Listeria monocytogenes protein p60 affects hemolytic activity and uptake of bacteria by macrophages

Bacillus subtilis strains expressing listeriolysin O (LLO) and simultaneously LLO and p60 protein were constructed. The effect of p60 protein on hemolytic activity and on the invasion of professional phagocytes was demonstrated in the absence of other virulence factors of L. monocytogenes. The hemolytic activity of LLO in the presence of p60 protein decreased which indicates that p60 promoted adhesion and subsequent invasion of professional phagocytes.     

Intracellular growth of Listeria monocytogenes insertional mutant deprived of protein p60

The study of the mutant strain described here demonstrates that several characteristics contribute to maximal virulence of pathogenic strains of L. monocytogenes. The invasion levels of L. monocytogenes JB1115, a p60-deficient strain, were the same as for the parent strain L. monocytogenes 1043S in J774 macrophage-like cells. The invasion level of Listeria strains in Int407 cells was 100 times lower than in J774 cells. In epithelial Int407 cells, the time of division of p60- strain L. monocytogenes JB1115 was 43% slower than for the parent strain. In this study, two lisosomotrophic agents, ammonium chloride and chlorquinoline were tested in experimental L. monocytogenes 1043S and p60-deprived mutant JB1115 infection in both cell lines. The presence of ammonium chloride increased the level of infection (calculated as number of gentamicin-resistant cells) of both Listeria strains, but in the case of infection by p60 mutant, the increased amount of ammonium chloride showed only a minimal effect on the number of isolated bacteria. In both cell lines treated with chlorquinoline we observed a decrease in the number of viable intracellular bacteria isolated from infected monolayers. Our observation of parental and mutated strains of Listeria showed that phospholipase activity also depends on the presence of p60 protein. Mutated strain showed 31.46% reduction of PI-PLC activity measured in normal growth conditions. Protein p60 plays a role not only in listeriolysin O mediated haemolytic activity but full phospholipase C activity is also dependent on the presence of the Iap protein.     

The iap gene of Listeria monocytogenes is essential for cell viability, and its gene product, p60, has bacteriolytic activity.

Expression of the iap gene of Listeria monocytogenes in the L. monocytogenes rough mutant RIII and in Bacillus subtilis DB104 caused the disruption of the cell chains which these two strains normally form under exponential growth conditions. The p60 protein produced by L. monocytogenes and B. subtilis DB104 also exhibited bacteriolytic activity detected in denaturing polyacrylamide gels containing heat-killed Micrococcus lysodeikticus. Purification of the p60 protein led to aggregation of p60 and loss of the cell chain disruption and bacteriolytic activities. A cysteine residue in the C-terminal part of p60 which is conserved in all p60-like proteins from the other Listeria species seems to be essential for both activities. The iap gene could not be inactivated without a loss of cell viability, indicating that p60 is an essential housekeeping protein for L. monocytogenes and probably also for other Listeria species. These data suggest that p60 possesses a murein hydrolase activity required for a late step in cell division.     

P45, an extracellular 45 kDa protein of Listeria monocytogenes with similarity to protein p60 and exhibiting peptidoglycan lytic activity

A monoclonal antibody obtained by immunization of mice with heat-killed cells of Listeria monocytogenes serotype 4d showed reactivity towards a protein (P45) from L. monocytogenes with an apparent molecular mass of 45 kDa. This protein was detected in the culture supernatant and at the cell surface of L. monocytogenes. Proteins cross-reacting with the monoclonal antibody were present in all Listeria strains investigated, except L. grayi. The structural gene was cloned in Escherichia coli and sequenced. Translation of the gene starts at a TTG initiation codon. The gene was found to code for a protein of 402 amino acid residues with a predicted molecular mass of 42.7 kDa. It has a signal peptide of 27 amino acid residues, resulting in a molecular mass for the mature polypeptide of 39.9 kDa. Protein database searches showed that this protein has 55% similarity and 38% identity to protein p60 of L. monocytogenes and exhibits significant sequence similarities to p54 from Enterococcus faecium and Usp45 from Lactococcus lactis. P45 was shown to have peptidoglycan lytic activity and the encoding gene was named spl (secreted protein with lytic property). Revision received: 11 August 1999 / Accepted: 8 September 1999     

Synthetic peptides derived from the Listeria monocytogenes p60 protein as antigens for the generation of polyclonal antibodies specific for secreted cell-free L. monocytogenes p60 proteins.

