Role of Listeriolysin O in Cell-to-Cell Spread of Listeria monocytogenes

Abstract

Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from a host vacuolar compartment and grows rapidly in the cytosol. Listeriolysin O (LLO) is a secreted pore-forming protein essential for the escape of L. monocytogenes from the vacuole formed upon initial internalization. However, its role in intracellular growth and cell-to-cell spread events has not been testable by a genetic approach. In this study, purified six-His-tagged LLO (HisLLO) was noncovalently coupled to the surface of nickel-treated LLO-negative mutants. Bound LLO mediated vacuolar escape in approximately 2% of the mutants. After 5.5 h of growth, cytosolic bacteria were indistinguishable from wild-type bacteria with regard to formation of pseudopod-like extensions, here termed listeriopods, and spread to adjacent cells. However, bacteria in adjacent cells failed to multiply and were found in double-membrane vacuoles. Addition of bound LLO to mutants lacking LLO and two distinct phospholipases C (PLCs) also resulted in spread to adjacent cells, but these triple mutants became trapped in multiple-membrane vacuoles that are reminiscent of autophagocytic vacuoles. These studies show that neither LLO nor the PLCs are necessary for listeriopod formation and uptake of bacteria into neighboring cells but that LLO is required for the escape of L. monocytogenes from the double-membrane vacuole that forms upon cell-to-cell spread.

The arsenal of virulence factors deployed byListeria monocytogenesto promote its cell infection cycle

“…Listeriolysin O (LLO) is the main bacterial determinant responsible for the escape of L. monocytogenes from primary and secondary vacuoles 113,114 and one of its major virulence factors. 115 The gene that encodes LLO (hly) was the first virulence gene identified in Listeria, and is part of a locus that contains the main virulence genes (prfA, plcA, hly, mpl, actA and plcB).…”

Harnessing Listeria monocytogenes to target tumors

“…6,80 As discussed earlier, LM enters the APC through phagocytosis, and escapes into the host cytosol by perforating the phagosomal membrane through the action of a cytolysin, listeriolysin O (LLO). 36,37 A sequence rich in proline, glutamic acid, serine and threonine (PEST) at the amino terminus of LLO is thought to control the production of LLO. 81 Others found that the γ-interferon-inducible lysosomal thiolreductase (GILT) is responsible for the activation of LLO in vivo.…”

“…36,37 Once in the cytosol, the endogenously produced TAA are processed, transported into the endoplasmic reticulum (ER) for loading onto major histocompatibility complex (MHC) class I molecules and presented as short peptides via the MHC class I pathway, resulting in the activation of CD8 T-cells. However, most LM are actually destroyed and processed in the phagolysosome.…”

Enhanced cytosolic delivery of plasmid DNA by a sulfhydryl-activatable listeriolysin O/protamine conjugate utilizing cellular reducing potential

“…45 The protein was purified using Ni-NTA superflow (Qiagen, CA) on an FPLC system (Bio-Rad, Biologic HR Chromatography System) as previously described. 46 Typically, from a 1 l culture, B20 mg of purified LLO was obtained.…”