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Stimulated by its physiological ligand, hepatocyte growth factor, the transmembrane receptor tyrosine kinase Met activates a signaling machinery that leads to mitogenic, motogenic, and morphogenic responses. Remarkably, the food-borne human pathogen Listeria monocytogenes also promotes autophosphorylation of Met through its virulence factor internalin B (InlB) and subsequently exploits Met signaling to induce phagocytosis into a broad range of host cells. Although the interaction between InlB and Met has been studied in detail, the signaling specificity of components involved in InlB-triggered cellular responses remains poorly characterized. The analysis of regulated phosphorylation events on protein kinases is therefore of particular relevance, although this could not as yet be characterized systematically by proteomics. Here, we implemented a new pyridopyrimidine-based strategy that enabled the efficient capture of a considerable subset of the human kinome in a robust one-step affinity chromatographic procedure. Additionally, and to gain functional insights into the InlB/Met-induced bacterial invasion process, a quantitative survey of the phosphorylation pattern of these protein kinases was accomplished. In total, the experimental design of this study comprises affinity chromatographic procedures for the systematic enrichment of kinases, as well as phosphopeptides; the quantification of all peptides based on the iTRAQ reporter system; and a rational statistical strategy to evaluate the quality of phosphosite regulations. With this improved chemical proteomics strategy, we determined and relatively quantified 143 phosphorylation sites detected on 94 human protein kinases. Interestingly, InlB-mediated signaling shows striking similarities compared with the natural ligand hepatocyte growth factor that was intensively studied in the past. In addition, this systematic approach suggests a new subset of protein kinases including Nek9, which are differentially phosphorylated after short time (4-min) treatment of cells with the Met-activating InlB(321). Thus, this quantitative phosphokinome study suggests a general, hypothesis-free concept for the detection of dynamically regulated protein kinases as novel signaling components involved in host-pathogen interactions.

Lipoprotein anchoring in bacteria is mediated by the prolipoprotein diacylglyceryl transferase (Lgt), which catalyzes the transfer of a diacylglyceryl moiety to the prospective N-terminal cysteine of the mature lipoprotein. Deletion of the lgt gene in the gram-positive pathogen Listeria monocytogenes (i) impairs intracellular growth of the bacterium in different eukaryotic cell lines and (ii) leads to increased release of lipoproteins into the culture supernatant. Comparative extracellular proteome analyses of the EGDe wild-type strain and the Delta lgt mutant provided systematic insight into the relative expression of lipoproteins. Twenty-six of the 68 predicted lipoproteins were specifically released into the extracellular proteome of the Delta lgt strain, and this proved that deletion of lgt is an excellent approach for experimental verification of listerial lipoproteins. Consequently, we generated Delta lgt Delta prfA double mutants to detect lipoproteins belonging to the main virulence regulon that is controlled by PrfA. Overall, we identified three lipoproteins whose extracellular levels are regulated and one lipoprotein that is posttranslationally modified depending on PrfA. It is noteworthy that in contrast to previous studies of Escherichia coli, we unambiguously demonstrated that lipidation by Lgt is not a prerequisite for activity of the lipoprotein-specific signal peptidase II (Lsp) in Listeria.

Pathogenic bacteria have to cope with defence mechanisms mediated by adaptive and innate immunity of the host cells. Cationic antimicrobial peptides (CAMPs) represent one of the most effective components of the host innate immune response. Here we establish the function of Lmo1695, a member of the VirR-dependent virulence regulon, recently identified in Listeria monocytogenes. Lmo1695 encodes a membrane protein of 98 kDa with strong homology to the multiple peptide resistance factor (MprF) of Staphylococcus aureus. Like staphylococcal MprF, we found that Lmo1695 is involved in the synthesis of the membrane phospholipid lysylphosphatidylglycerol (L-PG). In addition, Lmo1695 is also essential for lysinylation of diphosphatidylglycerol (DPG), another phospholipid widely distributed in bacterial membranes. A Deltalmo1695 mutant lacking the lysinylated phospholipids was particularly susceptible to CAMPs of human and bacterial origin. The mutant strain infected both epithelial cells and macrophages only poorly and was attenuated for virulence when tested in a mouse model of infection. Lmo1695 is a member of a growing list of survival factors which enable growth of L. monocytogenes in different environments.

The ability of Listeria monocytogenes to breach mucosal and endothelial barriers of the host during infection is a hallmark property mediated by the internalins (Inl) A and B. We examined the invasive property of several L. monocytogenes strains representing 13 serotypes. We found that invasiveness is a heterogeneous phenotype amongst L. monocytogenes serotype strains. Despite this, many of the poorly invasive and non-invasive strains of L. monocytogenes express internalins at levels comparable to those of invasive isolates. Introduction of the inlAB locus from EGD-e into several poorly invasive strains had no effect on their invasive properties. A strain from serotype 4b that exhibits highly invasive properties was further examined. Deletion of the inlAB locus abrogated invasion of this strain while reintroduction of the inlAB locus into this strain restored invasiveness. An analysis of regions flanking the inlAB locus revealed considerable differences in the strains studied. Our results suggest that efficacious entry of L. monocytogenes into eukaryotic cells is complex and requires additional factors apart from internalins. Data presented here also suggest that the inlAB locus was introduced into L. monocytogenes by horizontal gene transfer with subsequent deletion and rearrangements occurring during evolution of this species.

Listeria species are ubiquitous in the environment and often contaminate foods because they grow under conditions used for food preservation. Listeria monocytogenes, the human and animal pathogen, causes Listeriosis, an infection with a high mortality rate in risk groups such as immune-compromised individuals. Furthermore, L.monocytogenes is a model organism for the study of intracellular bacterial pathogens. The publication of its genome sequence and that of the non-pathogenic species Listeria innocua initiated numerous comparative studies and efforts to sequence all species comprising the genus. The Proteome database LEGER (http://leger2.gbf.de/cgi-bin/expLeger.pl) was developed to support functional genome analyses by combining information obtained by applying bioinformatics methods and from public databases to improve the original annotations. LEGER offers three unique key features: (i) it is the first comprehensive information system focusing on the functional assignment of genes and proteins; (ii) integrated visualization tools, KEGG pathway and Genome Viewer, alleviate the functional exploration of complex data; and (iii) LEGER presents results of systematic post-genome studies, thus facilitating analyses combining computational and experimental results. Moreover, LEGER provides an unpublished membrane proteome analysis of L.innocua and in total visualizes experimentally validated information about the subcellular localizations of 789 different listerial proteins.