All species of the genus Listeria secrete a major extracellular protein called p60. A comparison of the deduced amino acid sequences of all listerial p60 proteins previously indicated there were only a few regions which were unique to the pathogenic, food-borne species Listeria monocytogenes. Two of these p60 regions were chosen for the development of antibodies specific for the facultative intracellular species L. monocytogenes. Initially, these regions were characterized via epitope mapping, and this led to the development of two different synthetic peptides. Rabbits immunized with these synthetic peptides generated polyclonal antibodies that were then used in Western blot (immunoblot) analyses. Antiserum against peptide A (PepA) recognized the p60 protein in the supernatants collected from most L. monocytogenes serotypes except for several strains belonging to serotypes 4a and 4c. No p60-related protein was detected in the supernatants from other Listeria species with this anti-PepA antiserum. Antibodies raised against peptide D (PepD) reacted with p60 from all L. monocytogenes serotypes, including all 4a and 4c strains that were tested, and also showed no cross-reactivity with supernatant proteins from other Listeria species. Both antisera also detected p60 in supernatants of a large number of environmental isolates of L. monocytogenes. Besides Western blot analyses, these antisera to PepA and PepD reacted with secreted p60 in an enzyme-linked immunosorbent assay, indicating recognition of the native antigen in addition to the denatured form. These data suggest that synthetic peptides derived from the variable region of the L. monocytogenes p60 protein may be useful for the development of an immunological diagnostic assay.     

Immunological properties of partially purified material with monocytosis-producing activity from Listeria monocytogenes.

A fraction rich in a monocytosis-producing activity (MPA) has been obtained by a two-stage chromatographic separation from a saline extract (SE) of Listeria monocytogenes. Like SE, the purified material elicits monocytosis and decreases the halftime of circulating monocytes. The purification of MPA eliminates the following found in SE: the in vitro mitogenic activity, the in vitro adjuvant activity, the immunosuppressive activity, and the granulocytosis-promoting and the lymphopenia-inducing activities.     

Enhancement of the Listeria monocytogenes p60-specific CD4 and CD8 T cell memory by nonpathogenic Listeria innocua

The contact of T cells to cross-reactive antigenic determinants expressed by nonpathogenic environmental micro-organisms may contribute to the induction or maintenance of T cell memory. This hypothesis was evaluated in the model of murine Listeria monocytogenes infection. The influence of nonpathogenic L. innocua on the L. monocytogenes p60-specific T cell response was analyzed. We show that some CD4 T cell clones raised against purified p60 from L. monocytogenes cross-react with p60 purified from L. innocua. The L. monocytogenes p60-specific CD4 T cell clone 1A recognized the corresponding L. innocua p60 peptide QAAKPAPAPSTN, which differs only in the first amino acid residue. In vitro experiments revealed that after L. monocytogenes infection of APCs, MHC class I-restricted presentation of p60 occurs, while MHC class II-restricted p60 presentation is inhibited. L. innocua-infected cells presented p60 more weakly but equally well in the context of both MHC class I and MHC class II. In contrast to these in vitro experiments the infection of mice with L. monocytogenes induced a strong p60-specific CD4 and CD8 T cell response, while L. innocua infection failed to induce p60-specific T cells. L. innocua booster infection, however, expanded p60-specific memory T cells induced by previous L. monocytogenes infection. In conclusion, these findings suggest that infection with a frequently occurring environmental bacterium such as L. innocua, which is nonpathogenic and not adapted to intracellular replication, can contribute to the maintenance of memory T cells specific for a related intracellular pathogen.     

Simultaneous deficiency of both MurA and p60 proteins generates a rough phenotype in Listeria monocytogenes

We examined eight spontaneously occurring rough mutants of Listeria monocytogenes for their ability to express two previously reported autolysins, p60 and MurA. All mutants lack MurA expression and show strongly reduced levels of extracellular p60. One rough strain harbors a variant of the p60 protein with a partially truncated catalytic domain. In seven cases there were shifts in the localization of p60 to the membrane fraction. Mutations within the secA2 gene, encoding an auxiliary protein secretion system paralog, were previously shown to be involved in the smooth-rough phenotypic variation seen with Listeria strains. An isogenic DeltasecA2 EGDe deletion strain displays a strong pleiotropic reduction of p60 and MurA, in addition to a large number of secreted and surface proteins. However, we observed no apparent SecA2 dysfunction in several of the investigated strains as determined by direct sequencing of the secA2 gene and complementation of the DeltasecA2 mutant with the respective allele cloned from the rough mutant. To determine the gene products required for the smooth-rough transition, we created mutants lacking the individual iap and murA genes as well as a Deltaiap DeltamurA double mutant. The double mutant displays a rough phenotype and exhibits many of the properties seen with the DeltasecA2 mutant. Our results implicate p60 and MurA as important determinants in controlling the cell shape of L. monocytogenes. We also identified homologous MurA and SecA2 proteins in other Listeria species. The muramidase in two species, L. innocua and L. welshimeri, shows activity similar to that of the MurA protein in L. monocytogenes.     

