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Staphylococcus aureus is a major human pathogen. Superantigens (SAg) are important virulence factors in S. aureus, but the regulation of SAg gene expression is largely unknown. Using 2 sequenced S. aureus strains (COL and Newman) and 4 clinical isolates, regulation of gene expression was investigated in more detail for 12 SAgs. The SAg-encoding genes were expressed in a growth phase-dependent manner: while the egc operon was mainly transcribed at low optical densities, the transcription of seb was induced at high optical densities. The transcript levels of sea, sek, seq, sep, and tst-1 did not change significantly during growth. The T cell-mitogenic activity of supernatants correlated with the transcription data. SaeRS and σ(B) strongly influenced SAg gene transcription. σ(B) activated transcription of seh, tst-1, and of the egc operon. A possible σ(B)-dependent promoter was identified in front of the egc operon. In contrast, a loss of σ(B) enhanced the transcript level of seb, suggesting an indirect effect of the alternative sigma factor on the transcription of this gene. Transcriptional studies of an saeS mutant showed that the two-component system only activates transcription of seb. The influence of σ(B) and SaeRS on the expression of SAg genes was validated by T cell proliferation assays. For sigB mutants in different strains, different effects on the T cell-mitogenic potential were observed depending on the SAg gene repertoire of the isolates., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

The anaerobic metabolism of the opportunistic pathogen Pseudomonas aeruginosa is important for growth and biofilm formation during persistent infections. The two Fnr-type transcription factors Anr and Dnr regulate different parts of the underlying network in response to oxygen tension and NO. Little is known about all members of the Anr and Dnr regulons and the mediated immediate response to oxygen depletion. Comprehensive transcriptome and bioinformatics analyses in combination with a limited proteome analyses were used for the investigation of the P. aeruginosa response to an immediate oxygen depletion and for definition of the corresponding Anr and Dnr regulons. We observed at first the activation of fermentative pathways for immediate energy generation followed by induction of alternative respiratory chains. A solid position weight matrix model was deduced from the experimentally identified Anr boxes and used for identification of 170 putative Anr boxes in potential P. aeruginosa promoter regions. The combination with the experimental data unambiguously identified 130 new members for the Anr and Dnr regulons. The basis for the understanding of two regulons of P. aeruginosa central to biofilm formation and infection is now defined.

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.

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.

Recently, we identified a pyruvate fermentation pathway in Pseudomonas aeruginosa sustaining anaerobic survival in the absence of alternative anaerobic respiratory and fermentative energy generation systems (M. Eschbach, K. Schreiber, K. Trunk, J. Buer, D. Jahn, and M. Schobert, J. Bacteriol. 186:4596-4604, 2004). Anaerobic long-term survival of P. aeruginosa might be essential for survival in deeper layers of a biofilm and the persistent infection of anaerobic mucus plaques in the cystic fibrosis lung. Proteome analysis of P. aeruginosa cells during a 7-day period of pyruvate fermentation revealed the induced synthesis of three enzymes involved in arginine fermentation, ArcA, ArcB, and ArcC, and the outer membrane protein OprL. Moreover, formation of two proteins of unknown function, PA3309 and PA4352, increased by factors of 72- and 22-fold, respectively. Both belong to the group of universal stress proteins (Usp). Long-term survival of a PA3309 knockout mutant by pyruvate fermentation was found drastically reduced. The oxygen-sensing regulator Anr controls expression of the PPA3309-lacZ reporter gene fusion after a shift to anaerobic conditions and further pyruvate fermentation. PA3309 expression was also found induced during the anaerobic and aerobic stationary phases. This aerobic stationary-phase induction is independent of the regulatory proteins Anr, RpoS, RelA, GacA, RhlR, and LasR, indicating a currently unknown mechanism of stationary-phase-dependent gene activation. PA3309 promoter activity was detected in the deeper layers of a P. aeruginosa biofilm using a PPA3309-gfp (green fluorescent protein gene) fusion and confocal laser-scanning microscopy. This is the first description of an Anr-dependent, anaerobically induced, and functional Usp-like protein in bacteria.

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.