Proteomics is required to provide insight into any type of subproteome. While the workflow based on two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) can be applied for many subproteomes and comprises well-established strategies for data presentation and data analysis, the comprehensive investigation of membrane proteomes remains a challenging task. We present a number of procedures that provide an insight into such systems. We have established a novel protocol for the efficient preparation of membrane fractions, which is used here for the human pathogen Listeria monocytogenes that overcomes difficulties associated with ribosomes. Subsequently, we have used the combination of sodium dodecyl sulfate (SDS)-PAGE and liquid chromatography-tandem mass spectrometry for the characterization of the membrane proteome. Three hundred and one different membrane proteins could be identified, including 70 proteins that exhibited 2-15 transmembrane domains. However, a remarkably high ratio of proteins was detected in gel sections that were not in accordance with their expected migration behavior during SDS-PAGE. Protein identifications based on MASCOT significance criteria could be shown to be of high quality and therefore could not be the explanation of this observation. Consequently we have developed LaneSpector, a general visualization tool that allows the systematic comparison between apparent and calculated protein masses, which is routinely applicable to any high-throughput approach using a mass-dependent separation dimension prior to LC-MS/MS. The detailed presentation of the LaneSpector plot promotes the validation of the analytical process and might help to reveal relevant biological processes such as proteolysis or other post-translational modifications.

Cell-to-cell spread is a fundamental step in the infection cycle of Listeria monocytogenes that strictly depends on the formation of bacteria-induced protrusions. Since Listeria actin tails in the protrusions are tightly associated with the plasma membrane, we hypothesised that membrane-cytoskeleton linkers would be required for initiating and sustaining their formation and the subsequent cell-to-cell spread. We have found that ezrin, a member of the ezrin, radixin and moesin (ERM) family that functions as a key membrane-cytoskeleton linker, accumulates at Listeria protrusions. The ability of Listeria to induce protrusions and effectively spread between adjacent cells depends on the interaction of ERM proteins with both a membrane component such as CD44 and actin filaments. Interfering with either of these interactions or with ERM proteins phosphorylation not only reduces the number of protrusions but also alters their morphology, resulting in the formation of short and collapsed protrusions. As a consequence, Listeria cell-to-cell spread is severely impaired. Thus, ERM proteins are exploited by Listeria to escape the host immune response and to succeed in the development of the infection.

The surface subproteome of Listeria monocytogenes that includes many proteins already known to be involved in virulence and interaction with host cells has been characterized. A new method for the isolation of a defined surface proteome of low complexity has been established based on serial extraction of proteins by different salts at high concentration, and in all 55 proteins were identified by N-terminal sequencing and mass spectrometry. About 16% of these proteins are of unknown function and three proteins have no orthologue in the nonpathogenic L. innocua and might be involved in virulence mechanisms. Remarkably, a relatively high number of proteins with a function in the cytoplasmic compartment was identified in this surface proteome. These proteins had neither predicted or detectable signal peptides nor could any modification be observed except removal of the N-terminal methionine. Enolase (Lmo2455) is one of these proteins. It was shown to be present in the cell wall of the pathogen by immunoelectron microscopy and, along with heat shock factor DnaK (Lmo1473), elongation factor TU (Lmo2653), and glyceraldehyde-3-phosphate dehydrogenase (Lmo2459), it was found to be able to bind human plasminogen in overlay blots and surface plasmon resonance (SPR) experiments. The KD values of these interactions were determined by SPR measurements. The data indicate a possible role of these proteins as receptors for human plasminogen on the bacterial cell surface. The potential role of this recruitment of a host protease for extracellular invasion mechanisms is discussed.

The surface protein InlB of the pathogen Listeria monocytogenes promotes invasion of this bacterium into host cells by binding to and activating the receptor tyrosine kinase Met. The curved leucine-rich repeat (LRR) domain of InlB, which is essential for this process, contains a string of five surface-exposed aromatic amino acid residues positioned along its concave face. Here, we show that the replacement of four of these residues (F104, W124, Y170 or Y214) by serine leads to a complete loss of uptake of latex beads coated with InlB', a truncated functional variant of InlB. The mutants correspondingly display severely reduced binding to Met. To abrogate fully invasion of bacteria expressing full-length InlB, exchange of at least four aromatic amino acids is required. We conclude that InlB binds to Met through its concave surface of the LRR domain, and that aromatic amino acids are critical for binding and signalling before invasion.

We have examined the effect of covalently crosslinked profilin-actin (PxA), which closely matches the biochemical properties of ordinary profilin-actin and interferes with actin polymerization in vitro and in vivo, on Listeria monocytogenes motility. PxA caused a marked reduction in bacterial motility, which was accompanied by the detachment of bacterial tails. The effect of PxA was dependent on its binding to proline-rich sequences, as shown by the inability of PH133SxA, which cannot interact with such sequences, to impair Listeria motility. PxA did not alter the motility of a Listeria mutant that is unable to recruit Ena (Enabled)/VASP (vasodilator-stimulated phosphoprotein) proteins and profilin to its surface. Finally, PxA did not block the initiation of actin-tail formation, indicating that profilin-actin is only required for the elongation of actin filaments at the bacterial surface. Our findings provide further evidence that profilin-actin is important for actin-based processes, and show that it has a key function in Listeria motility.

Listeria monocytogenes, a food-borne bacterial pathogen, enters mammalian cells by inducing its own phagocytosis. The listerial protein internalin (InlA) mediates bacterial adhesion and invasion of epithelial cells in the human intestine through specific interaction with its host cell receptor E-cadherin. We present the crystal structures of the functional domain of InlA alone and in a complex with the extracellular, N-terminal domain of human E-cadherin (hEC1). The leucine rich repeat (LRR) domain of InlA surrounds and specifically recognizes hEC1. Individual interactions were probed by mutagenesis and analytical ultracentrifugation. These include Pro16 of hEC1, a major determinant for human susceptibility to L. monocytogenes infection that is essential for intermolecular recognition. Our studies reveal the structural basis for host tro-pism of this bacterium and the molecular deception L. monocytogenes employs to exploit the E-cadherin system.

