Purification and characterization of an extracellular 29-kilodalton phospholipase C from Listeria monocytogenes

Abstract

We purified and characterized an extracellular phospholipase produced by Listeria monocytogenes. This enzyme was separated as a homogeneous protein of 29 kDa by chromatography on DEAE-52 cellulose and Bio-Gel P100 columns. It is a zinc-dependent phospholipase C (PLC) that is mainly active at pH 6 to 7 and expresses lecithinase activity and a weaker sphingomyelinase activity. The exoenzyme also hydrolyzed phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin but not phosphatidylinositol. It was distinct from the 36-kDa phosphatidylinositol PLC produced by L. monocytogenes and from the L. ivanovii sphingomyelinase. The pure protein expressed a weak, calcium-independent hemolytic activity and was not toxic in mice. Western immunoblot analysis using a rabbit immune serum raised against the enzyme showed that all virulent strains of L. monocytogenes tested produced in the culture supernatant a 29-kDa PLC. In contrast, no proteins antigenically related to the 29-kDa PLC were detected in supernatants of L. ivanovii, L. seeligeri, L. innocua, or L. weishimeri. The role in virulence of the 29-kDa PLC specifically produced by L. monocytogenes remains to be established.

The smcL gene of Listeria ivanovii encodes a sphingomyelinase C that mediates bacterial escape from the phagocytic vacuole

“…a. This residual activity is attributable to the wide-substraterange phospholipase C, PlcB (or lecithinase), which has a weak SM-hydrolysing activity (Geoffroy et al, 1991) and is overproduced by wild-type L. ivanovii (Ripio et al, 1996).…”

Differential function of Listeria monocytogenes listeriolysin O and phospholipases C in vacuolar dissolution following cell-to-cell spread

“…7A). Several experiments have shown that PC and Sph can also be substrates for PC-PLC (Geoffroy et al, 1991;Goldfine et al, 1993;Montes et al, 2004). Therefore, in theory PC-PLC should be able to attack not only the inner membrane, but also the outer membrane of the spreading vacuole.…”

The metabolic regulator CodY links Listeria monocytogenes metabolism to virulence by directly activating the virulence regulatory gene prfA

“…The hemolytic activity assay was performed as described previously (Glomski et al, 2002): bacterial supernatants were treated with 5 mM DTT, serially diluted in phosphate-buffered saline (PBS), and incubated with 0.5% sheep red blood cell suspension (NovaMed ® ); hemolysis was measured by following the change in absorbance at 540 nm. The PI-PLC activity assay was adapted from Geoffroy et al (1991): 1 ml of sodium-cholate (58 mM), CaCl2 (10 mM) and 0.036 g phosphatidyl-inositol (P6636; Sigma ® ) were mixed with 7 ml NaCl (0.15 M). One hundred microliters of the assay solution was then mixed with 100 μl of bacterial supernatants and incubated in a plate reader at 37°C for 10 h, following turbidity assessment at 510 nm.…”

“…L. monocytogenes invades host cells either passively via phagocytosis or actively by expressing surface proteins that induce bacterial internalization (Cossart, 2011). Once inside the cell, L. monocytogenes is initially contained in a vacuole from which it rapidly escapes by expressing the pore-forming cytolysin listeriolysin O (LLO), two additional phospholipases, PlcA and PlcB, and components of the Com system (Mengaud et al, 1987;Goebel et al, 1988;Portnoy et al, 1988;Geoffroy et al, 1991;Leimeister-Wachter et al, 1991;Rabinovich et al, 2012). In the host cell cytosol, L. monocytogenes replicates and gains mobility via polymerization of host actin filaments, enabling the bacteria to spread from cell to cell without being exposed to the extracellular environment (Tilney and Portnoy, 1989).…”