Listeriolysin O and Two Listerial Phospholipases Allow Escape from the Phagocytic Vacuole

Hemolytic activity of L. monocytogenes is due to the action of a cytolysin called listeriolysin O (LLO), a secreted protein of 58-60 kDa, belonging to a family of thiol-activated, cholesterol-dependent, pore-forming toxins (CDTX) [24]. The gene encoding LLO, hly, was the first listerial virulence gene cloned [25] and codes for 529 amino acids including an N-terminal signal sequence and a highly conserved undecapeptide essential for hemolytic activity.

Nonhemolytic mutants are totally avirulent and are eliminated from the infected animal within a few hours [26]. They are unable to open the phagosome and hence unable to escape into the cytoplasm of the host cells [27]. The low pH optimum of LLO is in agreement with its function in the acidified phagosome. Detailed studies showed that this low pH optimum of LLO is critical for its function since it would otherwise damage the host cell upon escape of the bacteria into the pH-neutral cytoplasm (reviewed in Ref. [28]). In addition, a so-called PEST sequence identified in the N-terminal region of LLO [29] targets LLO to the proteasome degradation pathway, hence dramatically reducing its cytoplasmic half-life. Finally, the expression of LLO in the infected host cell is tightly controlled with strongly induced expression when the bacteria reside inside the phagosome [30].

A gene located immediately upstream of hly, called plcA, encodes a secreted phosphatidylinositol-specific phospholipase C (PI-PLC) of 34kDa which exhibits high homology to several gram-positive phospholipases [31]. The enzyme has been purified [32] and was found to be highly specific for phosphatidylinositol, with no other detectable activity.

Besides the highly specific PI-PLC, L. monocytogenes produces a second phospholipase C which hydrolyzes phosphatidylcholine (lecithin), and is thus a phos-phatidylcholine-specific phospholipase C (PC-PLC) or lecithinase [33], also called broad-spectrum phospholipase C. The 29-kDa mature PC-PLC has been purified [33] and is a zinc-dependent phospholipase, the gene of which, plcB, is part of the lecithinase operon [34]. Maturation of the 32-kDa precursor of PC-PLC occurs after secretion and is obviously accomplished by the metalloprotease Mpl of L. monocytogenes [35].

PI-PLC is required for the efficient escape of L. monocytogenes from the phagosome of some cell types [36]. It is assumed that PI-PLC acts in concert with lister-iolysin inside the acidified phagosomal vacuole of the host cell to mediate lysis of the vacuolar membrane. The role of PC-PLC in escaping from the primary vacuole is not clear and differs from cell type to cell type. In some human cell lines, where escape of L. monocytogenes occurs at low efficiency independent of LLO [37], PC-PLC is required for lysis of the phagocytic vacuole together with the metallopro-tease.

The metalloprotease Mpl of L. monocytogenes indirectly contributes to pathogeni-city and intracellular replication of the bacteria by proteolytic processing and hence activation of the PC-PLC proform [35]. Located downstream of the hly gene, the mpl gene [38] encoding the zinc-dependent Mpl is the first gene of the lecithinase operon [34, 38].

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