Listeria monocytogenes is a food-borne pathogen with a high mortality rate that has also emerged as a paradigm for intracellular parasitism. We present and compare the genome sequences of L. monocytogenes (2,944,528 base pairs) and a nonpathogenic species, L. innocua (3,011,209 base pairs). We found a large number of predicted genes encoding surface and secreted proteins, transporters, and transcriptional regulators, consistent with the ability of both species to adapt to diverse environments. The presence of 270 L. monocytogenes and 149 L. innocua strain-specific genes (clustered in 100 and 63 islets, respectively) suggests that virulence in Listeria results from multiple gene acquisition and deletion events.

The facultative intracellular human pathogenic bacterium Listeria monocytogenes actively recruits host actin to its surface to achieve motility within infected cells. The bacterial surface protein ActA is solely responsible for this process by mimicking fundamental steps of host cell actin dynamics. ActA, a modular protein, contains an N-terminal actin nucleation site and a central proline-rich motif of the 4-fold repeated consensus sequence FPPPP (FP(4)). This motif is specifically recognized by members of the Ena/VASP protein family. These proteins additionally recruit the profilin-G-actin complex increasing the local concentration of G-actin close to the bacterial surface. By using analytical ultracentrifugation, we show that a single ActA molecule can simultaneously interact with four Ena/VASP homology 1 (EVH1) domains. The four FP(4) sites have roughly equivalent affinities with dissociation constants of about 4 microm. Mutational analysis of the FP(4) motifs indicate that the phenylalanine is mandatory for ActA-EVH1 interaction, whereas in each case exchange of the third proline was tolerated. Finally, by using sedimentation equilibrium centrifugation techniques, we demonstrate that ActA is a monomeric protein. By combining these results, we formulate a stoichiometric model to describe how ActA enables Listeria to utilize efficiently resources of the host cell microfilament for its own intracellular motility.

Listeria monocytogenes is an opportunistic, food-borne human and animal pathogen. Host cell invasion requires the action of the internalins A (InlA) and B (InlB), which are members of a family of listerial cell-surface proteins. Common to these proteins are three distinctive N-terminal domains that have been shown to direct host cell-specific invasion for InlA and InlB. Here, we present the high-resolution crystal structures of these domains present in InlB and InlH, and show that they constitute a single "internalin domain". In this internalin domain, a central LRR region is flanked contiguously by a truncated EF-hand-like cap and an immunoglobulin (Ig)-like fold. The extended beta-sheet, resulting from the distinctive fusion of the LRR and the Ig-like folds, constitutes an adaptable concave interaction surface, which we propose is responsible for the specific recognition of the host cellular binding partners during infection., (Copyright 2001 Academic Press.)

The facultative intracellular, Gram-positive bacterium Listeria monocytogenes invades phagocytic and non-phagocytic cells from the tissues and organs of a wide variety of animals and humans. Here, we report the use of these bacteria as vehicles for gene transfer. Eukaryotic expression plasmids were introduced into the nucleus of host cells following lysis of the intracytosolic, plasmid-carrying bacteria with antibiotics. Cell lines of different tissues and species could be transfected in this way. We examined bacterial properties required for delivery of the expression plasmids and found that this was strictly dependent on the ability of these bacteria to both invade eukaryotic cells and egress from the vacuole into the cytosol of the infected host cells. Macrophage-like cell lines or primary, peritoneal macrophages proved to be almost refractory to Listeria-mediated gene transfer. Thus, attenuated L. monocytogenes represents a serious candidate for consideration as a DNA-transfer vehicle for in vivo somatic gene therapy. The potential for oral administration of L. monocytogenes and the ease in producing and cultivating recombinant strains are further attributes that make its use as a gene transfer vehicle attractive.

The recruitment of actin to the surface of intracellular Listeria monocytogenes and subsequent tail formation is dependent on the expression of the bacterial surface protein ActA. Of the different functional domains of ActA identified thus far, the N-terminal region is absolutely required for actin filament recruitment and intracellular motility. Mutational analysis of this domain which abolished actin recruitment by intracellular Listeria monocytogenes identified two arginine residues within the 146-KKRRK-150 motif that are essential for its activity. More specifically, recruitment of the Arp2/3 complex to the bacterial surface, as assessed by immunofluorescence staining with antibodies raised against the p21-Arc protein, was not obtained in these mutants. Consistently, treatment of infected cells with latrunculin B, which abrogated actin filament formation, did not affect association of ActA with p21-Arc at the bacterial surface. Thus, the initial recruitment of the Arp2/3 complex to the bacterial surface is independent of, and precedes, actin polymerisation. Our data suggest that binding of the Arp2/3 complex is mediated by specific interactions dependent on arginine residues within the 146-KKRRK-150 motif present in ActA.

Actin polymerisation is thought to drive the movement of eukaryotic cells and some intracellular pathogens such as Listeria monocytogenes. The Listeria surface protein ActA synergises with recruited host proteins to induce actin polymerisation, propelling the bacterium through the host cytoplasm [1]. The Arp2/3 complex is one recruited host factor [2] [3]; it is also believed to regulate actin dynamics in lamellipodia [4] [5]. The Arp2/3 complex promotes actin filament nucleation in vitro, which is further enhanced by ActA [6] [7]. The Arp2/3 complex also interacts with members of the Wiskott-Aldrich syndrome protein (WASP) [8] family - Scar1 [9] [10] and WASP itself [11]. We interfered with the targeting of the Arp2/3 complex to Listeria by using carboxy-terminal fragments of Scar1 that bind the Arp2/3 complex [11]. These fragments completely blocked actin tail formation and motility of Listeria, both in mouse brain extract and in Ptk2 cells overexpressing Scar1 constructs. In both systems, Listeria could initiate actin cloud formation, but tail formation was blocked. Full motility in vitro was restored by adding purified Arp2/3 complex. We conclude that the Arp2/3 complex is a host-cell factor essential for the actin-based motility of L. monocytogenes, suggesting that it plays a pivotal role in regulating the actin cytoskeleton.

