Abstract. The infective larvae of Necator americanus were shown to secrete all mechanistic classes of proteolytic enzymes with two overall pH optima of 6.5 and 8.5 using fluorescein isothiocyanate-labeled casein as the substrate. Since infective larvae are obligate skin penetrators, the effect of each of these enzyme classes against macromolecules derived from human skin was examined. Larval secretions were shown to degrade collagen types I, III, IV, and V, fibronectin, laminin, and elastin. All the skin macromolecules tested were hydrolyzed by aspartyl proteinase activity, which was inhibitable by pepstatin A. Collagen and elastin was also hydrolyzed by metalloproteinase activity, while the serine proteinase activity hydrolyzed only elastin. As a consequence of these experiments, the effect of proteinase inhibitors on the penetration of live larvae through hamster skin was tested. Larval penetration was significantly inhibited only by pepstatin A, confirming the importance of the aspartyl proteinase activity during the skin penetration process.Necator americanus is a human pathogen that invades the body by penetrating the skin. In 1982 Matthews 1 showed that cellular destruction of the skin during larval penetration through the epidermis was mediated by an undefined enzymatic process, optimally active at pH 8. Subsequently, Salafsky and others 2 developed a gelatin-agar membrane as a model for hookworm skin penetration and showed that serine, and possibly cysteinyl, proteinase activities were responsible for larval penetration through this type of membrane. The presence of cysteinyl and serine proteinases in larval secretions was confirmed using gelatin substrate sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 3 and it has been further demonstrated that live N. americanus larvae secrete a gelatinolytic metalloproteinase proteinase. 4 Despite the obvious presence of proteolytic enzymes in the excretory-secretory (ES) products of N. americanus larvae, an exact role for each of these proteinases has yet to be defined. Consequently, and given the fact that the larvae are obligate skin penetrators, we now describe attempts to assign a role during skin penetration for the previously described proteinases secreted by N. americanus larvae. The hydrolysis of a number of skin macromolecules has been studied and the enzymes responsible for their degradation have been implicated using standard proteinase inhibitors. Despite being a human parasite, N. americanus can also be maintained in the golden hamster. 5 Therefore, to confirm the data obtained using individual skin macromolecules, the effects of proteinase inhibitors on the penetration of live larvae through excised hamster skin was also studied.
MATERIALS AND METHODSPreparation of N. americanus larval ES products. Necator americanus was maintained in syngeneic DSN hamsters as described by Sen and Seth. 5 Infective larvae were cultured from fecal material by a method modified from Harada and Mori 6 and previously described by Kumar and Pritchard. ...