resistant snail, it is attacked and destroyed by hemocytes, whereas in a susceptible host it remains unaffected. We used 3 inbred strains of B. glabrata: 13-16-R1 and 10-R2, which are resistant to the PR-1 strain of S. mansoni, and M-line Oregon (MO), which is susceptible to PR-1. In an in vitro killing assay using plasma-free hemocytes from these strains, the rate of parasite killing corresponded closely to the rate by which S. mansoni sporocysts are killed in vivo. Hemocytes from resistant snails killed more than 80% of S. mansoni sporocysts within 48 hr, whereas sporocyst mortality in the presence of hemocytes from susceptible snails was <10%. Using this in vitro assay, we assessed the involvement of reactive oxygen species (ROS) produced by resistant hemocytes, during killing of S. mansoni sporocysts. Inhibition of NADPH oxidase significantly reduced sporocyst killing by 13-16-R1 hemocytes, indicating that ROS play an important role in normal killing. Reduction of hydrogen peroxide (H2O2) by including catalase in the killing assay increased parasite viability. Reduction of superoxide (O2−), however, by addition of superoxide dismutase or scavenging of hydroxyl radicals (·OH) and hypochlorous acid (HOCl) by addition of hypotaurine did not alter the rate of sporocyst killing by resistant hemocytes. We conclude that H2O2 is the ROS mainly responsible for killing.

Involvement of nitric oxide in killing of Schistosoma mansoni sporocysts by hemocytes from resistant Biomphalaria glabrata

In strains of the snail Biomphalaria glabrata (Gastropoda) that are resistant to the parasite Schistosoma mansoni (Trematoda), hemocytes in the hemolymph are responsible for elimination of S. mansoni sporocysts. The defensive role of reactive nitrogen species was investigated in in vitro interactions between hemocytes derived from the resistant 13-16-R1 strain of B. glabrata and the parasite. The nitric oxide synthase (NOS) inhibitor Nω-nitro-l-arginine methylester (l-NAME) and the nitric oxide (NO·) scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide reduced cell-mediated killing of S. mansoni sporocysts. To determine if peroxynitrite (ONOO−) is involved in killing, assays were run in the presence of the ONOO− scavengers uric acid and deferoxamine. These did not influence the rate of parasite killing, indicating that NO· is directly responsible for mediating cytotoxicity, but ONOO− is not. The combination of the NOS inhibitor l-NAME and catalase, an enzyme that detoxifies hydrogen peroxide (H2O2), reduced average sporocyst mortality to a greater extent than l-NAME alone. Killing of the sporocysts was, however, not totally inhibited. It is suggested that NO· and H2O2 are both involved in hemocyte-mediated toxicity of 13-16-R1 B. glabrata against S. mansoni sporocysts.

Respiratory burst of Biomphalaria glabrata hemocytes: Schistosoma mansoni-resistant snails produce more extracellular H2O2 than susceptible snails.

The production of reactive oxygen species by hemocytes from the gastropod Biomphalaria glabrata has been linked to their ability to kill the trematode parasite Schistosoma mansoni. For 2 laboratory strains of the snail, 1 resistant (13-16-R1) and 1 susceptible (MO) to the PR1 strain of S. mansoni, we compared hemocyte production of extracellular hydrogen peroxide when stimulated with the protein kinase C agonist phorbol myristate acetate (PMA). The time course of the PMA-induced response is similar in both strains with respect to onset, peak production, and termination of the respiratory burst. However, the magnitude of the response differs between strains, in that hemocytes from resistant snails generate significantly more hydrogen peroxide. These findings suggest that the capacity to produce hydrogen peroxide could be critical in determining susceptibility or resistance to S. mansoni.

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.

