Heparin-binding enhances extracellular listeriolysin O activity, overcoming cholesterol inhibition and pH dependence

The membranolytic toxin listeriolysin O (LLO), a member of the family of bacterial cholesterol-dependent cytolysins (CDC), is a key virulence factor of Listeria monocytogenes (Lm). Current evidence indicates that LLO is primarily a unique site-specific intracellular CDC, as it is highly active at the acidified pH of phagosomes but exhibits reduced activity and is prone to irreversible denaturation at physiological pH. Here, we demonstrate that LLO binds heparin and exploit this finding to affinity-purify the toxin in a single step. Heparin-binding increases LLO hemolytic activity at neutral pH manifold, and further amplifies the increased activity conferred by treatment with the thiol-reducing reagent DTT. Heparin-activated LLO is insensitive to inhibition by cholesterol, and its activity is not pH-dependent. We localized heparin binding to the C-terminal D4 membrane-interacting domain of LLO and used glycan arrays to define the minimal sulfated ligand engaged. Molecular docking studies suggest a heparin-binding site within this domain, and a LLO variant carrying a mutation (N508A) in this region no longer exhibited heparin-enhancing hemolytic activity. Engaging heparin enhances hemolysis and provides an additional layer of regulation of toxin function that ensures its optimal activity at physiological pH. As LLO is known to promote bacterial entry, and the sulfated heparin substructure recognized is present in many members of the family of cell surface heparan sulfates, our data suggest that this targeted tropism could contribute to the transition of the bacterium from extracellular compartments to intracellular niches.IMPORTANCEBacterial pore-forming cytolysins act extracellularly to disrupt host cell membranes. Listeriolysin (LLO), a cholesterol-dependent cytolysin (CDC) and a key virulence factor of Listeria monocytogenes, is distinguished by its site-specific intracellular activity, exhibiting maximal function at the acidic pH of phagosomes where the bacteria reside after uptake. At physiological pH and temperature, LLO exhibits reduced activity and susceptibility to denaturation. Here, we show that LLO binds heparin, a property that greatly enhances its hemolytic activity at neutral pH and overcomes cholesterol-dependent inhibition. We identified both the heparin binding site in LLO and the structure of the minimal interacting ligand engaged. The affinity of LLO for heparin enabled direct purification of highly active toxin from diverse naturally occurring isolates in a single step.