# Hookworm genes encoding intestinal excreted-secreted proteins are transcriptionally upregulated in response to the host’s immune system

## TL;DR

This study identifies hookworm genes that respond to the host's immune system, potentially helping the parasite survive and offering new targets for vaccines or therapies.

## Contribution

The study identifies 153 hookworm genes encoding excreted-secreted proteins that are upregulated in response to host immunity, suggesting novel virulence factors and vaccine targets.

## Key findings

- 153 excreted-secreted genes in A. ceylanicum are upregulated in normal hosts compared to immunosuppressed hosts.
- Immunoregulated genes are enriched for conserved and rapidly evolving proteins like CAP, ASPR, and TIMP.
- Male-biased expression is observed in 50.1% of positively immunoregulated intestinal genes.

## Abstract

Hookworms are intestinal parasitic nematodes that chronically infect ~500 million people. How hookworms successfully overcome host protective mechanisms is unclear, but it may involve hookworm proteins that digest host tissues, or counteract the host’s immune system, or both. To find such proteins in the zoonotic hookworm Ancylostoma ceylanicum, we used mass spectrometry to identify 565 genes encoding excreted-secreted (ES) proteins from adults, and used RNA-seq to identify genes expressed both in young adults (12 days post-infection) and in intestinal and non-intestinal tissues dissected from mature adults (19 days post-infection), infecting hamster hosts that either had normal immune systems or were immunosuppressed by dexamethasone. In adult A. ceylanicum, we observed 1,670 and 1,196 genes with intestine- and non-intestine-biased expression, respectively. Comparing hookworm gene activity in normal versus immunosuppressed hosts, we observed almost no changes of gene activity in 12-day young adults or non-intestinal 19-day adult tissues. However, in intestinal 19-day adult tissues, we observed 1,951 positively immunoregulated genes, and 137 genes that were negatively immunoregulated. Thus, immunoregulation was observed primarily in mature adult hookworm intestine directly exposed to host blood. Of positively immunoregulated intestinal genes, 50.1% (5.3-fold over background) also had male-biased expression, suggesting that male and female A. ceylanicum have different responses to the host immune system. We observed 153 ES genes showing positive immunoregulation in 19-day adult intestine, which disproportionately encoded CAP, ASPR, astacin, TIMP, TIL, ShK, and SCVP proteins, and that were enriched for ES gene orthologs in the dog hookworm Ancylostoma caninum, the human hookworm Necator americanus, or the related sheep parasite Haemonchus contortus. Such a mixture of rapidly evolving and conserved genes could comprise virulence factors enabling infection, provide new targets for vaccines against hookworm, and aid in developing therapies for immune-mediated diseases.

Hookworms chronically infect half a billion humans. They do this by partially suppressing their hosts’ immune systems with excreted or secreted (ES) proteins, which makes it difficult to protect against hookworm infections with vaccination or to cure them permanently with drugs. The hookworm Ancylostoma ceylanicum is a good laboratory model for this problem because it naturally infects both humans and other mammals. We used three approaches to define ES proteins of A. ceylanicum that might be crucial for host immunomodulation: we identified A. ceylanicum genes encoding ES proteins; we identified RNA expression levels of A. ceylanicum genes from intestines and non-intestinal tissues of adult hookworms; and we compared gene expression levels of hookworms infecting normal hamster hosts to those infecting hamsters immunosuppressed with dexamethasone. We found 153 genes in A. ceylanicum that encode ES proteins, are expressed in the intestine, and have stronger expression in normal hosts than in immunosuppressed hosts. These genes may be part of a feedback loop, where a hookworm dynamically responds to its host’s immune systems by upregulating these genes, excreting their protein products into their hosts’ bloodstreams, and immunomodulating their hosts. Some have relatives in other parasitic nematodes and may be an evolutionarily conserved set of virulence genes.