Hexachlorobenzene-induced porphyria in Japanese quail: effect of pretreatment with phenobarbital or β-naphthoflavone

In an effort to determine the role that metabolism by the cytochrome P-450 system plays in the development of hexachlorobenzene (HCB)-induced porphyria, Japanese quail were pretreated with either beta-naphthoflavone (BNF) or phenobarbital (PB) and then treated with HCB. PB or BNF pretreatment appeared to have no effect on the response of quail hepatic enzymes to HCB. There were no differences between the two groups in either the content of cytochrome P-450 or the activities of NADPH-cytochrome c reductase, glutathione transferase (microsomal or cytosolic), ethoxycoumarin-O-deethylase or ethoxyresorufin-O-deethylase following HCB treatment. These pretreatments did, however, markedly influence the development of porphyria in quail. BNF-treated birds had higher delta-aminolevulinic acid-synthetase (ALA-S) activities and developed porphyria much more rapidly than birds treated with HCB alone. Birds pretreated with PB did not exhibit porphyria even following 10 days of HCB. Although the ALA-S activities in this group were elevated slightly following HCB, they were about one-half of those seen in the BNF-pretreated HCB-treated group. These results may reflect a difference between the PB and BNF groups in the production of a porphyrogenic metabolite of HCB.

Cytochrome P-450 isozymes in salmonids determined with antibodies to purified forms of P-450 from rainbow trout

Abstract Purification of cytochromes P-450 from liver microsomes of β-naphthoflavone (BNF)-fed rainbow trout yielded three apparently homogeneous forms. The major form (LM4b)∗ appears to be a P-448 type of cytochrome. A minor form (LM4a), having properties very similar to LM4b, was also obtained. In addition, a P-450 form (LM2) was isolated, with properties quite different from LM4a or LM4b, including a high rate of aflatoxin B1 (AFB1) metabolism (Williams & Buhler, 1983c). Antibodies to all three forms were obtained from rabbits. The IgGs prepared against LM4a and LM4b both cross-reacted (forming lines of identity) equally well with both antigens on Ouchterlony plates. Rat P-448 cross-reacted (without lines of identity) with both LM4a- and LM4b-IgG. LM4b-IgG was much more effective than LM4a-IgG for inhibition of LM4a or LM4b reconstituted benzo[a]pyrene (BP) hydroxylase, suggesting that these two antibodies recognize different antigenic sites. The LM2-IgG did not cross-react with any of the other rat or trout cytochromes P-450 examined. Levels of LM2 and LM4b in microsomes from untreated, polychlorinated biphenyl (PCB), phenobarbital (PB) or BNF-treated trout were estimated with an immunological technique involving electrophoresis on SDS-PAGE, followed by transfer to nitrocellulose and staining with either LM 2 - or LM4b -IgG. The ratio of LM 4b LM 2 in microsomes from PCB- or BNF-treated rainbow trout was much higher than 1, whereas the reverse was true with microsomes from untreated rainbow trout. These results are consistent with previous observations (Vodicnik et al., 1982) that pretreatment with BNF induced the synthesis of a P-448 type cyytochrome, presumably responsible for the great increase in the metabolism and activation of BP seen in these fish. Conversely, pretreatment with PB did not affect the levels of either LM2 or LM4b. This specific immunological technique should make it possible to assay the levels of these P-450 and P-448 isozymes in various strains of rainbow trout and other species of fish. In addition, the effect of age, sex, diet and exposure to P-450 and P-448 inducers could be examined and, perhaps, utilized to predict the relative risk of certain populations to pollutants activated by these different isozymes.

Schistosoma mansoni sporocysts in culture: host plasma hemoglobin contributes to in vitro oxidative stress.

The initiation and promotion of sporocyst propagation and subsequent production of cercariae by intramolluscan larval stages of digenic trematodes are thought to depend on mollusc-derived factors. The ability to investigate this using in vitro cultures of Schistosoma mansoni sporocysts has been impeded by the fact that plasma from the host, Biomphalaria glabrata, becomes toxic to the parasite in long-term cultures. The present study identifies hemoglobin as the plasma component responsible for this toxicity. The addition of the enzyme catalase to sporocyst cultures neutralized the toxic effects of both purified hemoglobin and whole plasma, suggesting that the generation of H2O2 as a consequence of hemoglobin oxidation is the mechanism of plasma toxicity. Furthermore, cultures incubated in unconditioned schistosome medium with plasma plus catalase yielded significantly higher numbers of daughter sporocysts than cultures with media or plasma alone, but not higher than cultures with catalase alone. These latter results suggest that the oxidative environment and the antioxidant capacity of the media are critical factors for in vitro propagation of S. mansoni sporocysts.

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.