Identification of a new operon involved in Listeria monocytogenes virulence: its first gene encodes a protein homologous to bacterial metalloproteases.

The region flanking the transposon in a Tn1545-induced lecithinase-negative mutant of Listeria monocytogenes EGD was cloned and sequenced. The transposon had inserted in ORF D, the open reading frame previously identified downstream from hlyA, the gene encoding listeriolysin O. The complete sequence of ORF D from strain EGD has been determined as well as that of two other strains: LO28, a clinical isolate; and LM8, an epidemic strain. ORF D is 1,533 bp long and encodes a protein highly homologous to metalloproteases of bacilli, Serratia sp., Legionella pneumophila, and Pseudomonas aeruginosa. It was renamed prtA. Northern RNA blot analysis indicated that prtA is the first gene of a 6-kb operon, suggesting that the lecithinase-negative phenotype of the mutant might be due to a polar effect of the transposon insertion.     

Expression in Escherichia coli and sequence analysis of the listeriolysin O determinant of Listeria monocytogenes.

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 O. A recombinant cosmid, conferring a hemolytic phenotype to Escherichia coli, was shown to express listeriolysin O, by immunoblotting with a specific antiserum against listeriolysin O. 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 O would have 504 amino acids, in agreement with the molecular weight of purified listeriolysin O (58,000). The protein sequence is highly homologous to those of streptolysin O 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 O 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.     

Identification of the structural gene encoding the SH-activated hemolysin of Listeria monocytogenes: listeriolysin O is homologous to streptolysin O and pneumolysin.

By immunoblotting with an antiserum raised against purified listeriolysin O, we have detected the presence of a truncated protein of 52 kilodaltons in culture supernatants of a Tn1545-induced nonhemolytic mutant of Listeria monocytogenes (J.L. Gaillard, P. Berche, and P. Sansonetti, Infect. Immun. 52:50-55, 1986). The region of insertion of the transposon has been cloned and sequenced. The transposon had inserted in an open reading frame the listeriolysin O gene. The deduced amino acid sequence of this open reading frame revealed that listeriolysin O is homologous to streptolysin O and pneumolysin, although homologies were not detectable at the DNA level.     

A nonvirulent mutant of Listeria monocytogenes does not move intracellularly but still induces polymerization of actin.

Listeria monocytogenes has the capacity to penetrate and multiply within professional and nonprofessional phagocytic cells, such as the Caco-2 human enterocytelike cell line. It was shown recently that shortly after listeriae have been phagocytosed, the phagosomal membrane is dissolved, probably by the action of the bacterial cytolysin listeriolysin O. The listeriae, which are then lying obviously free in the cytoplasm, become surrounded by a coat of actin filaments within a few hours. Once formed, this layer of actin filaments is reorganized in an as yet unknown way to form polar tails, which seem to be associated to the generation of listerial movement inside the cytoplasm and in intercellular spread. By using transposon Tn916 mutagenesis, a bank of L. monocytogenes mutants was generated and subsequently screened by the plaque assay system in order to select an intracellular, nonmotile mutant of L. monocytogenes. One such mutant was identified. This mutant, called L. monocytogenes M117 Imt- (for intracellular motility), like the wild type, induced actin polymerization but was not able to rearrange the actin coat to generate movement and as a result remained entrapped within the actin cloud. In a mouse virulence assay, this strain was significantly reduced in virulence. L. monocytogenes M117 is the first example to date of a Listeria mutant which is still hemolytic and invasive but reduced in virulence.     

Sequences homologous to the listeriolysin O gene region of Listeria monocytogenes are present in virulent and avirulent haemolytic species of the genus Listeria

Various parts of the hlyA gene region of Listeria monocytogenes which encodes a major virulence factor, listeriolysin O, have been used to detect the presence of homologous sequences in other species of the genus Listeria. Under low-stringency hybridization conditions, sequences homologous to the hlyA gene and its 5' adjacent regions were detected in the haemolytic and pathogenic species L. ivanovii, and in the haemolytic but non-pathogenic species L. seeligeri. In contrast, the region located downstream from hlyA appeared specific to L. monocytogenes. None of the probes spanning the region revealed homologies between L. monocytogenes and the non-pathogenic and non-haemolytic members of the genus, L. innocua, L. murrayi and L. welshimeri. Among various strains of L. monocytogenes tested, the gene hlyA and its 3' adjacent region appeared well-conserved. In contrast, a restriction length polymorphism was detected in the region located upstream from hlyA with no obvious correlation with the haemolytic phenotype or the serovar of the strains tested.     

