Dissection of the mechanisms of cytolytic and antibacterial activity of lysenin, a defence protein of the annelid Eisenia fetida

The body fluid of earthworms is known to contain a variety of cytolytic and antibacterial activities to combat potential pathogens that may migrate from the environment into the body cavity. In the annelid Eisenia fetida, a multi-gene family exists that give rise to several isoforms, which display hemolytic and antibacterial activity. The hemolytic activity of lysenin, a major isoform, is known to be strictly dependend on sphingomyelin. As bacteria are devoid of sphingomyelin, the nature of the antibacterial activity of lysenin-like proteins appeared obscure. Here, we report the recombinant expression of lysenin, a defined single entity, which exerted hemolytic, antibacterial and membrane-permeabilizing activity comparable to that of the natural counterpart. Experiments using fluorescence resonance energy transfer spectroscopy with liposomes and planar lipid bilayers demonstrated membrane insertion and single channel fluctuations in the presence of sphingomyelin. By monitoring the lipid specificity of lysenin and its molecular organization on different target cell membranes, it became evident that oligomerization to stable pore-like structures occurs on erythrocytes but does not occur on bacterial membranes. However, bacterial membranes became permeabilized by lysenin, albeit much slower. Accordingly, lysenin appears to display sphingomyelin-dependent and sphingomyelin-independent activities to kill various foreign intruders of the earthworm's coelomic cavity.