Extracellular proteins of bacterial pathogens play a crucial role in the infection of the host. Here we present the first comprehensive validation of the secretory subproteome of the Gram positive pathogen Listeria monocytogenes using predictive bioinformatic and experimental proteomic approaches. The previous original signal peptide (SP) prediction (Glaser et al., Science 2001, 294, 849-852) has been greatly improved by an in-depth analysis using seven different bioinformatic tools. Subsequent careful classification of the resulting data gives a probability dependent annotation of 121 putatively secreted proteins of which 45 are novel. Complementary proteomic analysis using both two-dimensional gel electrophoresis/matrix assisted laser desorption/ionization mass spectrometry and high performance liquid chromatography/electrospray ionization-mass spectrometry has identified 105 proteins in the culture supernatant of L. monocytogenes. Among these, we were able to detect all the currently known virulence factors with an SP showing the importance of this subproteome and demonstrating the reliability of the techniques used. The comparison between the L. monocytogenes wildtype and the nonpathogenic species Listeria innocua was performed to reveal proteins probably involved in pathogenicity and/or the adaptation to their respective lifestyles. In addition to the eight known virulence factors, all of which have no orthologous genes in L. innocua, eight additional proteins have been identified that exhibit the typical key feature defining the known listerial virulence factors. Further significant differences between the two species are evident in the group of cell wall and secretory proteins that warrant further study. Our investigation clearly demonstrates that the major difference between the pathogenic and nonpathogenic species, noted in the comparative genome analysis, manifests itself strongest in the secretome.

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 proteomes of cultured Pseudomonas aeruginosa isolates from chronically infected cystic fibrosis (CF) lungs were compared by using genetically divergent clones and isogenic morphotypes of one strain. Cellular extracts gave very similar protein patterns in two-dimensional gels, suggesting that the conserved species-specific core genome encodes proteins that are expressed under standard culture conditions in vitro. In contrast, the protein profiles of extracts of culture supernatants were dependent on the growth phase, and there were significant differences between clones. The profiles also varied within clonally related morphotypes from one CF patient, including a hyperpiliated small-colony variant. Mass spectrometry revealed that this variant overexpressed proteins secreted by the type I secretion system (including proteins involved in iron acquisition) and by the type III secretion system. Furthermore, the proteins in the supernatant extracts from the small-colony variant which were recognized by sera from different CF patients varied greatly. We concluded that the secretome expression is a sensitive measure of P. aeruginosa strain variation.

The facultative intracellular bacterium Burkholderia pseudomallei induces actin rearrangement within infected host cells leading to formation of actin tails and membrane protrusions. To investigate the underlying mechanism we analysed the contribution of cytoskeletal proteins to B. pseudomallei-induced actin tail assembly. By using green fluorescent protein (GFP)-fusion constructs, the recruitment of the Arp2/3 complex, vasodilator-stimulated phosphoprotein (VASP), Neural Wiskott-Aldrich syndrome protein (N-WASP), zyxin, vinculin, paxillin and alpha-actinin to the surface of B. pseudomallei and into corresponding actin tails was studied. In addition, antibodies against the same panel of proteins were used for immunolocalization. Whereas the Arp2/3 complex and alpha-actinin were incorporated into B. pseudomallei-induced actin tails, none of the other proteins were detected in these structures. The overexpression of an Arp2/3 binding fragment of the Scar1 protein, shown previously to block actin-based motility of Listeria, had no effect on B. pseudomallei tail formation. Infections of either N-WASP- or Ena/VASP-defective cells showed that these proteins are not essential for B. pseudomallei-induced actin polymerization. In conclusion, our results suggest that B. pseudomallei induces actin polymerization through a mechanism that differs from those evolved by Listeria, Shigella, Rickettsia or vaccinia virus.

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 gram-positive bacterium Listeria monocytogenes is the causative agent of listeriosis, a highly fatal opportunistic foodborne infection. Pregnant women, neonates, the elderly, and debilitated or immunocompromised patients in general are predominantly affected, although the disease can also develop in normal individuals. Clinical manifestations of invasive listeriosis are usually severe and include abortion, sepsis, and meningoencephalitis. Listeriosis can also manifest as a febrile gastroenteritis syndrome. In addition to humans, L. monocytogenes affects many vertebrate species, including birds. Listeria ivanovii, a second pathogenic species of the genus, is specific for ruminants. Our current view of the pathophysiology of listeriosis derives largely from studies with the mouse infection model. Pathogenic listeriae enter the host primarily through the intestine. The liver is thought to be their first target organ after intestinal translocation. In the liver, listeriae actively multiply until the infection is controlled by a cell-mediated immune response. This initial, subclinical step of listeriosis is thought to be common due to the frequent presence of pathogenic L. monocytogenes in food. In normal individuals, the continual exposure to listerial antigens probably contributes to the maintenance of anti-Listeria memory T cells. However, in debilitated and immunocompromised patients, the unrestricted proliferation of listeriae in the liver may result in prolonged low-level bacteremia, leading to invasion of the preferred secondary target organs (the brain and the gravid uterus) and to overt clinical disease. L. monocytogenes and L. ivanovii are facultative intracellular parasites able to survive in macrophages and to invade a variety of normally nonphagocytic cells, such as epithelial cells, hepatocytes, and endothelial cells. In all these cell types, pathogenic listeriae go through an intracellular life cycle involving early escape from the phagocytic vacuole, rapid intracytoplasmic multiplication, bacterially induced actin-based motility, and direct spread to neighboring cells, in which they reinitiate the cycle. In this way, listeriae disseminate in host tissues sheltered from the humoral arm of the immune system. Over the last 15 years, a number of virulence factors involved in key steps of this intracellular life cycle have been identified. This review describes in detail the molecular determinants of Listeria virulence and their mechanism of action and summarizes the current knowledge on the pathophysiology of listeriosis and the cell biology and host cell responses to Listeria infection. This article provides an updated perspective of the development of our understanding of Listeria pathogenesis from the first molecular genetic analyses of virulence mechanisms reported in 1985 until the start of the genomic era of Listeria research.