Transcriptional mapping and nucleotide sequence of the Listeria monocytogenes hlyA region reveal structural features that may be involved in regulation

DNA sequence analysis of the regions adjacent to the hlyA gene, which encodes listeriolysin O, an essential virulence factor of Listeria monocytogenes, revealed the presence of two open reading frames (ORFs): ORF D located 304 base pairs downstream from hlyA, and ORF U located 224 base pairs upstream from and in opposite direction to hlyA. Promoter mapping performed with RNAs extracted from cells growing exponentially in rich medium showed that the three ORFs are independently transcribed. hlyA is transcribed from two promoters separated by 10 base pairs (P1 hlyA and P2 hlyA). ORF U is transcribed in the opposite direction from an adjacent promoter. These two promoter regions are separated by a palindromic sequence T-T-A-A-C-A-A/T-T-G-T-T-A-A. This palindrome was also found upstream from the ORF D promoter, suggesting that all three genes are similarly regulated.     

Pathogenicity and immunogenicity of a mutant strain of Listeria monocytogenes in the chicken infection model

Listeria monocytogenes has been exploited as a vaccine carrier based upon its ability to induce a strong cell-mediated immune response. At present, the safety of live, attenuated L. monocytogenes vaccines in patients is being studied in clinical trials. L. monocytogenes is also an attractive vaccine vector for use in poultry; however, the pathogenicity and immunogenicity of this organism in poultry remain to be fully elucidated. In this study, we investigated the pathogenicity and immunogenicity of an actA- and plcB-deficient L. monocytogenes strain, yzuLM4ΔactA/plcB, and its wild-type parent strain, yzuLM4, in an avian infection model. The results showed that the wild-type strain could infect ISA brown chickens, causing serious tissue disruptions, including various degrees of degeneration, necrotic lesions, and inflammatory cell infiltration in the liver, spleen, heart, and kidney. However, the mutant strain showed reduced virulence in embryonated eggs compared with that of the parent strain (the 50% lethal dose [LD(50)] was 3 logs higher). The mutant strain also showed low virulence in chickens and was rapidly eliminated by the host. There were no obvious pathological changes in tissue sections, but the mutant strain still retained the ability to stimulate high levels of antibody against the protein listeriolysin O (LLO). Booster immunization with the mutant strain led to rapid bacterial clearance from the livers and spleens of chickens challenged by the intramuscular route or the oral route. Collectively, our data suggest that the wild-type serotype 1/2a L. monocytogenes strain can cause serious disease in chickens but the mutant strain with a deletion of the actA and plcB genes is less virulent but induces a strong immune response. This mutant strain of L. monocytogenes is therefore a promising candidate as a safe and effective vector for the delivery of heterologous antigens to prevent zoonosis and infectious disease in poultry.     

Detection of listeriolysin, the thiol-dependent hemolysin in Listeria monocytogenes, Listeria ivanovii, and Listeria seeligeri.

The listeriolysin gene from a weakly hemolytic but virulent strain of Listeria monocytogenes serotype 1/2a was cloned in Escherichia coli K-12. Recombinants were identified on the basis of their cross-reactivities to hyperimmune antisera raised against streptolysin O and listeriolysin. Low levels of hemolytic activity were detected in crude lysates of strains harboring the listeriolysin gene. In DNA hybridization studies with five DNA probes that encoded the listeriolysin gene and surrounding sequences, highly homologous listeriolysin genes were found to be present in the species L. monocytogenes, Listeria ivanovii, and Listeria seeligeri. Immunoblotting performed with affinity-purified antibody to listeriolysin allowed the detection of this protein in supernatants of all three species. This study demonstrates for the first time that listeriolysin is produced by L. seeligeri and documents the genetic homology between the various listeriolysins produced by Listeria spp. Sequences unique to the species L. monocytogenes were found to be located downstream of the listeriolysin gene. Furthermore, the restriction fragment length polymorphisms detected with probes flanking the hlyA gene may be useful epidemiological markers in identifying and distinguishing virulent Listeria strains from each other.     

Roles of Listeria monocytogenes virulence factors in survival: virulence factors distinct from listeriolysin are needed for the organism to survive an early neutrophil-mediated host defense mechanism.

