Entomopathogenic nematodes (EPNs) are unique parasites due to their symbiosis with entomopathogenic bacteria and their ability to kill insect hosts quickly after infection. It is widely believed that EPNs rely on their bacterial partners for killing hosts. Here we disproved this theory by demonstrating that the in vitro activated infective juveniles (IJs) of Steinernema carpocapsae (a well-studied EPN species) release venom proteins that are lethal to several insects including Drosophila melanogaster. We confirmed that the in vitro activation is a good approximation of the in vivo process by comparing the transcriptomes of individual in vitro and in vivo activated IJs. We further analyzed the transcriptomes of non-activated and activated IJs and revealed a dramatic shift in gene expression during IJ activation. We also analyzed the venom proteome using mass spectrometry. Among the 472 venom proteins, proteases and protease inhibitors are especially abundant, and toxin-related proteins such as Shk domain-containing proteins and fatty acid- and retinol-binding proteins are also detected, which are potential candidates for suppressing the host immune system. Many of the venom proteins have conserved orthologs in vertebrate-parasitic nematodes and are differentially expressed during IJ activation, suggesting conserved functions in nematode parasitism. In summary, our findings strongly support a new model that S. carpocapsae and likely other Steinernema EPNs have a more active role in contributing to the pathogenicity of the nematode-bacterium complex than simply relying on their symbiotic bacteria. Furthermore, we propose that EPNs are a good model system for investigating vertebrate- and human-parasitic nematodes, especially regarding the function of excretory/secretory products.

Parasitic helminths release molecular effectors into their hosts and these effectors can directly damage host tissue and modulate host immunity. Excreted/secreted proteins (ESPs) are one category of parasite molecular effectors that are critical to their success within the host. However, most studies of nematode ESPs rely on in vitro stimulation or culture conditions to collect the ESPs, operating under the assumption that in vitro conditions mimic actual in vivo infection. This assumption is rarely if ever validated. Entomopathogenic nematodes (EPNs) are lethal parasites of insects that produce and release toxins into their insect hosts and are a powerful model parasite system. We compared transcriptional profiles of individual Steinernema feltiae nematodes at different time points of activation under in vitro and in vivo conditions and found that some but not all time points during in vitro parasite activation have similar transcriptional profiles with nematodes from in vivo infections. These findings highlight the importance of experimental validation of ESP collection conditions. Additionally, we found that a suite of genes in the neuropeptide pathway were downregulated as nematodes activated and infection progressed in vivo , suggesting that these genes are involved in host-seeking behavior and are less important during active infection. We then characterized the ESPs of activated S . feltiae infective juveniles (IJs) using mass spectrometry and identified 266 proteins that are released by these nematodes. In comparing these ESPs with those previously identified in activated S . carpocapsae IJs, we identified a core set of 52 proteins that are conserved and present in the ESPs of activated IJs of both species. These core venom proteins include both tissue-damaging and immune-modulating proteins, suggesting that the ESPs of these parasites include both a core set of effectors as well as a specialized set, more adapted to the particular hosts they infect.

Most nematodes are small worms that lack enough RNA for regular RNA-seq protocols without pooling hundred to thousand of individuals. We have adapted the Smart-seq2 protocol in order to sequence the transcriptome of an individual worm. While developed for individual Steinernema carpocapsae and Caenorhabditis elegans larvae as well as embryos, the protocol should be adaptable for other nematode species and small invertebrates. In addition, we describe how to analyze the RNA-seq results using the Galaxy online environment. We expect that this method will be useful for the studying gene expression variances of individual nematodes in wild type and mutant backgrounds.

LRIG1, a member of the LRIG family of transmembrane leucine rich repeat-containing proteins, is a negative regulator of receptor tyrosine kinase signaling and a tumor suppressor. LRIG1 expression is broadly decreased in human cancer and in breast cancer, low expression of LRIG1 has been linked to decreased relapse-free survival. Recently, low expression of LRIG1 was revealed to be an independent risk factor for breast cancer metastasis and death. These findings suggest that LRIG1 may oppose breast cancer cell motility and invasion, cellular processes which are fundamental to metastasis. However, very little is known of LRIG1 function in this regard. In this study, we demonstrate that LRIG1 is down-regulated during epithelial to mesenchymal transition (EMT) of human mammary epithelial cells, suggesting that LRIG1 expression may represent a barrier to EMT. Indeed, depletion of endogenous LRIG1 in human mammary epithelial cells expands the stem cell population, augments mammosphere formation and accelerates EMT. Conversely, expression of LRIG1 in highly invasive Basal B breast cancer cells provokes a mesenchymal to epithelial transition accompanied by a dramatic suppression of tumorsphere formation and a striking loss of invasive growth in three-dimensional culture. LRIG1 expression perturbs multiple signaling pathways and represses markers and effectors of the mesenchymal state. Furthermore, LRIG1 expression in MDA-MB-231 breast cancer cells significantly slows their growth as tumors, providing the first in vivo evidence that LRIG1 functions as a growth suppressor in breast cancer.

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