In this study, we analyzed whether the actin-based motility of intracellular Listeria monocytogenes is controlled by the small GTP-binding proteins of the Rho- and Ras-subfamilies. These signalling proteins are key regulatory elements in the control of actin dynamics and their activity is essential for the maintenance of most cellular microfilament structures. We used the Clostridium difficile toxins TcdB-10463 and TcdB-1470 to specifically inactivate these GTP-binding proteins. Treatment of eukaryotic cells with either of these toxins led to a dramatic breakdown of the normal actin cytoskeleton, but did not abrogate the invasion of epithelial cells by L. monocytogenes and had no effect on the actin-based motility of this bacterial parasite. Our data indicate that intracellular Listeria reorganize the actin cytoskeleton in a way that circumvents the control mechanisms mediated by the members of the Rho- and Ras-subfamilies that can be inactivated by the TcdB-10463 and TcdB-1470 toxins.

Intracellular propulsion of Listeria monocytogenes is the best understood form of motility dependent on actin polymerization. We have used in vitro motility assays of Listeria in platelet and brain extracts to elucidate the function of the focal adhesion proteins of the Ena (Drosophila Enabled)/VASP (vasodilator-stimulated phosphoprotein) family in actin-based motility. Immunodepletion of VASP from platelet extracts and of Evl (Ena/VASP-like protein) from brain extracts of Mena knockout (-/-) mice combined with add-back of recombinant (bacterial or eukaryotic) VASP and Evl show that VASP, Mena, and Evl play interchangeable roles and are required to transform actin polymerization into active movement and propulsive force. The EVH1 (Ena/VASP homology 1) domain of VASP is in slow association-dissociation equilibrium high-affinity binding to the zyxin-homologous, proline-rich region of ActA. VASP also interacts with F-actin via its COOH-terminal EVH2 domain. Hence VASP/ Ena/Evl link the bacterium to the actin tail, which is required for movement. The affinity of VASP for F-actin is controlled by phosphorylation of serine 157 by cAMP-dependent protein kinase. Phospho-VASP binds with high affinity (0.5 x 10(8) M-1); dephospho-VASP binds 40-fold less tightly. We propose a molecular ratchet model for insertional polymerization of actin, within which frequent attachment-detachment of VASP to F-actin allows its sliding along the growing filament.

ActA, an essential virulence factor of Listeria monocytogenes, is an integral membrane protein that is required for intracellular motility, cell-to-cell spread, and rapid dissemination of the bacteria in the infected host. To reveal cytotoxic T cell responses against ActA we introduced a recombinant soluble form of ActA into the MHC class I-processing compartment of APC using a variant of listeriolysin mutated within its immunodominant MHC class I epitope. With this experimental system we demonstrate that T cells are induced against ActA during a sublethal infection with L. monocytogenes. However, adoptively transferred cytotoxic CD8+ T cells specific for ActA did not protect mice against a subsequent challenge with this pathogen. This was due to an inability of APC to present ActA by either MHC class I or class II molecules as long as ActA remained tethered to the surface of intracellular viable bacteria. ActA was only presented when L. monocytogenes were engineered to secrete ActA or when the bacteria were killed by antibiotics during the assay. These findings raise questions on the general use of membrane proteins of pathogens as candidates for subunit vaccines.

The property of listeriolysin (LLO) to introduce soluble passenger proteins into the cytosol of antigen-presenting cells allows the induction of CD8+ cytotoxic T cells against such antigens. To overcome the potential problem of presentation of the immunodominant epitope LL091-99 by H-2Kd, a variant LLO92A was established in which Tyr 92 was replaced by Ala. Immunization of BALB/c mice with purified LLO92A failed to stimulate cytotoxic T cells specific for either the epitope LLO91-99 or for any other LLO-derived peptide. Injection of mixtures of purified LLO92A and soluble nucleoprotein (NP) of influenza virus into mice resulted in a strong cytotoxic T cell response exclusively directed against NP. The LLO92A variant was successfully used to generate, propagate and characterize a CD8 T cell line specific for the membrane-bound virulence factor ActA of Listeria monocytogenes. Interestingly, wildtype ActA bound to the surface of live L. monocytogenes was not presented by MHC class I molecules to the CD8+ T cell line.

Entry of Listeria monocytogenes into nonphagocytic cells requires the inlAB gene products. InlA and InlB are bacterial cell wall-associated polypeptides that can be released by sodium dodecyl sulfate treatment. By applying more gentle extraction methods, we have purified InlB in its native form. Treatment of bacteria with various nondenaturating agents including mutanolysin, thiol reagents, sodium chloride, and detergents like Triton X-100 or 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate did not release substantial amounts of InlB from the bacterial cell wall. Instead, InlB was nearly quantitatively extracted in a solubilized form by treatment of bacteria with 1 M Tris-Cl or other protonated amines at pH 7.5. However, the reduced solubility of the extracted InlB in low-salt buffers hampered further biochemical purification. A panel of monoclonal antibodies against listerial Tris-Cl extracts containing InlB was therefore produced to generate reagents for use in affinity chromatography. One of the monoclonal antibodies enabled purification of the InlB protein to homogeneity with relatively high yields. When added externally, purified InlB associated with the surface of noninvasive bacteria such as Listeria innocua or an L. monocytogenes inlB2 mutant, where it promoted entry of these strains into Vero cells >300- and 17-fold, respectively. This effect was even more dramatic for HeLa cells, where the observed invasion was increased about 9,000- and 4,000-fold, respectively. The availability of purified native, invasion-competent InlB will allow analysis of the molecular basis of InlB-mediated entry into tissue culture cell lines in greater detail.

