<Emphasis Type="Italic">Listeria </Emphasis><Emphasis Type="Italic">monocytogenes</Emphasis> internalins bind to the human intestinal mucin MUC2

Listeria monocytogenes cross the intestinal barrier causing systemic infections with high mortality rates. Intestinal infection triggers release of intestinal mucus. We show that three L. monocytogenes internalins, InlB, InlC and InlJ all bound to MUC2 (the major component of intestinal mucus), but not to the cell surface mucin MUC1. Binding was strongest to InlB>InlC>InlJ (P < 0.001). Listerial internalins are characterized by their internalin domain, composed by leucine rich repeats (LRR) followed by an immunogloblin-like region. We report here that the internalin domain of the InlJ protein also bound MUC2, suggesting that an internalin domain is sufficient to bind to MUC2.     

InlA- but not InlB-mediated internalization of Listeria monocytogenes by non-phagocytic mammalian cells needs the support of other internalins

To determine the contribution of the previously identified internalins, InlA, InlB, InlC, InlE, InlG, and InlH, to internalization of Listeria monocytogenes by non-professional phagocytic mammalian cells, we constructed mutants with various combinations of deletions in the respective inl genes. Internalization of these mutants into the epithelial-like Caco-2 and the microvascular endothelial HBMEC cell lines were studied. Deletion of the inlGHE gene cluster, or of the single genes, led to a two to fourfold increased internalization by HBMEC and other non-phagocytic mammalian cells. Invasion into HBMEC was totally blocked in the absence of InlB, and InlB-dependent internalization did not require the presence of any of the other internalins. Internalization by Caco-2 cells was reduced to a level of about 1% in the absence of InlA and InlB, and was most efficient in the presence of InlA, InlB and InlC and in the absence of InlG, InlH and InlE. InlB and InlA, in each case in the absence of the other internalins, led (compared with the wild-type strain) to reduced internalization of about 20% and less than 10% respectively. InlA-dependent internalization (in the absence of InlB) required the additional function of InlC and InlGHE. The deletion of inlGHE enhanced the expression of InlA and InlB. The increased amount of InlA led to an increase in early association of L. monocytogenes with Caco-2 cells without enhancing its uptake in the absence of the other internalins, whereas the larger amount of InlB did not enhance early association of L. monocytogenes with HBMEC but led to an increase in internalization of L. monocytogenes. The results suggest that InlB is able to induce phagocytosis in HBMEC and (at a lower efficiency) in Caco-2 cells by itself, but InlA needs the supportive functions of the other internalins to trigger phagocytosis. None of these internalins seems to be required for cell-to-cell spread by L. monocytogenes, as shown by microinjection of Caco-2 cells with appropriate inl mutants.     

Birth-and-death evolution of the internalin multigene family in Listeria

The birth-and-death model of multigene family evolution describes patterns of gene origination, diversification and loss within multigene families. Since it was first developed in the 1990s, the model has been found to characterize a large number of eukaryotic multigene families. In this paper, we report for the first time a bacterial multigene family that undergoes birth-and-death evolution. By analyzing the evolutionary relationships among internalins, a relatively large and diverse family of genes associated with key virulence functions in Listeria, we demonstrate the importance of birth-and-death evolution in the diversification of this important bacterial pathogen. We also detected two instances of lateral gene transfer within the internalins, but the estimated frequency would have been much higher had it not been analyzed within the context of birth-and-death evolutionary dynamics and a phenomenon that we term "paralog-sorting", which involves the unequal transmittal of gene duplicates during or subsequent to the speciation process. As such, in addition to providing the first demonstration of birth-and-death evolution within a bacterial multigene family, our results indicate that the extent of lateral transfer in bacterial multigene families should be re-examined in the light of birth-and-death evolution.     