Avirulent mutant strains of Listeria monocytogenes which fail to produce phosphatidylinositol-specific phospholipase C, or which produce reduced amounts of hemolytic listeriolysin O, are incapable of causing progressive infection in normal mice. However, both strains can grow progressively in mice that have been rendered incapable of focusing neutrophils at sites of infection as a result of being treated with monoclonal antibody 5C6, specific for the type 3 complement receptor of myelomonocytic cells. In 5C6-treated mice, phospholipase C-negative and listeriolysin-defective mutant strains of L. monocytogenes, like the wild-type strain, give rise in the liver to large numbers of discrete foci of infected hepatocytes that retain their morphological integrity during the first 24 h, despite their large bacterial burden. In normal mice, in contrast, sites of infection in the liver are indicated by discrete focal accumulations of neutrophils that occupy the space originally occupied by infected hepatocytes. It is apparent that in normal mice neutrophils function to lyse infected hepatocytes and thereby to release L. monocytogenes for ingestion and killing by neutrophils themselves and by macrophages. However, whereas a proportion of wild-type organisms survive this early mechanism of defense to give rise to progressive infection, the phospholipase C-negative organisms are totally eliminated. On the basis of these and other results, it is suggested that virulence factors other than listeriolysin are needed by L. monocytogenes to counteract the early neutrophil-mediated mechanism of defense. Listeriolysin, itself, is an intrinsic virulence factor that allows L. monocytogenes to survive and multiply in a proportion of the fixed phagocytes of the liver (permissive phagocytes) and which enables the organism to go on to infect and replicate in adjacent hepatocytes. It was found that a mutant strain of L. monocytogenes incapable of producing any listeriolysin was incapable of establishing progressive infection, even in 5C6-treated mice.     

Characterization of Listeria monocytogenes expressing anthrolysin O and phosphatidylinositol-specific phospholipase C from Bacillus anthracis

Two virulence factors of Listeria monocytogenes, listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC), mediate escape of this pathogen from the phagocytic vacuole of macrophages, thereby allowing the bacterium access to the host cell cytosol for growth and spread to neighboring cells. We characterized their orthologs from Bacillus anthracis by expressing them in L. monocytogenes and characterizing their contribution to bacterial intracellular growth and cell-to-cell spread. We generated a series of L. monocytogenes strains expressing B. anthracis anthrolysin O (ALO) and PI-PLC in place of LLO and L. monocytogenes PI-PLC, respectively. We found that ALO was active at both acidic and neutral pH and could functionally replace LLO in mediating escape from a primary vacuole; however, ALO exerted a toxic effect on the host cell by damaging the plasma membrane. B. anthracis PI-PLC, unlike the L. monocytogenes ortholog, had high activity on glycosylphosphatidylinositol-anchored proteins. L. monocytogenes expressing B. anthracis PI-PLC showed significantly decreased efficiencies of escape from a phagosome and in cell-to-cell spread. We further compared the level of cytotoxicity to host cells by using mutant strains expressing ALO in combination either with L. monocytogenes PI-PLC or with B. anthracis PI-PLC. The results demonstrated that the mutant strain expressing the combination of ALO and B. anthracis PI-PLC caused less damage to host cells than the strain expressing ALO and L. monocytogenes PI-PLC. The present study indicates that LLO and L. monocytogenes PI-PLC has adapted for L. monocytogenes intracellular growth and virulence and suggests that ALO and B. anthracis PI-PLC may have a role in B. anthracis pathogenesis.     

Role of Listeria monocytogenes exotoxins listeriolysin and phosphatidylinositol-specific phospholipase C in activation of human neutrophils

Polymorphonuclear leukocytes (PMN) are essential for resolution of infections with Listeria monocytogenes. The present study investigated the role of the listerial exotoxins listeriolysin (LLO) and phosphatidylinositol-specific phospholipase C (PlcA) in human neutrophil activation. Different Listeria strains, mutated in individual virulence genes, as well as purified LLO were used. Coincubation of human neutrophils with wild-type L. monocytogenes provoked PMN activation, occurring independently of phagocytosis events, with concomitant elastase secretion, leukotriene generation, platelet-activating factor (PAF) synthesis, respiratory burst, and enhanced phosphoinositide hydrolysis. Degranulation and leukotriene formation were noted to be solely dependent on LLO expression, as these features were absent when the LLO-defective mutant EGD- and the avirulent strain L. innocua were used. These effects were fully reproduced by a recombinant L. innocua strain expressing LLO (INN+) and by the purified LLO molecule. LLO secretion was also required for PAF synthesis. However, wild-type L. monocytogenes was more potent in eliciting PAF formation than mutants expressing LLO, suggesting the involvement of additional virulence factors. This was even more obvious for phosphoinositide hydrolysis and respiratory burst: these events were provoked not only by INN+ but also by the LLO-defective mutant EGD- and by a recombinant L. innocua strain producing listerial PlcA. We conclude that human neutrophils react to extracellularly provided listerial exotoxins by rapid cell activation. Listeriolysin is centrally involved in triggering degranulation and lipid mediator generation, and further virulence factors such as PlcA apparently contribute to trigger neutrophil phosphoinositide hydrolysis and respiratory burst. In this way, listerial exotoxins may influence the host defense against infections with L. monocytogenes.     