DNA sequence heterogeneity in the gene encoding a 60-kilodalton extracellular protein of Listeria monocytogenes and other Listeria species.

Chromosomal DNA sequences from the 60 kilodalton protein gene of Listeria monocytogenes, amplified by the polymerase chain reaction, were used for restriction fragment length polymorphism differentiation of L. monocytogenes serotypes and other Listeria species. All 24 strains of L. monocytogenes examined produced an extracellular protein of molecular weight 60,000 (p60) as determined by Western blot analysis. Four of six other Listeria species had a protein that cross-reacted to antibodies to p60, but all differed in molecular weight, ranging from approximately 50,000 to 65,000. The gene encoding p60 was amplified from chromosomal DNA in all strains using polymerase chain reaction with a single primer pair. Restriction enzyme digestion with HindIII of the amplified product revealed a restriction pattern that was distinct between serotypes 1/2a and either 4b or 1/2b of L. monocytogenes. Of the other Listeria species, four strains that produced a cross-reacting protein likewise produced a polymerase chain reaction amplification product with the primer pair. Listeria innocua alone had a restriction pattern similar to that of Listeria monocytogenes serotype 4b and 1/2b. Genotypic heterogeneity, as revealed by DNA amplification and restriction endonuclease digestion of the p60 open reading frame, correlates with "electrophoretic type" grouping and may be related to differences in virulence mechanisms of Listeria monocytogenes and other Listeria species.     

Contribution of hemolysin and phospholipase activity to cytolytic properties and viability of Listeria monocytogenes

The life cycle of Listeria monocytogenes strongly depends on cytolysin which facilitates the pathogen's escape from the endosome. This process is very ineffective in the absence of phospholipases. In this study we investigated the molecular mechanism of cooperation between hemolysin and phospholipases. We propose a model involving interactions between proteins that increase the affinity of cytolysins for the membrane as well direct enzymatic enhancement of lysis.     

The p60 and NamA autolysins from Listeria monocytogenes contribute to host colonization and induction of protective memory

Inducing long‐term protective memory CD8⁺ T‐cells is a desirable goal for vaccines against intracellular pathogens. However, the mechanisms of differentiation of CD8⁺ T‐cells into long‐lived memory cells capable of mediating protection of immunized hosts remain incompletely understood. We have developed an experimental system using mice immunized with wild type (WT) or mutants of the intracellular bacterium Listeria monocytogenes (Lm) that either do or do not develop protective memory CD8⁺ T‐cells. We previously reported that mice immunized with Lm lacking functional SecA2, an auxiliary secretion system of gram‐positive bacteria, did not differentiate functional memory CD8⁺ T‐cells that protected against a challenge infection with WT Lm. Herein we hypothesized that the p60 and NamA autolysins of Lm, which are major substrates of the SecA2 pathway, account for this phenotype. We generated Lm genetically deficient for genes encoding for the p60 and NamA proteins, ΔiapΔmurA Lm, and further characterized this mutant. Δp60ΔNamA Lm exhibited a strong filamentous phenotype, inefficiently colonized host tissues, and grew mostly outside cells. When Δp60ΔNamA Lm was made single unit, cell invasion was restored to WT levels during vaccination, yet induced memory T‐cells still did not protect immunized hosts against recall infection. Recruitment of blood phagocytes and antigen‐presenting cell activation was close to that of mice immunized with ΔActA Lm, which develop protective memory. However, key inflammatory factors involved in optimal T‐cell programming such as IL‐12 and type I IFN (IFN‐I) were lacking, suggesting that cytokine signals may largely account for the observed phenotype. Thus, altogether, these results establish that p60 and NamA secreted by Lm promote primary host cell invasion, the inflammatory response and the differentiation of functional memory CD8⁺ T‐cells, by preventing Lm filamentation during growth and subsequent triggering of innate sensing mechanisms.     

