Secretion of Metalloproteases by Living Infective Larvae of Necator americanus

Abstract

Fresh living third-stage larvae of Necator americanus released a significant amount of label within 2 hr of their incubation on 125I-labeled gelatin-coated polystyrene plastic plates. This protease activity was primarily susceptible to o-phenanthroline, which identifies the activity as predominantly metalloprotease.

Proteases in Blood-Feeding Nematodes and Their Potential as Vaccine Candidates

“…ES degraded IgG, IgA and IgM (but not IgD or IgE) at the Fc region, this being inhibited by PMSF indicative of the action of a serine protease. 69 Taken together these studies suggest that proteases are used by hookworm larvae to establish infection, by the adults for feeding purposes and by both stages to evade the host immune response. Further evidence for the latter role was provided when it was shown that adult N. americanus ES products inhibited eosinophil recruitment in vivo by proteolytic degradation of eotaxin.…”

Necator americanus (human hookworm) aspartyl proteinases and digestion of skin macromolecules during skin penetration.

“…APMSF ϭ 4-(amidinophenyl)methane sulfonyl fluoride; Phe ϭ 1,10-phenanthroline; Pep A ϭ pepstatin A. mar and Pritchard. 3,4 In addition to these activities, the presence of aspartyl proteinase activity has been demonstrated for the first time Using FITC-labeled casein as the substrate, the pH optimum of the overall proteinase activity secreted by N. americanus larvae was shown to be pH 6.5, with a lesser peak of activity at pH 8 ( Figure 1). At pH 6.5 and pH 8, the proteinase activities were characterized using a number of proteinase inhibitors (Table 1) At pH 6.5, the predominant proteinase activity was shown to be aspartyl in nature.…”

“…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.…”

Hookworm (Necator americanus) Larval Enzymes Disrupt Human Vascular Endothelium

“…Using protease inhibition assays, 21 we were able to confirm the presence of serine, cysteinyl, aspartyl, and metalloproteases in both EF and ES products, as described before. 1,18 Consequently, the use of inhibitors in combination resulted in the most significant reduction in protease activity in both EF and ES products, although complete inhibition was not achieved with inhibitor cocktails even though each inhibitor was quality controlled against its cognate enzyme class, Figure 7. Graphs showing effects of the apical/basal administration of larval products on human umbilical vein endothelial cell (HUVEC) secretions of ( A ) IL-6 and ( B ) IL-8.…”

“…[1][2][3] As part of the infection process, larvae are required to enter the vasculature, presumably by disrupting the vascular endothelium, as has been shown for other parasites such as Dirofilaria immitis 4 and Schistosoma mansoni . 5 The vascular endothelium forms the intact, innermost layer of the vessel wall, which acts as a physical barrier to maintain vascular homeostasis and regulate vascular permeability.…”