Listeria monocytogenes causes rhombencephalitis in humans and animals and also affects the fetus in utero, causing disseminated sepsis. In both instances, the infection occurs by the crossing of endothelial cells lining a physiological barrier, the blood-brain barrier or the transplacental barrier. In this study, the ability of L. monocytogenes wild-type EGD to invade human umbilical vein endothelial cells (HUVECs) was evaluated using wild-type bacteria and isogenic Listeria mutants. Here, we show that invasion of HUVECs by L. monocytogenes is dependent on the expression of the internalin B gene product. This was demonstrated in several ways. First, L. monocytogenes strains lacking the inlB gene did not invade HUVECs. Secondly, avid invasion was obtained when a strain deleted for inlAB was complemented with a plasmid harbouring inlB only, whereas strains expressing inlA did not enter HUVECs. Thirdly, entry of wild-type EGD could be blocked effectively with antibodies to InlB. Fourthly, cell binding assays and flow cytometry with HUVECs showed binding of purified InlB, but not InlA, suggesting a tropism of InlB for this cell type. Finally, physical association of purified native InlB with the surface of non-invasive mutants dramatically increased their ability to invade HUVECs. In laser-scanning confocal microscopy, binding of InlB was observed as focal and localized patches on the cell surface of HUVECs. Qualitative examination of the entry process by scanning electron microscopy revealed that both wild-type EGD and a recombinant strain overexpressing only InlB enter HUVECs in a similar fashion. The entry process was polarized, involved single bacteria and occurred over the entire surface of endothelial cells.

The function of the ActA protein of Listeria monocytogenes has been partially elucidated. These results illustrate the sophistication with which intracellular pathogens like Listeria use the host cell to their advantage, and have provided new insights into some of the molecular mechanisms of complex cell functions such as actin-promoted cell motility. The clarification of these processes is of fundamental importance not only for understanding elementary processes such as development and growth, but also for the treatment of both diseases caused by cytopathogenic bacteria such as Listeria and pathophysiological processes arising from disorders in cell motility and cell adhesion.

An attenuated strain of S. typhimurium has been used as a vehicle for oral genetic immunization. Eukaryotic expression vectors containing truncated genes of ActA and listeriolysin--two virulence factors of Listeria monocytogenes--have been used to transform S. typhimurium aroA. Multiple or even single oral immunizations with such transformants induced excellent cellular and humoral responses. In addition, protective immunity was induced with listeriolysin transformants. The quality of the responses suggested a transfer of plasmid DNA from the bacterial carrier to the host. Such transfer was unequivocally shown in vitro with primary peritoneal macrophages. We describe a highly versatile system for antigen delivery, identification of protective antigens for vaccination, and efficient generation of antibodies against the product of open reading frames present on virtually any DNA segment.

The X-ray crystal structure of the phosphatidylinositol-specific phospholipase C (PI-PLC) from the human pathogen Listeria monocytogenes has been determined both in free form at 2.0 A resolution, and in complex with the competitive inhibitor myo-inositol at 2.6 A resolution. The structure was solved by a combination of molecular replacement using the structure of Bacillus cereus PI-PLC and single isomorphous replacement. The enzyme consists of a single (beta alpha)8-barrel domain with the active site located at the C-terminal side of the beta-barrel. Unlike other (beta alpha)8-barrels, the barrel in PI-PLC is open because it lacks hydrogen bonding interactions between beta-strands V and VI. myo-Inositol binds to the active site pocket by making specific hydrogen bonding interactions with a number of charged amino acid side-chains as well as a coplanar stacking interaction with a tyrosine residue. Despite a relatively low sequence identity of approximately 24%, the structure is highly homologous to that of B.cereus PI-PLC with an r.m.s. deviation for 228 common C alpha positions of 1.46 A. Larger differences are found for loop regions that accommodate most of the numerous amino acid insertions and deletions. The active site pocket is also well conserved with only two amino acid replacements directly implicated in inositol binding.

We have examined the human humoral immune response directed against proteins of Listeria monocytogenes in both healthy individuals and listeriosis patients. Two major targets for an antibody response were found in individuals that did not suffer from listeriosis: listeriolysin (Hly) and the recently described internalin-related protein (IrpA). In contrast, the humoral response in listeriosis patients appears to be more heterogeneous and included Hly, IrpA, InlB, and ActA as major targets.

The ActA protein of the intracellular pathogen Listeria monocytogenes induces a dramatic reorganization of the actin-based cytoskeleton. Two profilin binding proteins, VASP and Mena, are the only cellular proteins known so far to bind directly to ActA. This interaction is mediated by a conserved module, the EVH1 domain. We identify E/DFPPPPXD/E, a motif repeated 4-fold within the primary sequence of ActA, as the core of the consensus ligand for EVH1 domains. This motif is also present and functional in at least two cellular proteins, zyxin and vinculin, which are in this respect major eukaryotic analogs of ActA. The functional importance of the novel protein-protein interaction was examined in the Listeria system. Removal of EVH1 binding sites on ActA reduces bacterial motility and strongly attenuates Listeria virulence. Taken together we demonstrate that ActA-EVH1 binding is a paradigm for a novel class of eukaryotic protein-protein interactions involving a proline-rich ligand that is clearly different from those described for SH3 and WW/WWP domains. This class of interactions appears to be of general importance for processes dependent on rapid actin remodeling.

We have examined the influence of listeriolysin O (LLO), the hemolysin secreted by the pathogenic bacterium Listeria monocytogenes, on major histocompatibility complex class II-dependent T cell activation. Stimulation of T cells by native antigens but not by peptides is inhibited upon pretreatment of antigen-presenting cells (APC) with LLO. Experiments presented here reveal that this inhibition is not due to a lack in processing of antigen by APC but is the result of an irreversible inactivation of T cells that recognize antigen on LLO-treated APC. Incubation of mixtures of two different T cells where only one antigen was presented on LLO-treated APC suggested that T cell inactivation is antigen specific. The inactivation was dominant and could be observed even in the presence of amounts of synthetic peptides that normally lead to T cell responses. This condition is reminiscent of the T cell inhibition observed when antagonistic and stimulatory peptides are added to APC at the same time. Our results thus reveal a novel type of interference by pathogens with antigen presentation and T cell stimulation that could give the pathogen a decisive advantage in dissemination and disease.