Characterization of an aromatic amino acid-dependent Listeria monocytogenes mutant: attenuation, persistence, and ability to induce protective immunity in mice.

A transposon insertion mutant of Listeria monocytogenes was shown to be deficient in prephenate dehydratase, an enzyme acting late in the pathway for biosynthesis of phenylalanine. This mutant had reduced virulence in mice. The mutant and parent strains persisted to the same extent in the tissues of infected mice and elicited similar degrees of splenomegaly. Mice vaccinated with the mutant were protected significantly from subsequent challenge with virulent L. monocytogenes.     

Stress-Induced ClpP Serine Protease of Listeria monocytogenes Is Essential for Induction of Listeriolysin O-Dependent Protective Immunity

The stress-induced protease ClpP is required for virulence of the facultative intracellular pathogen Listeria monocytogenes. We previously found that in the absence of ClpP, the virulence of this pathogen was strongly reduced, mainly due to the decreased production of functional listeriolysin O (LLO), a major immunodominant virulence factor promoting intracellular growth. In this work, a clpP deletion mutant of L. monocytogenes was used to study the generation of anti-Listeria protective immunity. We found that ClpP is required for the intracellular growth of L. monocytogenes in resident macrophages in vivo. Mice infected with doses as high as 10(6) clpP mutant bacteria were not protected against a lethal challenge of wild-type bacteria and did not develop any detectable LLO-specific cytolytic T cells or antibodies, suggesting that the amount of LLO produced in infected mice under these conditions was too low to induce a specific immune response. However, in contrast to the results obtained with a mutant with a disrupted hly gene, this lack of protection was overcome by inoculation of very high infecting doses of clpP mutant bacteria (5 x 10(8)), thus producing sufficient amounts of LLO to stimulate anti-Listeria immunity. The role of ClpP was confirmed by showing that anti-Listeria immunity was restored in mice infected with a clpP-complemented mutant. These results indicate that the stress-induced serine protease ClpP is a potential target for modulating the presentation of protective antigens such as LLO and thereby the immune response against L. monocytogenes.     

Detection of genes coding for listeriolysin and Listeria monocytogenes antigen A (ImaA) in Listeria spp. by the polymerase chain reaction.

Two pairs of synthetic oligonucleotide primers were used in a polymerase chain reaction (PCR) protocol to detect targeted sequences in genes coding for listeriolysin O and Listeria monocytogenes antigen A (ImaA). Strains of Listeria spp. used in this study were isolated from clinical specimens, contaminated foods, and environmental sources. Primers were targeted to internal regions of the genes coding for listeriolysin (hlyA) and Listeria antigen (ImaA) and amplification fragments were detected after the PCR by agarose gel electrophoresis. PCR was performed using nucleic acids extracted from a collection of 74 strains of Listeria spp. including 18 reference strains, 41 L. monocytogenes, nine L. innocua, five L. seeligeri and one L. ivanovii, encompassing representative sources, serovars, and enzyme electrophoretic types. Although the listeriolysin gene was found exclusively in L. monocytogenes, some strains of serovar 4c were negative. Simultaneous presence of both genes was restricted to L. monocytogenes strains of serovars 1/2, 3, and 4. The ImaA gene was identified in five of 10 L. innocua strains and one L. ivanovii isolated from pork. Strains of L. seeligeri, L. welshimeri, and L. grayi were negative for both genes. The detection limits in the PCR were found to be 10 pg of nucleic acids for the hlyA gene and 1 pg for the ImaA gene.     

The zinc metalloprotease of Listeria monocytogenes is required for maturation of phosphatidylcholine phospholipase C: direct evidence obtained by gene complementation.

The maturation of the 33-kDa proenzyme to the 29-kDa phosphatidylcholine phospholipase C (PC-PLC) of Listeria monocytogenes requires the production of the zinc metalloprotease encoded by mpl, the proximal gene of the lecithinase operon. We recently described a low-virulence lecithinase-deficient mutant of L. monocytogenes EGD-SmR, designated JL762, generated by a single insertion of transposon Tn1545 in mpl. This mutant failed to produce the 29-kDa PC-PLC, an exoenzyme probably involved in cell-to-cell spreading. The role of the product of the mpl gene in production of PC-PLC was investigated in trans-complementation experiments. The entire mpl gene was cloned in a plasmid able to replicate in L. monocytogenes. This recombinant plasmid was introduced into JL762 and restored the lecithinase phenotype on egg yolk agar and the production of the active 29-kDa PC-PLC in culture supernatants and partially restored the level of virulence. These results demonstrate that zinc-dependent metalloprotease of L. monocytogenes is involved in the virulence of this bacteria at least through its action on PC-PLC.     