A spontaneous genomic deletion in Listeria ivanovii identifies LIPI-2, a species-specific pathogenicity island encoding sphingomyelinase and numerous internalins

Listeria ivanovii differs from the human pathogen Listeria monocytogenes in that it specifically affects ruminants, causing septicaemia and abortion but not meningo-encephalitis. The genetic characterization of spontaneous L. ivanovii mutants lacking the virulence factor SmcL (sphingomyelinase) led us to identify LIPI-2, the first species-specific pathogenicity island from Listeria. Besides SmcL, this 22 kb chromosomal locus encodes 10 internalin (Inl) proteins: i-InlB1 and -B2 are large/surface-associated Inls similar to L. monocytogenes InlB; i-InlE to -L are small/excreted (SE)-Inls, i-InlG being a tandem fusion of two SE-Inls. Except i-inlB1, all LIPI-2 inl genes are controlled by the virulence regulator, PrfA. LIPI-2 is inserted into a tRNA locus and is unstable - half of it deleting at approximately 10(-4) frequency with a portion of contiguous DNA. The spontaneous mutants were attenuated in vivo in mice and lambs and showed impaired intracellular growth and apoptosis induction in bovine MDBK cells. Targeted knock-out mutations associated the virulence defect with LIPI-2 genes. The region between the core genome loci ysnB-tRNA(arg) and ydeI flanking LIPI-2 contained different gene complements in the different Listeria spp. and even serovars of L. monocytogenes, including remnants of the PSA bacteriophage int gene in serovar 4b, indicating it is a hot spot for horizontal genome diversification. LIPI-2 is conserved in L. ivanovii ssp. ivanovii and londoniensis, suggesting an early acquisition during the species' evolution. LIPI-2 is likely to play an important role in the pathogenic and host tropism of L. ivanovii.     

Exploitation of host cell cytoskeleton and signalling during Listeria monocytogenes entry into mammalian cells

Deciphering how Listeria monocytogenes exploits the host cell machinery to invade mammalian cells during infection is a key issue for the understanding how this food-borne pathogen causes a pleiotropic disease ranging from gastro-enteritis to meningitis and abortions. Using multidisciplinary approaches, essentially combining bacterial genetics and cell biology, we have identified two bacterial proteins critical for entry into target cells, InlA and InlB. Their cellular ligands have been also identified: InlA interacts with the adhesion molecule E-cadherin, while InlB interacts with the receptor for the globular head of the complement factor C1q (gC1q-R), with the hepatocyte growth factor receptor (c-Met) and with glycosaminoglycans (including heparan sulphate). The dynamic interaction between these cellular receptors and the actin cytoskeleton is currently under investigation. Several intracellular molecules have been recognized as key effectors for Listeria entry into target cells, including catenins (implicated in the connection of E-cadherin to actin) and the actin depolymerising factor/cofilin (involved in the rearrangement of the cytoskeleton in the InlB-dependent internalisation pathway). At the organism level, species specificity has been discovered concerning the interaction between InlA and E-cadherin, leading to the generation of transgenic mice expressing the human E-cadherin, in which the critical role of InlA in the crossing of the intestinal barrier has been clearly determined. Listeria appears as an instrumental model for addressing critical questions concerning both the complex process of bacterial pathogenesis and also fundamental molecular processes, such as phagocytosis.     

Involvement of Intronic Sequences in Cell-Specific Expression of the Peripherin Gene

Peripherin is an intermediate filament protein expressed in restricted populations of neurons. Our previous study of the chromatin structure of the mouse peripherin gene in cells that do or do not express peripherin suggested that the region located between -1,500 and +800 bp of the gene could be involved in its cell specificity. In the present work, we performed an in vitro functional analysis of the 5' flanking region of the mouse peripherin gene and observed that this region up to 9 kb contained both enhancer and inhibiting activities; however, it was insufficient to achieve a complete extinction of reporter gene expression in peripherin-negative cells. Furthermore, analysis of the first three introns with the 5' flanking sequences of the gene showed that intron I greatly increased specificity of the gene expression. Intron I also conferred the same properties to thymidine kinase heterologous promoter. DNase I footprinting experiments performed with intron I revealed at least two protected regions (Inl A and Inl B). Inl A encompasses an AP-2-like binding site that interacted with both neuroblast and fibroblast nuclear factors, as well as with the recombinant AP-2alpha protein. However, gel shift experiments suggested that the interacting nuclear factors are distinct from AP-2alpha itself and probably belong to the AP-2 family. Inl B perfectly matched the consensus binding site for Sp1 and specifically interacted with nuclear protein factors that showed the same binding properties as the Sp1 family members. Fine deletion analysis of intron I indicated that the Inl A element alone is responsible for its enhancing properties, whereas a region located between +789 and +832 gives to intron I its silencer activity.     