Distinct protein patterns associated with Listeria monocytogenes InlA- or InlB-phagosomes

Internalization of Listeria monocytogenes into non-phagocytic cells is mediated by the interactions between the two bacterial invasion proteins InlA (internalin) and InlB and their cellular surface receptors E-cadherin and c-Met. To get an insight into all the cellular components necessary for uptake and early intracellular life, we undertook a global proteomic characterization of the early listerial phagosome in the human epithelial cell line LoVo. First, we proceeded to an immunocytochemical characterization of intracellular marker recruitment to phagosomes containing latex beads coated with InlA or InlB. E-cadherin and c-Met were, as expected, rapidly recruited to the phagosomal formation site. Phagosomes subsequently acquired the early endosomal antigen 1 (EEA1) and the lysosomal-associated membrane protein 1 (LAMP1), while presenting a more delayed enrichment of the lysosomal hydrolase cathepsin D. Early phagosomes devoid of lysosomal, endoplasmic reticulum and Golgi enzymatic activities could then be isolated by subcellular fractionation of LoVo cells. Two-dimensional gel electrophoresis (2DPAGE) revealed differences between the protein profiles of InlA- or InlB-phagosomes and those of early/late endosomes or lysosomes of naive LoVo cells. One major protein specifically recruited on the InlB-phagosomes was identified by mass spectrometry as MSF, a previously reported member of the septin family of GTPases. MSF forms filaments that co-localize with the actin cytoskeleton in resting cells and it is recruited to the entry site of InlB-coated beads. These results suggest that MSF is a putative effector of the InlB-mediated internalization of L. monocytogenes into host cells.     

Membrane fusion induced by the catalytic activity of a phospholipase C/sphingomyelinase from Listeria monocytogenes

Listeria monocytogenes is a bacterium responsible for localized and generalized infections in humans and animals. It has the ability to spread from the cytoplasm of an infected cell to neighboring cells without becoming exposed to the extracellular space. The bacterium secretes a phospholipase C (PLC(LM)) that is active on glycerophospholipids, e.g., phosphatidylcholine, and on sphingomyelin; thus, PLC(LM) should be described more appropriately as a phospholipase C/sphingomyelinase. We have obtained PLC(LM) free from a frequent contaminant, listeriolysin O, using an improved purification procedure. PLC(LM) has been assayed on large unilamellar liposomes of defined lipid composition. The enzyme is activated by K(+) and Mg(2+), and readily degrades phospholipids in bilayer form, in the absence of detergents. Enzyme activity is accompanied by important changes in the structure of the phospholipid vesicles, namely, vesicle aggregation, intervesicular mixing of lipids, and mixing of aqueous contents, with very low leakage of vesicular contents. The data are interpreted as indicative of PLC(LM)-induced vesicle fusion. This is confirmed by the demonstration of intervesicular mixing of inner monolayer lipids, using a novel procedure. The observation of PLC(LM)-induced membrane fusion suggests a mechanism for the cell-to-cell propagation of the bacterium, which requires disruption of a double-membrane vacuole.     

Structural and functional properties of the p60 proteins from different Listeria species.

The major extracellular protein p60 of Listeria monocytogenes seems to be required for this microorganism's adherence to and invasion of 3T6 mouse fibroblasts but not for adherence to human epithelial Caco-2 cells. Western blot analysis with polyclonal antibodies against p60 of L. monocytogenes indicated the presence of cross-reacting proteins in the culture supernatants of all Listeria species. Protein p60 of L. monocytogenes could restore adhesion of the L. monocytogenes mutant RIII (impaired in the synthesis of p60) to mouse fibroblasts more efficiently than that of Listeria grayi. The amino acid sequences of the p60-related proteins of L. innocua, L. ivanovii, L. seeligeri, L. welshimeri, and L. grayi indicated highly conserved regions of about 120 amino acids at both the N-terminal and the C-terminal ends. The middle portions of these proteins, consisting of about 240 amino acids, varied considerably. These parts include the repeat domain consisting of repetitions of Thr (T) and Asn (N) which was present only, albeit in different arrangements, in the p60 proteins of L. monocytogenes and L. innocua. The p60-related proteins of L. grayi, L. ivanovii, L. seeligeri, and L. welshimeri each contained an insertion of 54 amino acids which was absent in the p60 proteins of L. monocytogenes and L. innocua.     

Physical properties of nisin-incorporated gelatin and corn zein films and antimicrobial activity against Listeria monocytogenes

Edible films of gelatin and corn zein were prepared by incorporating nisin to the film-forming solutions. Corn zein film with nisin of 12,000 IU/ml had an increase of 11.6 MPa in tensile strength compared with the control, whereas gelatin film had a slight increase with the increase of nisin concentration added. Water vapor permeability for both corn zein and gelatin films decreased with the increase of nisin concentration, thus providing a better barrier against water. Antimicrobial activity against Listeria monocytogenes increased with the increase of nisin concentration, resulting in 1.4 log cycle reduction for corn zein film and 0.6 log cycle reduction for gelatin film at 12,000 IU/ml. These results suggest that incorporation of nisin into corn zein and gelatin films improve the physical properties of the films as well as antimicrobial activity against pathogenic bacteria during storage, resulting in extension of the shelf life of food products by providing with antimicrobial edible packaging films.