Reduced virulence of a Listeria monocytogenes phospholipase-deficient mutant obtained by transposon insertion into the zinc metalloprotease gene.

A phospholipase-deficient mutant, termed JL762, was obtained from a virulent strain of Listeria monocytogenes by screening a bank of 5,000 Tn1545 transposon-induced mutants on 2.5% egg yolk brain heart infusion agar. As previously shown (J. Mengaud, C. Geoffroy, and P. Cossart, Infect. Immun. 59:1043-1049, 1991), the transposon insertion took place inside the gene mpl, which encodes a zinc metalloprotease. By Western blot (immunoblot) analysis, we showed that loss of phospholipase activity was associated with loss of a 29-kDa zinc-dependent phosphatidylcholine-phospholipase C (PC-PLC) in culture supernatant of JL762 and of EGD-SmR incubated with ion chelator. As the parental strain, JL762 still produced in supernatants approximately 33-kDa proteins antigenically closely related to the 29-kDa PC-PLC. These results strongly suggest that the zinc metalloprotease of L. monocytogenes might play a role in the maturation of the 29-kDa PC-PLC. Although the uptake and the intracellular growth of bacteria were not affected in vitro, we found that the virulence of mutant JL762 was strongly impaired in the mouse.     

Listeriolysin O secreted by Listeria monocytogenes into the host cell cytosol is degraded by the N-end rule pathway

The intracellular pathogen Listeria monocytogenes escapes from a phagosomal compartment into the cytosol by secreting the pore-forming cytolysin listeriolysin O (LLO). During the proliferation of L. monocytogenes bacteria in the mammalian cell cytosol, the secreted LLO is targeted for degradation by the ubiquitin system. We report here that LLO is a substrate of the ubiquitin-dependent N-end rule pathway, which recognizes LLO through its N-terminal Lys residue. Specifically, we demonstrated by reverse-genetic and pharmacological methods that LLO was targeted for degradation by the N-end rule pathway in reticulocyte extracts and mouse NIH 3T3 cells and after its secretion by intracellular bacteria into the mouse cell cytosol. Replacing the N-terminal Lys of LLO with a stabilizing residue such as Val increased the in vivo half-life of LLO but did not strongly affect the intracellular growth or virulence of L. monocytogenes. Nevertheless, this replacement decreased the virulence of L. monocytogenes by nearly twofold, suggesting that a destabilizing N-terminal residue of LLO may stem from positive selection during the evolution of this and related bacteria. A double mutant strain of L. monocytogenes in which upregulated secretion of LLO was combined with a stabilizing N-terminal residue was severely toxic to infected mammalian cells, resulting in reduced intracellular growth of bacteria and an approximately 100-fold-lower level of virulence. In summary, we showed that LLO is degraded by the N-end rule pathway and that the degradation of LLO can reduce the toxicity of L. monocytogenes during infection, a property of LLO that may have been selected for its positive effects on fitness during the evolution of L. monocytogenes.     

Synthesis of species-specific stress proteins by virulent strains of Listeria monocytogenes.

Listeriolysin is a virulence factor that appears to be necessary for the intracellular survival of Listeria monocytogenes. As shown in this investigation, listeriolysin is produced in only small amounts by clinical isolates of L. monocytogenes belonging to the serogroup 1/2a, but its synthesis can be induced by heat shock and to a lesser extent by oxidative stress. In addition to about 15 heat shock proteins that appear to be common to L. monocytogenes and Listeria species that are nonpathogenic for humans, at least five heat shock proteins are specifically coinduced with listeriolysin in all L. monocytogenes strains under heat shock conditions but not in the other Listeria species. One type of L. monocytogenes mutant blocked in the expression of listeriolysin failed to synthesize several of these specific heat shock proteins.     

Listeriolysin is a potent inducer of the phosphatidylinositol response and lipid mediator generation in human endothelial cells.

The impact of Listeria monocytogenes listeriolysin O (LLO) secretion on phosphoinositide metabolism and mediator (platelet-activating factor and prostaglandin I2) generation was investigated in human umbilical vein endothelial cells. Wild-type L. monocytogenes, purified LLO, and an L. innocua strain engineered to secrete LLO all elicited a strong response, whereas mutant strains defective in LLO production were ineffective. Thus, human umbilical vein endothelial cell stimulation by listeriae is linked to production of LLO.