Study of immunomodulatory activity and anti-coagulation factors from the salivary gland of the black fly, Simulium vittatum
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Vector arthropods modulate many physiological responses of hosts in order to acquire a blood meal. They create wounds that initiate hosts’ hemostatic responses to stop bleeding. To counteract this response, vectors inject saliva containing many anti-hemostatic factors. However, injection of saliva, (recognized as foreign by the hosts) also elicits host immune responses to remove such materials, which may reduce the anti-hemostatic activity. Vector saliva therefore contains factors that modulate host immune responses. As vectors create wounds and modulate hosts’ physiological responses, they may serendipitously aid establishment of pathogenic infections. Black flies belong to the Simuliidae, a family of lower Diptera, and are important vectors of onchocerciasis (river blindness) and vesicular stomatitis virus, but also cause a direct impact including death of animals due to simuliotoxicosis. However, the effects of black fly salivary gland components have not been extensively investigated. This dissertation describes immunomodulatory properties of the salivary glands of the black fly, Simulium vittatum, using mouse models. Black fly salivary gland extract (SGE) inhibited mitogen-stimulated T-cell and B-cell proliferation. T-cell proliferation was inhibited more strongly than B-cell proliferation. SGE induced apoptosis specifically in CD4+ and CD8+ T cells. The component responsible for the inhibition was determined to be a protein larger than 50 kDa. SGE enhanced production of nitric oxide (NO) from LPS-stimulated macrophages at low concentrations and inhibited it at a high concentration. As NO-producing macrophages can inhibit T-cell proliferation, altogether these results imply that SGE specifically inhibits the T-cell mediated adaptive immune response. NO-enhancing effects of SGE on macrophages could also explain the fatal effect of simuliotoxicosis. This dissertation also describes functions of putative anti-coagulation factors in S. vittatum salivary glands. Two Kunitz proteins (SV-66 and SV-170) were expressed in a bacterial system, and recombinant SV-66 exhibited anti-coagulation activity. Mutant forms revealed that the residues in the reactive-site loop (RSL) are important in the anti-coagulation activity, as is typical for canonical Kunitz family proteins. SV-66 inhibited a wide range of serine proteases, but with relatively weak affinity. These results indicate that SV-66 could inhibit coagulation as well as modulating local inflammation.