Listeria monocytogenes is driven through infected host cytoplasm by a comet tail of actin filaments that serves to project the bacterium out of the cell surface, in pseudopodia, to invade neighboring cells. The characteristics of pseudopodia differ according to the infected cell type. In PtK2 cells, they reach a maximum length of approximately 15 microm and can gyrate actively for several minutes before reentering the same or an adjacent cell. In contrast, the pseudopodia of the macrophage cell line DMBM5 can extend to >100 microm in length, with the bacteria at their tips moving at the same speed as when at the head of comet tails in bulk cytoplasm. We have now isolated the pseudopodia from PtK2 cells and macrophages and determined the organization of actin filaments within them. It is shown that they possess a major component of long actin filaments that are more or less splayed out in the region proximal to the bacterium and form a bundle along the remainder of the tail. This axial component of filaments is traversed by variable numbers of short, randomly arranged filaments whose number decays along the length of the pseudopodium. The tapering of the tail is attributed to a grading in length of the long, axial filaments. The exit of a comet tail from bulk cytoplasm into a pseudopodium is associated with a reduction in total F-actin, as judged by phalloidin staining, the shedding of alpha-actinin, and the accumulation of ezrin. We propose that this transition reflects the loss of a major complement of short, random filaments from the comet, and that these filaments are mainly required to maintain the bundled form of the tail when its borders are not restrained by an enveloping pseudopodium membrane. A simple model is put forward to explain the origin of the axial and randomly oriented filaments in the comet tail.

The expression of all virulence factors in Listeria monocytogenes characterized to date is controlled by the virulence regulator protein, PrfA. To identify further PrfA-regulated proteins, we examined supernatants of L. monocytogenes EGD harboring additional copies of the PrfA regulator for the presence of novel proteins. This led to the identification and biochemical purification of a hitherto uncharacterized PrfA-dependent 30-kDa protein (A. Lingnau, T. Chakraborty, K. Niebuhr, E. Domann, and J. Wehland, Infect. Immun. 64:1002-1006, 1996). Oligonucleotide primers derived from internal peptide sequences of this protein allowed the cloning and determination of the entire sequence of the respective gene. The protein comprised 297 amino acids with strong overall homology to the internalins, InlA and InlB, particularly in the region harboring the leucine-rich repeats. The gene has been designated irpA for internalin-related protein A gene. Transcriptional studies revealed that the gene was monocistronic and, like the inlA and inlB genes, was transcribed by PrfA-dependent and PrfA-independent mechanisms. Monoclonal antibodies raised against IrpA indicated that it was produced by L. monocytogenes but not by the nonpathogenic species Listeria innocua. To examine the role of IrpA in pathogenesis, we constructed an isogenic in-frame deletion mutant that removed all but 116 amino acids of the IrpA protein. This mutant was neither defective for invasion into many tissue culture cell lines nor did it demonstrate reduced intracellular survival. However, in vivo studies using the mouse infection model revealed that the irpA mutant showed reduced virulence compared to the parental strain. These results suggest a role for IrpA during disseminated infection by L. monocytogenes.

The surface-bound ActA polypeptide of the intracellular bacterial pathogen Listeria monocytogenes acts as a nucleator protein, generating the actin cytoskeleton around intracellularly motile bacteria. In this work, we examined the functional similarity of ActA from Listeria ivanovii (iActA) ATCC 19119 to its L. monocytogenes counterpart. The amino acid sequence of iActA predicts a molecular mass of 123 kDa and harbors eight proline-rich repeats. For functional analysis, various iActA derivatives and hybrid constructs of L. ivanovii and L. monocytogenes ActA polypeptides were transiently expressed in epithelial cells and examined for recruitment of host microfilament proteins by a mitochondrial targeting assay. As has been demonstrated with ActA, iActA also spontaneously inserted into the surface of mitochondria and induced recruitment of actin, alpha-actinin, and the vasodilator-stimulated phosphoprotein (VASP) to these subcellular organelles. By comparison of amino-terminally truncated iActA derivatives for their ability to recruit cytoskeletal proteins, a region essential for actin filament accumulation was identified between amino acid residues 290 and 325. Such derivatives, however, retained their ability to bind VASP. Replacement of the proline-rich repeats in ActA with those of iActA also resulted in VASP recruitment. Hence, despite the limited overall sequence homology between ActA and iActA, the two molecules consist of at least two similar domains: a highly positively charged N-terminal domain that is directly involved in actin filament recruitment and a proline-rich repeat region required for VASP binding.

Infection of a murine-spleen dendritic cell line by Listeria monocytogenes was found to induce cell death through apoptosis. To characterize the bacterial product(s) involved in induction of apoptosis, dendritic cells were infected with the L. monocytogenes EGD strain and several isogenic mutants deficient in the production of individual listerial virulence factors. The ability to induce cellular apoptosis was retained by all mutants tested, except the prfA and delta hly mutants, both of which are unable to produce listeriolysin. Apoptosis was also induced by purified listeriolysin suggesting that this protein directly induces apoptosis. Purified recombinant listeriolysins rendered either weakly haemolytic by a C-484 to S mutation, or nonhaemolytic by a W-491 to A mutation exhibited little or no capacity to induce apoptosis, indicating that both activities are associated within the same protein region. Treatment with purified listeriolysin or L. monocytogenes infection also triggers apoptosis in explanted bone-marrow dendritic cells. Thus invasion of dendritic cells by L. monocytogenes, which results in cell death, may play an important role in the pathogenesis of listerial infections by impairing immune responses, hindering bacterial clearance and promoting spread of the infection.

Monoclonal antibodies were generated against a 30-kDa protein fraction derived from culture supernatants of a Listeria monocytogenes strain complemented with additional copies of the prfA regulator gene. Several of the antibodies reacted specifically with a hitherto unidentified, secreted 30-kDa polypeptide. By immunoblot analysis, the expression of this 30kDa polypeptide was found to be dependent on the presence of the PrfA regulator protein. Microsequencing of peptides derived from the partially purified 30-kDa protein revealed homologies to the InlA and InlB polypeptides of L. monocytogenes, which are required for the internalization of the bacteria into nonphagocytic cell lines. This prompted us to term the 30-kDa polypeptide internalin-related protein (Irp). Irp-specific monoclonal antibodies cross-reacted with a 24-kDa polypeptide present in culture supernatants of Listeria ivanovii, indicating the existence of an Irp-related protein in this pathogenic Listeria species.

