Cardiac glycosides are a prime example of highly toxic plant secondary compounds, which block an essential transmembrane carrier in animals, the Na,K-ATPase. Nevertheless, over 100 insect species from diverse orders are known to feed on plants containing these compounds and in many cases these toxins are additionally sequestered without ill effect. We investigated whether the insects' adaptations for handling cardiac glycosides are based on a single physiological mechanism or whether various strategies have evolved across groups. We analyzed gene sequences of the Na, K-ATPase a-subunit from cardiac glycoside-adapted insects and screened for amino-acid substitutions which could alter the affinity of the enzyme toward cardiac glycosides. In representatives from five insect orders, separated by over 300 million years of evolutionary divergence, we uncovered amino-acid substitutions at identical positions. Especially striking is the convergent substitution of a histidine for the conserved asparagine at position 122, which we report here for the first time in a sawfly, Monophadnus latus Costa (Hymenoptera: Tenthredinidae), and which was previously observed in the orders Lepidoptera, Coleoptera, Hemiptera, and Diptera. Prior in vitro expression and enzyme assays indicated that this substitution as well as combined substitutions with other residues result in a strongly increased cardenolide resistance of the Na,K-ATPase. The substitutions to threonine 111 and histidine 122 observed in M. latus are highly effective and were previously known only in lygaeid bugs. However, not all insects dealing with dietary cardenolides rely on target-site insensitivity as a mechanism of resistance. An impermeable gut or the exclusion of cardenolides from the nervous tissue with the greatest expression of Na,K-ATPase by the perineurium, the insect blood brain barrier, apparently represent alternative strategies. Immuno-histochemical data presented here support the existence of P-glycoprotein-like efflux transporters in insect gut membranes that might prevent the uptake of allelochemicals like cardenolides.

Monarch butterflies, Danaus plexippus, migrate long distances over which they encounter host plants that vary broadly in toxic cardenolides. Remarkably little is understood about the mechanisms of sequestration in Lepidoptera that lay eggs on host plants ranging in such toxins. Using closely-related milkweed host plants that differ more than ten-fold in cardenolide concentrations, we mechanistically address the intake, sequestration, and excretion of cardenolides by monarchs. We show that on high cardenolide plant species, adult butterflies saturate in cardenolides, resulting in lower concentrations than in leaves, while on low cardenolide plants, butterflies concentrate toxins. Butterflies appear to focus their sequestration on particular compounds, as the diversity of cardenolides is highest in plant leaves, lower in frass, and least in adult butterflies. Among the variety of cardenolides produced by the plant, sequestered compounds may be less toxic to the butterflies themselves, as they are more polar on average than those in leaves. In accordance with this, results from an in vitro assay based on inhibition of Na + /K + ATPase (the physiological target of cardenolides) showed that on two milkweed species, including the high cardenolide A. perennis, extracts from butterflies have lower inhibitory effects than leaves when standardized by cardenolide concentration, indicating selective sequestration of less toxic compounds from these host plants. To understand how ontogeny shapes sequestration, we examined cardenolide concentrations in caterpillar body tissues and hemolymph over the course of development. Caterpillars sequestered higher concentrations of cardenolides as early instars than as late instars, but within the fifth instar, concentration increased with body mass. Although it appears that large amounts of sequestration occurs in early instars, a host switching experiment revealed that caterpillars can compensate for feeding on low cardenolide host plants with substantial sequestration in the fifth instar. We highlight commonalities and striking differences in the mechanisms of sequestration depending on host plant chemistry and developmental stage, which have important implications for monarch defense.

Translocation of virulence effector proteins through the type III secretion system (T3SS) is essential for the virulence of many medically relevant Gram‐negative bacteria. The T3SS ATPases are conserved components that specifically recognize chaperone–effector complexes and energize effector secretion through the system. It is thought that functional T3SS ATPases assemble into a cylindrical structure maintained by their N‐terminal domains. Using size‐exclusion chromatography coupled to multi‐angle light scattering and native mass spectrometry, we show that in the absence of the N‐terminal oligomerization domain the Salmonella T3SS ATPase InvC can form monomers and dimers in solution. We also present for the first time a 2.05 å resolution crystal structure of InvC lacking the oligomerization domain (InvCΔ79) and map the amino acids suggested for ATPase intersubunit interaction, binding to other T3SS proteins and chaperone–effector recognition. Furthermore, we validate the InvC ATP‐binding site by co‐crystallization of InvCΔ79 with ATPγS (2.65 å) and ADP (2.80 å). Upon ATP‐analogue recognition, these structures reveal remodeling of the ATP‐binding site and conformational changes of two loops located outside of the catalytic site. Both loops face the central pore of the predicted InvC cylinder and are essential for the function of the T3SS ATPase. Our results present a fine functional and structural correlation of InvC and provide further details of the homo‐oligomerization process and ATP‐dependent conformational changes underlying the T3SS ATPase activity.

The type III secretion system (T3SS) is a large, transmembrane protein machinery used by various pathogenic gram‐negative bacteria to transport virulence factors into the host cell during infection. Understanding the structure of T3SSs is crucial for future developments of therapeutics that could target this system. However, much of the knowledge about the structure of T3SS is available only for Salmonella, and it is unclear how this large assembly is conserved across species. Here, we combined cryo‐electron microscopy, cross‐linking mass spectrometry, and integrative modeling to determine the structure of the T3SS needle complex from Shigella flexneri. We show that the Shigella T3SS exhibits unique features distinguishing it from other structurally characterized T3SSs. The secretin pore complex adopts a new fold of its C‐terminal S domain and the pilotin MxiM[SctG] locates around the outer surface of the pore. The export apparatus structure exhibits a conserved pseudohelical arrangement but includes the N‐terminal domain of the SpaS[SctU] subunit, which was not present in any of the previously published virulence‐related T3SS structures. Similar to other T3SSs, however, the apparatus is anchored within the needle complex by a network of flexible linkers that either adjust conformation to connect to equivalent patches on the secretin oligomer or bind distinct surface patches at the same height of the export apparatus. The conserved and unique features delineated by our analysis highlight the necessity to analyze T3SS in a species‐specific manner, in order to fully understand the underlying molecular mechanisms of these systems. The structure of the type III secretion system from Shigella flexneri delineates conserved and unique features, which could be used for the development of broad‐range therapeutics.