Listerial invasion protein internalin B promotes entry into ileal Peyer's patches in vivo

Listeria monocytogenes (Lm) invades the host intestine using listerial invasion proteins, internalins. The in vivo role of internalin A (InlA) and internalin B (InlB) is reported here. Intragastric (i.g.) administration and ligated loop assays with ΔinlB-Lm demonstrated that a lack of InlB significantly attenuates the invasive ability of Lm into various organs. On the other hand, InlA(m)-Lm expressing a mutant InlA with two substitutions, S192N and Y369S, which has been reported to increase the affinity of InlA to mouse E-cadherin, resulted in little increase in intestinal infection according to both ligated loop and i.g. infection assays. Lm preferentially enters ileal Peyer's patch (PP) via M cells and ΔinlB-Lm showed severely reduced ability to invade though these cells. The present results reveal the importance of InlB, which accelerates listerial invasion into M cells on ileal PPs in vivo.     

Successive post-translational modifications of E-cadherin are required for InlA-mediated internalization of Listeria monocytogenes

Listeria monocytogenes surface proteins internalin (Inl)A and InlB interact with the junctional protein E-cadherin and the hepatocyte growth factor (HGF) receptor Met, respectively, on the surface of epithelial cells to mediate bacterial entry. Here we show that InlA triggers two successive E-cadherin post-translational modifications, i.e. the Src-mediated tyrosine phosphorylation of E-cadherin followed by its ubiquitination by the ubiquitin-ligase Hakai. E-cadherin ubiquitination induces the recruitment of clathrin that is required for optimal bacterial internalization. We also show that the initial clustering of E-cadherin at the bacterial entry site requires caveolin, a protein normally involved in clathrin-independent endocytosis. Strikingly clathrin and caveolin are also recruited at the site of entry of E-cadherin-coated sepharose beads and functional experiments demonstrate that these two proteins are required for bead entry. Together these results not only document how the endocytosis machinery is recruited and involved in the internalization of a zippering bacterium, but also strongly suggest a functional link between E-cadherin endocytosis and the formation of adherens junctions in epithelial cells.     

Transduction of fimbriation demonstrating common ancestry in FIRN strains of Salmonella typhimurium.

The production of fimbriate (Fim+) recombinants was observed in transductional crosses between different pairs of wild-type strains of different biotypes of Salmonella typhimurium. Fim+ recombinants were readily produced in transductions from Fim+ donor strains to Fim- recipient strains and, less frequently, between some pairs of Fim- strains, for example, between almost any strain of the firn biogroup (Fim- Inl- Rha- Bxyl-) and many strains of the non-FIRN Fim- biogroup. None of numerous crosses between different pairs of FIRN strains gave Fim+ recombinants, suggesting that the fim mutation was present at the same intragenic site in all FIRN strains. FIRN strains are thought to have descended from a single ancestral FIRN bacterium which originated by a series of mutations from a strain of the common biotype 1a (Fim+ Inl+ Rha+ Bxyl+). Two FIRN-like (Fim- Inl+ Rha- Bxyl-) strains that did not yield Fim+ recombinants in crosses with FIRN strains were probably wild-type Inl+ mutants from FIRN strains.     

Crystal structure and standardized geometric analysis of InlJ, a listerial virulence factor and leucine-rich repeat protein with a novel cysteine ladder

We report on the crystal structure of the internalin domain of InlJ, a virulence-associated surface protein of Listeria monocytogenes, at 2.7-Å resolution. InlJ is a member of the internalin family of listerial cell surface proteins characterized by a common N-terminal domain. InlJ bears 15 leucine-rich repeats (LRRs), the same number as in InlA, the prototypical internalin family member. The LRRs of InlJ differ from those of other internalins by having 21, rather than 22, residues and by replacing 1 LRR-defining hydrophobic residue with a conserved cysteine. These cysteines stack to form an intramolecular ladder and regular hydrophobic interactions in consecutive repeats. Analyzing the curvature, twist, and lateral bending angles of InlJ and comparing these with several other LRR proteins, we provide a systematic geometric comparison of LRR protein structures (http://bragi2.helmholtz-hzi.de/Angulator/). These indicate that both cysteine and asparagine ladders stabilize the LRR fold, whereas substitutions in some repeat positions are more likely than others to induce changes in LRR geometry.     

