ABSTRACT: Hookworms are parasitic nematodes that chronically infect 170-440 million people, despite healthy human immune systems ending most infections quickly. How hookworms successfully overcome host resistance is unclear, but is thought to involve hookworm proteins that either digest host tissues or dampen the host's immune system. To find such proteins in the model hookworm Ancylostoma ceylanicum, we identified hookworm genes encoding proteins excreted into the environment, genes expressed in the intestine, and genes transcriptionally upregulated in response to a normal host immune system. To maximize our accuracy, we repredicted 33,190 protein-coding genes in A. ceylanicum with a BUSCO completeness of 95.3%. We collected excreted-secreted (ES) proteins from two sets of adult hookworms in vitro, and used mass spectrometry to identify 565 A. ceylanicum genes encoding ES proteins; we also used RNA-seq to identify A. ceylanicum genes expressed both in young adults (12 days post-infection) and in intestines and non-intestinal tissues dissected from mature adults (19 days post-infection), with hamster hosts that either had normal immune systems or were immunosuppressed by dexamethasone. In adult A. ceylanicum, we observed 2,145 and 1,694 genes with intestine- and non-intestine-biased expression, respectively. Comparing hookworm gene activity in normal versus immunosuppressed hosts, we observed essentially no changes of gene activity in non-blood-feeding 12-day young adults or non-intestinal 19-day adult tissues. However, in intestinal 19-day adult tissues, we observed 671 genes upregulated at least two-fold in normal hosts versus immunosuppressed hosts (positively immunoregulated), and 75 genes that were negatively immunoregulated; thus, immunoregulation was observed only in mature adult hookworm intestine, directly exposed to host blood. Of the positively immunoregulated intestinal genes, 590 (87.9%) showed male-biased gene expression (9.8-fold greater overlap than chance), suggesting that male and female hookworms have different responses to host immune systems; of the other 81 non-male-biased genes, 53 encoded ES proteins, a 38-fold greater overlap than expected by chance. Of these 53 immunoregulated ES genes, 26 had ES gene homologs in the closely related hookworm Ancylostoma caninum, five in the human hookworm Necator americanus, and four in the more distantly related strongylid parasite Haemonchus contortus. Such a mixture of rapidly evolving and conserved genes could serve as virulence factors enabling infection.
Erich Schwarz 