Internalization of Listeria monocytogenes into nonphagocytic cell lines in vitro requires the products of the inlAB locus (J.-L. Gaillard, P. Berche, C. Frehel, E. Gouin, and P. Cossart, Cell 65:1127-1141, 1991). By generating isogenic mutants with a chromosomal in-frame deletion in either inlA or inlB, we have identified InlA and InlB as surface-bound proteins of L. monocytogenes with molecular weights of 88,000 and 65,000, respectively. These results were obtained with monoclonal antibodies raised against either protein and corroborated by N-terminal end sequencing of InlA and InlB. By immunoblot analysis, the production of both polypeptides was found to be strongly dependent on growth temperature and, particularly for InlB, on the presence of the PrfA regulator protein. Expression of InlA was not strictly dependent on the presence of the PrfA regulator protein. Transcription analysis of the inlAB locus revealed that the inlA gene was transcribed by several promoters, of which only one is PrfA dependent. This PrfA-dependent inlA promoter, which contains two base substitutions within its putative PrfA DNA-binding palindrome, is responsible for transcription of both inlA and inlB genes. A hitherto unrecognized promoter located 51 bp upstream of the GTG start codon of the inlB gene was also detected. Hence, inlA and inlB are transcribed both individually and in an operon by PrfA-dependent and -independent mechanisms. Tissue culture invasion assays employing various epithelial cell lines demonstrated that both InlA and InlB are required for invasion. In vivo studies using the mouse infection model revealed that both internalin mutants were attenuated for virulence.

In this study, the interaction of murine dendritic cells with Listeria monocytogenes was investigated. Dendritic cells are efficient antigen-presenting cells, play a key role in the immune response, and are capable of migrating over substantial distances between sites of infection and lymphoid tissues. L. monocytogenes EGD invaded dendritic cells, escaped from phagosomes into the cytoplasm, and there directed actin nucleation, polymerization, and polarization in a typical fashion, thereby achieving intracellular movement and cell-to-cell spread. The internalization process appears to be independent of the inl locus. Interestingly, an intact microtubular function was essential for efficient uptake, whereas in a previous report, microtubule disruption did not affect bacterial spread in Caco-2 cells. The results obtained also suggest that L. monocytogenes binds to glycosylated receptors of dendritic cells. Uptake of Listeria cells was mediated by a protein kinase-dependent transducing phosphorylation signal that induces the actin polymerization-dependent phagocytic process. To achieve efficient uptake, de novo protein synthesis of eukaryotic and prokaryotic cells is also required. Despite the killing of dendritic cells, wild-type bacteria were found to persist in small numbers in some cells for at least 24 h. When different isogenic mutants of the EGD strain were analyzed for their capability to interact with dendritic cells, it was observed that some virulence-attenuated mutants (i.e., prfA and delta hly) persisted in large numbers for even longer times. Invasion of dendritic cells by L. monocytogenes, which in turn could result in either cell death or persistent infection, might have an important role in the pathogenesis of listeriosis, leading to impaired immune responses with inefficient bacterial clearance and/or promoting bacterial spread.

Background: Several intracellular pathogens, including Listeria monocytogenes, use components of the host actin-based cytoskeleton for intracellular movement and for cell-to-cell spread. These bacterial systems provide relatively simple model systems with which to study actin-based motility. Genetic analysis of L. monocytogenes led to the identification of the 90 kD surface-bound ActA polypeptide as the sole bacterial factor required for the initiation of recruitment of host actin filaments. Numerous host actin-binding proteins have been localized within the actin-based cytoskeleton that surrounds Listeria once it is inside a mammalian cell, including alpha-actinin, fimbrin, filamin, villin, ezrin/radixin, profilin and the vasodilator-stimulated phosphoprotein, VASP. Only VASP is known to bind directly to ActA. We sought to determine which regions of the ActA molecule interact with VASP and other components of the host microfilament system., Results: We used the previously developed mitochondrial targeting assay to determine regions of the ActA protein that are involved in the recruitment of the host actin-based cytoskeleton. By examining amino-terminally truncated ActA derivatives for their ability to recruit cytoskeletal proteins, an essential element for actin filament nucleation was identified between amino acids 128 and 151 of ActA. An ActA derivative from which the central proline-rich repeats were deleted retained its ability to recruit filamentous actin, albeit poorly, but was unable to bind VASP., Conclusions: Our studies reveal the initial interactions that take place between invading Listeria and host microfilament proteins. The listerial ActA polypeptide contains at least two essential sites that are required for efficient microfilament assembly: an amino-terminal 23 amino-acid region for actin filament nucleation, and VASP-binding proline-rich repeats. Hence, ActA represents a prototype actin filament nucleator. We suggest that host cell analogues of ActA exist and are important components of structures involved in cell motility.

Experimental infections of mice with strains of Listeria spp. isolated from contaminated food sources allowed discrimination of strains into those either exhibiting high, attenuated or low virulence. Compared to the highly virulent L. monocytogenes strain EGD, an attenuated strain such as L99 persisted for shorter times (5 versus 10 days) in the infected host. Using a tissue culture cell model of infection, we found that, although strain L99 was capable of accumulating actin like its virulent counterpart following invasion, it was unable to generate the polarized actin tails required for intracellular and cell-to-cell movement. Immunoblot analysis using specific antiserum to the ActA polypeptide, a molecule that is necessary for movement of the bacterium within the eucaryotic cell, indicated that a slightly truncated form of this polypeptide was produced in the L99 strain. Despite its reduced virulence, the attenuated strain L99 was just as effective in generating protection in immune mice as the highly virulent strains, albeit with a 1000-fold higher infective dose. Based on the results obtained from this study, we suggest that one of the mechanisms accounting for widespread resistance in humans to infection by Listeria may be due to asymptomatic infections by naturally occurring strains attenuated for virulence.