InlA and InlC2 of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> Serotype 4b Are Two Internalin Proteins Eliciting Humoral Immune Responses Common to Listerial Infection of Various Host Species

The internalins InlA and InlC2 are encoded proteins from two strongly immunoreactive clones recently identified by differential immunoscreening of a Listeria monocytogenes serotype 4b genomic expression library during the search of the gene products of L. monocytogenes specifically induced in vivo during infection (Yu WL, Dan H, Lin M. J Med Microbiol 56:888–895, 2007). In this study, we examined the humoral immune response against InlA and InlC2 in various L. monocytogenes-infected hosts using Western blots. InlA and InlC2 were recognized by antibodies in experimentally infected rabbits but not by antisera from rabbits immunized with the heat-killed bacterium. Similar strong immunological reactions to InlA and InlC2 were seen with antisera from infected guinea pigs, cattle, and sheep but not with those from the animals (guinea pigs or sheep) receiving heat-killed bacteria. This study provides the first experimental evidence that InlA and InlC2 are the in vivo induced or upregulated antigens for humoral immune responses that are common to listerial infection of various host species. These two immunogenic proteins may thus be explored as reagents for the laboratory diagnosis of listeriosis or candidates for vaccine development.     

Surface proteins of gram-positive bacteria and mechanisms of their targeting to the cell wall envelope.

The cell wall envelope of gram-positive bacteria is a macromolecular, exoskeletal organelle that is assembled and turned over at designated sites. The cell wall also functions as a surface organelle that allows gram-positive pathogens to interact with their environment, in particular the tissues of the infected host. All of these functions require that surface proteins and enzymes be properly targeted to the cell wall envelope. Two basic mechanisms, cell wall sorting and targeting, have been identified. Cell well sorting is the covalent attachment of surface proteins to the peptidoglycan via a C-terminal sorting signal that contains a consensus LPXTG sequence. More than 100 proteins that possess cell wall-sorting signals, including the M proteins of Streptococcus pyogenes, protein A of Staphylococcus aureus, and several internalins of Listeria monocytogenes, have been identified. Cell wall targeting involves the noncovalent attachment of proteins to the cell surface via specialized binding domains. Several of these wall-binding domains appear to interact with secondary wall polymers that are associated with the peptidoglycan, for example teichoic acids and polysaccharides. Proteins that are targeted to the cell surface include muralytic enzymes such as autolysins, lysostaphin, and phage lytic enzymes. Other examples for targeted proteins are the surface S-layer proteins of bacilli and clostridia, as well as virulence factors required for the pathogenesis of L. monocytogenes (internalin B) and Streptococcus pneumoniae (PspA) infections. In this review we describe the mechanisms for both sorting and targeting of proteins to the envelope of gram-positive bacteria and review the functions of known surface proteins.     

The 213-amino-acid leucine-rich repeat region of the Listeria monocytogenes InlB protein is sufficient for entry into mammalian cells, stimulation of PI 3-kinase and membrane ruffling

The Listeria monocytogenes InlB protein is a 630-amino-acid surface protein that mediates entry of the bacterium into a wide variety of cell types, including hepatocytes, fibroblasts and epithelial cells such as Vero, HEp-2 and HeLa cells. Invasion stimulates host proteins tyrosine phosphorylation, PI 3-kinase activity and rearrangements in the actin cytoskeleton. We previously showed that InlB is sufficient for entry of InlB-coated latex beads into cells and recent results indicate that purified InlB can stimulate PI 3-kinase activity and is thus the first bacterial agonist of this lipid kinase. In this study, we identified the region of InlB responsible for entry and stimulation of signal transduction events. Eight monoclonal antibodies directed against InlB were raised and, of those, five inhibited bacterial entry. These five antibodies recognized epitopes within the leucine-rich repeat (LRR) region and/or the inter-repeat (IR) region. InlB-staphylococcal protein A (SPA) fusion proteins and recombinant InlB derivatives were generated and tested for their capacity to mediate entry into cultured mammalian cells. All the InlB derivatives that carried the amino-terminal 213-amino-acid LRR region conferred invasiveness to the normally non-invasive bacterium L. innocua or to inert latex beads and the corresponding purified polypeptides inhibited bacterial entry. In addition, the 213-amino-acid LRR region was able to stimulate PI 3-kinase activity and changes in the actin cytoskeleton (membrane ruffling). These properties were not detected with purified internalin, another invasion protein of L. monocytogenes that displays LRRs similar to those of InlB. Taken together, these results show that the first 213 amino acids of InlB are critical for its specific properties.     

