Hydra actinoporin-like toxin-1, an unusual hemolysin from the nematocyst venom of Hydra magnipapillata which belongs to an extended gene family

• The Hydra genome encodes six Hydra actinoporin-like toxin (HALT) genes, all of which are expressed in laboratory populations.
• Hydra's HALTs are phylogenetically distinct from anemone actinoporins.
• HALT genes differ from hemolytic and lethal anemone toxins in several important sequence features.
• We expressed and purified HALT-1, a toxin previously found in the nematocysts used for prey capture (stenoteles).
• HALT-1 reveals lower hemolytic activity, a larger pore size and different lipid affinity compared to Equinatoxin II.

Cnidarians rely on their nematocysts and the venom injected through these unique weaponry systems to catch prey and protect themselves from predators. The development and physiology of the nematocysts of Hydra magnipapillata, a classic model organism, have been intensively studied, yet the composition and biochemical activity of their venom components are mostly unknown. Here, we show that hydra actinoporin-like toxins (HALTs), which have previously been associated with Hydra nematocysts, belong to a multigene family comprising six genes, which have diverged from a single common ancestor. All six genes are expressed in a population of Hydra magnipapillata. When expressed recombinantly, HALT-1 (Δ-HYTX-Hma1a), an actinoporin-like protein found in the stenoteles (the main penetrating nematocysts used in prey capture), reveals hemolytic activity, albeit about two-thirds lower than that of the anemone actinoporin equinatoxin II (EqTII, Δ-AITX-Aeq1a). HALT-1 also differs from EqTII in the size of its pores, and likely does not utilize sphingomyelin as a membrane receptor. We describe features of the HALT-1 sequence which may contribute to this difference in activity, and speculate on the role of this unusual family of pore-forming toxins in the ecology of Hydra.