Abstract
The cholesterol dependent cytolysins (CDCs) are a family of pore-forming toxins produced by a wide range of bacteria. Some CDCs are important virulence factors for their cognate organisms, but their activity must be tightly regulated to ensure they operate at appropriate times and within the appropriate subcellular compartments. pH-dependent activity has been described for several CDCs, but the mechanism of such regulation has been studied in depth only for listeriolysin O (LLO), which senses environmental pH through a triad of acidic residues that mediate protein unfolding. Here we present data supporting a distinct mechanism for pH-dependence for inerolysin (INY), the CDC produced by Lactobacillus iners. Inerolysin (INY) has an acidic pH optimum with loss of activity at neutral pH. INY pH-dependence is characterized by reversible loss of pore formation with preservation of membrane binding. Fluorescent membrane probe assays indicated that INY insertion into host cell membranes, but not oligomerization, was defective at neutral pH. These data support the existence of a newly appreciated form of CDC pH-dependence functioning at a late stage of pore formation.
Highlights
Pore-forming toxin (PFT) production by bacterial species is a common feature of pathogenic bacteria and can play prominent roles in colonization, invasion and disease[1]
The use of pH as a regulatory factor is common among proteins[7,23], allowing for rapid regulation of their activity via conformational changes. pH dependent activity has been identified for a number of cholesterol dependent cytolysins (CDCs), including ILY, VLY, INY, perfringolysin O (PFO), and LLO8,13,14
Our prior work demonstrated that the basis of pH dependence in INY was likely distinct from the unfolding-based mechanism observed in listerolysin O (LLO)
Summary
Pore-forming toxin (PFT) production by bacterial species is a common feature of pathogenic bacteria and can play prominent roles in colonization, invasion and disease[1]. Because the charge present on these amino acids can be altered rapidly by pH and because toxin activity is dependent on the secondary and tertiary structure of the protein, changes in environmental pH may influence toxin activity. Studies have demonstrated that a number of toxins that are trafficked through the endosomal compartment become partially unfolded, exposing hydrophobic residues and facilitating insertion into membranes, including diphtheria toxin[5] as well as Clostridium difficile toxin B6 This feature has been noted for anthrax toxin, in which low pH induces unfolding of lethal factor (LF) and edema factor (EF). This partial unfolding event is required for the passage of these two factors through the heptameric pore formed by protective antigen (PA)[7] These rapid alterations of secondary/tertiary structure are critical for proper activity of these various toxins. We show that the mechanism for pH dependence in INY involves blockade at a stage that follows membrane binding and toxin oligomerization
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