Effective cell-to-cell spreading of the facultative intracellular pathogen Listeria monocytogenes requires the interaction between bacteria and the microfilament system of the host cell. By recruiting actin filaments into a 'comet tail' localized at one pole of the bacterial cell wall, Listeria become mobile and propel themselves through the cytoplasm. They create protrusions at the plasma membrane that can invaginate adjacent cells. In this work, we have analysed the structural composition of Listeria-recruited microfilaments in various epithelial cell lines by immunofluorescence microscopy. The microfilament-crosslinking proteins alpha-actinin, fimbrin and villin were localized around bacteria as soon as actin filaments could be detected on the bacterial surface. Surprisingly, the same was found for ezrin/radixin, proteins involved in linking microfilaments to the plasma membrane. We found that in a polarized cell line derived from brush border kidney epithelium (LLC-PK1), the actin filaments surrounding intracytoplasmic motile bacteria show the same immunoreactivity as the brush border-like microvilli, when analysed by a specific actin antibody. The successful invasion of polarized LLC-PK1 islets is vectorial, i.e. it progresses predominantly from the periphery of the islets towards the centre. Infection of the peripheral cells is sufficient for infiltration of the entire cellular islets, without any further contact with the extracellular milieu. This is in contrast to nonpolarized epithelial sheets, which can be invaded from the apical surface of any individual cell. The importance of active bacterial motility in this vectorial spreading is emphasized by our finding that an isogenic Listeria mutant that is unable to recruit actin filaments cannot colonize polarized epithelial layers but accumulates in the peripheral cells of the islets.

Listeria monocytogenes, a facultative intracellular pathogen, employs actin and other microfilament-associated proteins to move through the host cell cytoplasm. Isogenic mutants of L. monocytogenes lacking the surface-bound ActA polypeptide no longer interact with cytoskeletal elements and are, as a consequence, non-motile (Domann et al., 1992, EMBO J., 11, 1981-1990; Kocks et al., 1992, Cell, 68, 521-531). To investigate the interaction of ActA with the microfilament system in the absence of other bacterial factors, the listerial actA gene was expressed in eukaryotic cells. Immunofluorescence studies revealed that the complete ActA, including its C-terminally located bacterial membrane anchor, colocalized with mitochondria in transfected cells. When targeted to mitochondria, the ActA polypeptide recruited actin and alpha-actinin to these cellular organelles with concomitant reorganization of the microfilament system. Removal of the internal proline-rich repeat region of ActA completely abrogated interaction with cytoskeletal components. Our results identify the ActA polypeptide as a nucleator of the actin cytoskeleton and provide the first insights into the molecular nature of such controlling elements in microfilament organization.

Expression of listeriolysin, a major virulence factor of pathogenic Listeria monocytogenes, is positively regulated by the pleiotropic virulence regulator PrfA. In this study, we demonstrate that L. monocytogenes strains lacking the prfA regulator gene produce listeriolysin in small, albeit detectable, amounts when analyzed in a hemolysin assay and by immunoblots with listeriolysin-specific monoclonal antibodies. Transcriptional analysis revealed the existence of a PrfA-independent promoter that was responsible for the hemolytic activity expressed by these strains.

The ActA protein of the gram-positive pathogen Listeria monocytogenes is a 90-kDa polypeptide required for interaction of the bacteria with components of the host cell microfilament system to generate intra- and intercellular movement. To study the localization, distribution, and expression of the ActA polypeptide in L. monocytogenes grown either in broth culture or in infected tissue culture cells, we first isolated ActA by monoclonal antibody-based immunoaffinity chromatography. Polyclonal rabbit antisera raised against purified ActA revealed that ActA was associated with the cell wall and exposed on the surface of the bacteria, readily accessible to ActA antibodies. In contrast, a C-terminally truncated ActA1 polypeptide expressed by the isogenic actA1 mutant was detected only in the supernatant fluids. Immunofluorescence microscopy and electron microscopic studies using immunogold labeling showed that ActA was present on the surface of the bacteria infecting PtK2 and J774 cells at all stages of the infection cycle and was not found to be associated with the actin "tail" of individual bacteria. For the isogenic actA1 mutant strain, which grew as microcolonies within infected cells, only diffuse staining of the secreted ActA1 polypeptide in the host cytoplasm was observed. The ActA polypeptide therefore appears to be required in the initiation of actin accumulation by the bacterium and is apparently not directly involved in the generation of the actin tail. Analysis of strains of several L. monocytogenes serotypes indicated microheterogeneity in the molecular weights of the ActA polypeptides of individual strains and led to the detection of a serotype 3a strain that does not produce ActA.

The ability of Listeria monocytogenes to move within the cytosol of infected cells and their ability to infect adjacent cells is important in the development of infection foci leading to systemic disease. Interaction with the host cell microfilament system, particularly actin, appears to be the basis for propelling the bacteria through the host cell cytoplasm to generate the membraneous protrusions whereby cell-to-cell spread occurs. The actA locus of L.monocytogenes encodes a 90 kDa polypeptide that is a key component of bacterium-host cell microfilament interactions. Cloning of the actA gene allowed the identification of its gene product and permitted construction of an isogenic mutant strain defective in the production of the ActA polypeptide. Sequencing of the region encoding the actA gene revealed that it was located region encoding the actA gene revealed that it was located between the metalloprotease (mpl) and phosphatidylcholine-specific phospholipase C (plcB) genes. Within the cytoplasm of the infected cells, the mutant strain grew as microcolonies, was unable to accumulate actin following escape from the phagocytic compartment and was incapable of infecting adjacent cells. It was also dramatically less virulent, demonstrating that the capacity to move intracellularly and spread intercellularly is a key determinant of L.monocytogenes virulence. Like all other virulence factors described for this microorganism, expression of the ActA polypeptide is controlled by the PrfA regulator protein. The primary sequence of this protein appeared to be unique with no extended homology to known protein sequences. However, an internal repeat sequence showed strong regional homology to a sequence from within the hinge region of the cytoskeletal protein vinculin.