Comprehensive analysis of the secreted proteome of adult necator americanus hookworms

The human hookworm Necator americanus infects more than 400 million people worldwide, contributing substantially to the poverty in these regions. Adult stage N. americanus live in the small intestine of the human host where they inject excretory/secretory (ES) products into the mucosa. ES products have been characterized at the proteome level for a number of animal hookworm species, but until now, the difficulty in obtaining sufficient live N. ameri-canus has been an obstacle in characterizing the secretome of this important human patho-gen. Herein we describe the ES proteome of N. americanus and utilize this information along with RNA Seq data to conduct the first proteogenomic analysis of a parasitic helminth, significantly improving the available genome and thereby generating a robust description of the parasite secretome. The genome annotation resulted in a revised prediction of 3,425 fewer genes than initially reported, accompanied by a significant increase in the number of exons and introns, total gene length and the percentage of the genome covered by genes. Almost 200 ES proteins were identified by LC-MS/MS with SCP/TAPS proteins, ‘hypotheti-cal’ proteins and proteases among the most abundant families. These proteins were compared to commonly used model species of human parasitic infections, including Ancylostoma caninum, Nippostrongylus brasiliensis and Heligmosomoides polygyrus. SCP/ TAPS proteins are immunogenic in nematode infections, so we expressed four of those identified in this study in recombinant form and showed that they are all recognized to vary-ing degrees by serum antibodies from hookworm-infected subjects from a disease-endemic area of Brazil. Our findings provide valuable information on important families of proteins with both known and unknown functions that could be instrumental in host-parasite interac-tions, including protein families that might be key for parasite survival in the onslaught of robust immune responses, as well as vaccine and diagnostic targets.