Inerolysin, a Cholesterol-Dependent Cytolysin Produced by Lactobacillus iners

Abstract

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 Å 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...

Comparative Functional Genomics of Lactobacillus spp. Reveals Possible Mechanisms for Specialization of Vaginal Lactobacilli to Their Environment

“…This likely corresponds to inerolysin (52), a broad-range cholesterol-dependent cytolysin. This toxin forms pores in epithelial cells and activates proinflammatory signaling and so is suggested to be a virulence factor for L. iners (55). In support of this is the fact that this cytolysin is upregulated 6-fold under bacterial vaginosis conditions (56).…”

Association between Obesity and Cervical Microflora Dominated by Lactobacillus iners in Korean Women

“…Inerolysin, a pore-forming toxic protein, activates p38 mitogen-activated protein kinase and allows the entry of molecules from the extracellular space into the cytoplasm of epithelial cells. Cytolysin may serve a defensive function by killing immune cells and may make new anatomical niches available by disrupting epithelial barriers (28). What is remarkable is that the distribution of cholesterol in the cell membranes may be important for cytolysin binding (29).…”

“…The atypical characteristics of L. iners also include a protein produced by the species. Clinical isolates of L. iners were shown to produce inerolysin, a cholesterol-dependent cytolysin, during growth in vitro, whereas this protein was absent in other Lactobacillus species (28). Inerolysin, a pore-forming toxic protein, activates p38 mitogen-activated protein kinase and allows the entry of molecules from the extracellular space into the cytoplasm of epithelial cells.…”

Current views on vaginal microbiota and its impact on pregnancy outcomes

“…Эта особенность данного вида является одним из факторов распространенности БВ у таких женщин. R. Rampersaud et al (2011) выделили и охарактеризовали токсин инеролизин, вы-деляемый L. iners, аналогичный интермедили-зину Streptococcus intermedius и вагинолизину G. vaginalis, роль которых в патогенезе дисбио-тических процессов доказана [19]. При этом происходит гибель других видов лактобацилл и повышение рН вагинальной среды.…”