The lecithinase gene of the intracellular pathogen Listeria monocytogenes, plcB, was identified in a 5,648-bp DNA fragment which expressed lecithinase activity when cloned into Escherichia coli. This fragment is located immediately downstream of the previously identified gene mpl (prtA). It contains five open reading frames, named actA, plcB, and ORFX, -Y, and -Z, which, together with mpl, form an operon, since a 5.7-kb-long transcript originates from a promoter located upstream of mpl (J. Mengaud, C. Geoffroy, and P. Cossart, Infect. Immun. 59:1043-1049, 1991). A second promoter was detected in front of actA which encodes a putative membrane protein containing a region of internal repeats. plcB encodes the lecithinase, a predicted 289-amino-acid protein homologous to the phosphatidylcholine-specific phospholipases C of Bacillus cereus and Clostridium perfringens (alpha-toxin). plcB mutants produce only small plaques on fibroblast monolayers, and an electron microscopic analysis of infected macrophages suggests that lecithinase is involved in the lysis of the two-membrane vacuoles that surround the bacteria after cell-to-cell spread. On the opposite DNA strand, downstream of the operon, three more open reading frames, ldh, ORFA, and ORFB, were found. The deduced amino acid sequence of the first one is homologous to lactate dehydrogenases. Low-stringency Southern hybridization experiments suggest that these three open reading frames lie outside of the L. monocytogenes virulence region: mpl and actA were specific for L. monocytogenes, sequences hybridizing to plcB were detected in L. ivanovii and L. seeligeri, and sequences hybridizing to ORFX, -Y, and -Z were found in L. innocua. In contrast to this, sequences hybridizing to ldh or ORFB were detected in all Listeria species (including the nonpathogenic ones).

We purified and characterized an extracellular hemolysin produced by Listeria monocytogenes. Hemolysin production was greatly enhanced by growing bacteria in resin (Chelex)-treated medium. This hemolysin was separated as a homogeneous protein of 60,000 daltons by using thiol-disulfide exchange affinity chromatography. This protein was a sulflydryl-activated toxin, termed listeriolysin 0, which shared the classical properties of other bacterial sulfhydryl-activated toxins: (i) inhibition by very low amounts of cholesterol; (ii) activation by reducing agents and suppression of the lytic activity by oxidation; (iii) antigenic cross-reactivity with streptolysin 0. However, listeriolysin 0 differed remarkably from the other sulfhydryl-activated toxins in that its cytolytic activity towards erythrocytes from various animal species was maximum at low pH (-5.5) and was undetectable at pH 7.0. This suggests that the lytic activity of the toxin in host tissues might be better expressed in the acidic microenvironment, including macrophage phagosomes where bacteria presumably replicate. Listeriolysin 0 was lethal to mice (50% lethal dose of ca. 0.8 ,ug) and induced a rapid inflammatory reaction when injected intradermally. These results favor the view that listeriolysin 0 might play a major role during intracellular replication of L. monocytogenes, ultimately promoting death of infected macrophages.

To evaluate the role of hemolysin production in the virulence of Listeria monocytogenes, we have undertaken the analysis of the chromosomal region containing hlyA, the gene coding for listeriolysin 0. A recombinant cosmid, conferring a hemolytic phenotype to Escherichia coli, was shown to express listeriolysin 0, by immunoblotting with a specific antiserum against listeriolysin 0. The presence of hlyA on the cosmid was demonstrated by DNA hybridization with a probe previously shown to contain part of hlyA. The complete nucleotide sequence of hlyA has been determined. The deduced protein sequence reveals the presence of a putative 25-amino-acid signal sequence: the secreted form of listeriolysin 0 would have 504 amino acids, in agreement with the molecular weight of purified listeriolysin 0 (58,000). The protein sequence is highly homologous to those of streptolysin 0 and pneumolysin. A peptide of 11 amino acids conserved in the three proteins contains the unique cysteine known to be essential for lytic activity. By DNA-DNA hybridization, the listeriolysin 0 gene was detected in all L. monocytogenes strains tested, even in the nonhemolytic type strain. The gene was absent in other species of the genus Listeria.

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.

Twenty-six strains belonging to the five main species of the genus Listeria were examined for production of thiol-dependent exotoxins. All strains of L. monocytogenes cultured in charcoal-treated broth secreted a haemolytic factor at a level ranging from 200 to 800 haemolytic units (HU) ml-1, except for the strain EGD (1500 HU ml-1) and the type strain CIP 82110T (10 HU ml-1). The haemolytic activity reached a maximum level by 8-10 h and then rapidly declined as soon as bacterial exponential growth ceased. The titres of haemolytic activity were markedly reduced when bacteria were grown in charcoal-untreated broth. The haemolytic factor produced by L. monocytogenes strains was characterized as listeriolysin O (Mr about 60,000), a member of the group of thiol-dependent exotoxins. Strains of Listeria ivanovii also produced high levels of thiol-dependent exotoxin (about 2500 HU ml-1), in both charcoal-treated and untreated broth. Small amounts of haemolytic factor (about 9-30 HU ml-1) were also produced by Listeria seeligeri in charcoal-treated broth. The haemolysin produced by L. seeligeri was identified for the first time as a thiol-dependent exotoxin of Mr about 60,000, antigenically related to listeriolysin O. As expected, we failed to detect thiol-dependent exotoxin in the two nonhaemolytic species, Listeria innocua and Listeria welshimeri.