Lactobacillus iners is a common constituent of the human vaginal microbiota. This species was only recently characterized due to its fastidious growth requirements and has been hypothesized to play a role in the pathogenesis of bacterial vaginosis. Here we present the identification and molecular characterization of a protein toxin produced by L. iners. The L. iners genome encodes an open reading frame with significant primary sequence similarity to intermedilysin (ILY; 69.2% similarity) and vaginolysin (VLY; 68.4% similarity), the cholesterol-dependent cytolysins from Streptococcus intermedius and Gardnerella vaginalis, respectively. Clinical isolates of L. iners produce this protein, inerolysin (INY), during growth in vitro, as assessed by Western analysis. INY is a pore-forming toxin that is activated by reducing agents and inhibited by excess cholesterol. It is active across a pH range of 4.5 to 6.0 but is inactive at pH 7.4. At sublytic concentrations, INY activates p38 mitogen-activated protein kinase and allows entry of fluorescent phalloidin into the cytoplasm of epithelial cells. Unlike VLY and ILY, which are human specific, INY is active against cells from a broad range of species. INY represents a new target for studies directed at understanding the role of L. iners in states of health and disease at the vaginal mucosal surface.The cholesterol-dependent cytolysins (CDCs) are a family of protein toxins produced by a wide range of Gram-positive bacteria. CDCs share several characteristics, including a fourdomain structure, a requirement for membrane cholesterol for efficient activity, and an ability to form large pores in host cells, exceeding 150 Ă
in diameter (38). In general, soluble CDC monomers are secreted into the extracellular environment and bind to target cell membranes through direct recognition of cholesterol or, in the cases of the human-specific toxins vaginolysin (VLY) from Gardnerella vaginalis and intermedilysin (ILY) from Streptococcus intermedius, via recognition of human CD59 on the target cell surface (12,14,21). Following membrane association, CDCs oligomerize to form a prepore structure, a process that is dependent upon the availability of cholesterol (22)(23)(24)34). In many cases, CDCs are required for virulence for their cognate organisms, and rather than acting solely as cytolytic toxins, CDCs may have more sophisticated roles in disease pathogenesis (15,18,30). Understanding CDC evolution and host specificity is of considerable interest and has been limited by incomplete knowledge of the diversity of the CDC family. In particular, characterization of cytolysins most closely related to those in which host specificity has evolved may provide additional insights into the mechanism and effects of such restriction.Lactobacillus iners is a relatively recently recognized member of the human vaginal microbiota (9, 33, 42) that was initially overlooked because of its inability to grow on de ManRogosa-Sharpe agar, which is normally used to isolate vaginal lactobacilli. In healthy women...