Surface Proteins of Gram-Positive Bacteria and Mechanisms of Their Targeting to the Cell Wall Envelope

The cell wall envelope of gram-positive bacteria is a macromolecular, exoskeletal organelle that is assembled and turned over at designated sites. The cell wall also functions as a surface organelle that allows gram-positive pathogens to interact with their environment, in particular the tissues of the infected host. All of these functions require that surface proteins and enzymes be properly targeted to the cell wall envelope. Two basic mechanisms, cell wall sorting and targeting, have been identified. Cell well sorting is the covalent attachment of surface proteins to the peptidoglycan via a C-terminal sorting signal that contains a consensus LPXTG sequence. More than 100 proteins that possess cell wall-sorting signals, including the M proteins of Streptococcus pyogenes, protein A of Staphylococcus aureus, and several internalins of Listeria monocytogenes, have been identified. Cell wall targeting involves the noncovalent attachment of proteins to the cell surface via specialized binding domains. Several of these wall-binding domains appear to interact with secondary wall polymers that are associated with the peptidoglycan, for example teichoic acids and polysaccharides. Proteins that are targeted to the cell surface include muralytic enzymes such as autolysins, lysostaphin, and phage lytic enzymes. Other examples for targeted proteins are the surface S-layer proteins of bacilli and clostridia, as well as virulence factors required for the pathogenesis of L. monocytogenes (internalin B) and Streptococcus pneumoniae (PspA) infections. In this review we describe the mechanisms for both sorting and targeting of proteins to the envelope of gram-positive bacteria and review the functions of known surface proteins.     

Deciphering the biodiversity of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> lineage III strains by polyphasic approaches

Listeria monocytogenes is a foodborne pathogen of humans and animals. The majority of human listeriosis cases are caused by strains of lineages I and II, while lineage III strains are rare and seldom implicated in human listeriosis. We revealed by 16S rRNA sequencing the special evolutionary status of L. monocytogenes lineage III, which falls between lineages I and II strains of L. monocytogenes and the non-pathogenic species L. innocua and L. marthii in the dendrogram. Thirteen lineage III strains were then characterized by polyphasic approaches. Biochemical reactions demonstrated 8 biotypes, internalin profiling identified 10 internal-in types clustered in 4 groups, and multilocus sequence typing differentiated 12 sequence types. These typing schemes show that lineage III strains represent the most diverse population of L. monocytogenes, and comprise at least four subpopulations IIIA-1, IIIA-2, HIB, and IIIC. The in vitro and in vivo virulence assessments showed that two lineage IIIA-2 strains had reduced pathogenicity, while the other lineage III strains had comparable virulence to lineages I and II. The HIB strains are phylogenetically distinct from other sub-populations, providing additional evidence that this sublineage represents a novel lineage. The two biochemical reactions L-rhamnose and L-lactate alkalinization, and 10 internalins were identified as potential markers for lineage III subpopulations. This study provides new insights into the biodiversity and population structure of lineage III strains, which are important for understanding the evolution of the L. mono-cytogenes-L. innocua clade.     

Attachment Strength of Listeria monocytogenes and its Internalin-Negative Mutants

A single cell of Listeria monocytogenes attached on food contact surfaces can be a potential source of cross-contamination in a food-processing plant. To see whether internalin A (InlA) and B (InlB), major surface proteins on L. monocytogenes, play a significant role in the attachment process, wild-type L. monocytogenes EGD (LM_EGD) and its isogenic internalin-negative mutants (LM_EGDΔinlA, LM_EGDΔinlB, and LM_EGDΔinlAB) were used to determine attachment strength on inert glass surface. Western blot analysis using InlA and InlB antibodies confirmed the absence of InlA in LM_EGDΔinlA, InlB in LM_EGDΔinlB, and both InlA and InlB in LM_EGDΔinlAB. Regardless of initial attachment numbers, LM_EGD which expressed both InlA and InlB proteins exhibited the strongest attachment strength while the double mutant (LM_EGDΔinlAB) exhibited the weakest. The two single mutants (LM_EGDΔinlA and LM_EGDΔinlB) that expressed only one type of the internalins were shown to have intermediate attachment strength. These results suggest that both InlA and InlB expression play a significant role in the attachment strength of L. monocytogenes on glass surface.