D3324: Toxin diversity contributes to target specificity in defensive bacterial symbiosis

How symbiont defenses distinguish friend from foe can influence fundamental dynamics in the host-parasite arms race. Bacterial toxins are frequently implicated as defensive effectors but the molecular basis of parasite specificity and host avoidance is not understood. Members of the genus Spiroplasma defend fruit fly hosts against parasitic nematodes and wasps through the activity of a protein toxin related to ricin and Shiga toxin that inactivates a conserved ribosomal stem-loop required for protein translation. Throughout Spiroplasma, ribosome-inactivating proteins (RIPs) exhibit dynamic evolution including duplication, domain swapping, and mobilization via plasmids. The resulting diversity of RIPs has been proposed to contribute to observed differences in defensive capabilities among closely related Spiroplasma strains; however, it is unknown if target specificity is determined by the toxins or through the action of other symbiont-encoded factors. We hypothesized that specificity is mediated by domains encoded within RIP toxins. To test this hypothesis, we purified a panel of Spiroplasma RIPs that together represent the diversity within the genus using an E. coli expression system. For each toxin, we measured N-glycosidase activity on free ribosomes and ribosomes within live insect cells. Consistent with our hypothesis, we report two levels of target specificity are mediated by RIPs, 1) at the ribosome itself, and 2) at the cell membrane and/or other barriers to the intracellular environment. These results suggest RIPs mediate both toxicity and specificity in parasite-host interactions and highlight multiple potential routes to evolution of resistance by parasites.