Souadkia, Nahed
(2010)
Hookworms and the vascular endothelium.
PhD thesis, University of Nottingham.
Abstract
Background. Necator americanus is one of the major causes of human hookworm infection, affecting over 800 million people worldwide. Hookworm infections cause gastro-intestinal bleeding, anaemia and iron deficiency, and are associated with high rates of morbidity, especially in children. Although chemotherapy has proven effective, high rates of reinfection are reported in socioeconomically developing countries, possibly due to the short-term efficacy of anthelmintic drugs in addition to individual predisposition to these infections, raising interests in developing suitable alternatives to chemotherapy which are capable of providing complete, long-term protection against hookworms. Understanding of the molecular mechanisms used by Necator americanus larvae to penetrate the human skin and the vasculature would therefore aid the development of effective vaccines against this important pathogen.
Methods. First, Necator americanus larval exsheathing fluid (EF) and excretory/secretory products (ES) were profiled using gel electrophoresis and enzyme assays. Protease inhibitors against the main protease classes were used to determine which proteases are present in larval products. Second, the interaction of larval EF and ES products with human skin and extracellular matrix (ECM) macromolecules including collagens I, III, IV and V, fibronectin and laminin was investigated using western blots and protein separation by gel electrophoresis. Third, the impact of Necator americanus larval EF and ES on the endothelial barrier was examined using human umbilical vein endothelial cells (HUVEC). Permeability, an essential endothelial barrier function, was assessed during treatment with larval products, using transendothelial electrical resistance (TEER), and post-treatment using albumin-tracer flux. Finally, at the cellular level, responses to treatment with larval products were assessed by investigating molecular changes at cell-cell vascular endothelial (VE)-cadherin junctions and actin filaments, and by determining levels of secreted inflammatory cytokines, IL-6 and IL-8, and vascular endothelial growth factor (VEGF) in the culture medium.
Results. It would appear that a repertoire of larval proteases, including serine, cysteine, aspartyl and metalloprotinases, caused partial degradation of skin macromolecules, collagens I, III, IV and laminin while fibronectin was fully degraded. Proteolysis of skin- and ECM macromolecules was related to the characteristic presence of proteolytic enzymes in larval products. The presence of transglutaminase activity was confirmed in both EF and ES products.
Larval proteases caused a dose related increase in endothelial permeability, characterised by a decrease in monolayer resistance (TEER) with increased permeation of albumin tracer, which was minimal in the presence of a cocktail of protease inhibitors. These barrier changes were associated with disruption of junctional VE-cadherin and F-actin, the formation of intercellular gaps and an increase in endothelial secretion of IL-6 and IL-8.
Conclusions. Necator americanus larvae produce a repertoire of proteolytic enzymes which could play an important role in negotiating the skin and breaching the endothelium to gain access to the host’s blood circulation.
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