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.

Combined applications of affinity purification procedures and mass-spectrometric analyses (affinity mass spectrometry or affinity-directed mass spectrometry) have gained broad interest in various fields of biological sciences. We have extended these techniques to the purification and analysis of closely related peptides from complex mixtures and to the characterization of binding motifs and relative affinities in protein-protein interactions. The posttranslational modifications in the carboxy-terminal region of porcine brain tubulin are used as an example for the applicability of affinity mass spectrometry in the characterization of complex patterns of related peptides. We also show that affinity mass spectrometry allows the mapping of sequential binding motifs of two interacting proteins. Using the ActA/Mena protein-protein complex as a model system, we show that we can selectively purify Mena-binding peptides from a tryptic digest of ActA. The results from this assay are compared to data sets obtained earlier by classical methods using synthetic peptides and molecular genetic experiments. As a further expansion of affinity mass spectrometry, we have established an internally standardized system that allows comparison of the affinities of related ligands for a given protein. Here the affinities of two peptide ligands for the monoclonal tubulin-specific antibody YL1/2 are determined in terms of half-maximal competition., (Copyright 1999 Academic Press.)

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.

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.

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.

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.

Exotoxins such as listeriolysin (LLO) and phosphatidylinositol-specific phospholipase C (PIcA) have been implicated in listerial infection and sepsis. Employing different Listeria strains, mutated in individually known virulence genes, we examined exotoxin-related induction of endothelial cell signaling. Listeria monocytogenes was a potent inductor of phosphatidylinositol (PtdIns) metabolism in HUVEC. This effect was completely absent in a LLO-negative strain. Using a recombinant Listeria innocua strain, engineered to produce high levels of LLO, PtdIns metabolism was restored to approximately 30% of that produced by the parental L. monocytogenes strain. A recombinant L. innocua strain expressing only PIcA did not induce any PtdIns metabolism. Even higher than wild-type levels of PtdIns hydrolysis products were, however, evoked when engineered bacteria secreted both LLO and PIcA. These effects occurred in the absence of bacterial uptake by the endothelial cells. Corresponding results were observed with regard to endothelial diacylglycerol (DAG) generation. The amplification of endothelial cell signaling could be reproduced by engaging purified LLO and PIcA in the absence of bacteria. In these experiments, the unrelated pore-forming agent staphylococcal alpha-toxin, a very weak stimulus for endothelial phosphoinositide metabolism by itself, substituted for LLO to allow marked PtdIns hydrolysis when co-applied with PIcA. We conclude that the listerial exotoxins LLO and PIcA cooperate to provoke potent second messenger synthesis in endothelial cells, in the absence of cell invasion by the bacteria. This is an impressive example of synergism between a pore-forming and an enzymatic bacterial exotoxin in provoking cell signaling and inflammatory events.

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.

Listeriolysin, the hemolysin of the pathogenic species Listeria monocytogenes, was expressed in the non-pathogenic species Listeria innocua. Coexpression of the positive regulatory factor prfA in the plasmid vector in conjunction with the structural gene hly increased the expression over 500-fold. Purification from supernatant fluids was achieved by two steps of ion exchange chromatography. The procedure resulted in over 60% yield of a hemolytically active, homogeneous 58 kDa protein which was used to produce monospecific antibodies. As shown by immunoblot the purified listeriolysin was free of p60, a highly immunogenic protein of similar size also produced by Listeria spp., which otherwise would interfere with immunoassays. Listeriolysin retained full activity for more than 6 months at -70 degrees C.

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.

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.

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.