Role of listeriolysin O in cell-to-cell spread of Listeria monocytogenes

Listeria monocytogenes is a facultative intracellular bacterial pathogen th at escapes from a host vacuolar compartment and grows rapidly in the cytoso l, Listeriolysin O (LLO) is a secreted pore-forming protein essential for t he escape oft. monocytogenes from the vacuole formed upon initial internali zation. However, its role in intracellular growth and cell-to-cell spread e vents has not been testable by a genetic approach. In this study, purified six-sis-tagged LLO (HisLLO) was noncovalently coupled to the surface of nic kel-treated LLO-negative mutants. Bound LLO mediated vacuolar escape in app roximately 2% of the mutants. After 5.5 h of growth, cytosolic bacteria wer e indistinguishable from wild-type bacteria with regard to formation of pse udopod-like extensions, here termed listeriopods, and spread to adjacent ce lls. However, bacteria in adjacent cells failed to multiply and were found in double-membrane vacuoles. Addition of bound LLO to mutants lacking LLO a nd two distinct phospholipases C (PLCs) also resulted in spread to adjacent cells, but these triple mutants became trapped in multiple-membrane vacuol es that are reminiscent of autophagocytic vacuoles, These studies show that neither LLO nor the PLCs are necessary for listeriopod formation and uptak e of bacteria into neighboring cells but that LLO is required for the escap e oft. monocytogenes from the double-membrane vacuole that forms upon cell- to-cell spread.