CD44-independent activation of the Met signaling pathway by HGF and InlB

Listeria monocytogenes is a facultative intracellular Gram-positive bacterium responsible for listeriosis. It is able to invade, survive and replicate in phagocytic and non-phagocytic cells. The L. monocytogenes surface protein InlB interacts with c-Met, the hepatocyte growth factor (HGF) receptor, inducing bacterial internalization in numerous non-phagocytic cells. As InlB and HGF are known to trigger similar signaling pathways upon c-Met activation, we investigated the role of CD44, and more specifically its isoform CD44v6, in bacterial internalization in non-phagocytic cells. Indeed, CD44, the hyaluronic acid transmembrane receptor, and more specifically its isoform CD44v6 have been reported as necessary for the activation of c-Met upon the interaction with either the endogenous ligand HGF or the L. monocytogenes surface protein InlB. Our results demonstrate that, in the cell lines that we used, CD44 receptors play no role in the activation of c-Met, neither during L. monocytogenes entry, nor upon HGF activation. Furthermore, none of the CD44 isoforms was recruited at the L. monocytogenes entry site, and depletion by siRNA of total CD44 or of CD44v6 isoform did not reduce bacterial infections. Conversely, the overexpression of CD44 or CD44v6 had no significant effect on L. monocytogenes internalization. Together our results reveal that the activation of c-Met can be largely CD44-independent.     

The InlB protein of Listeria monocytogenes is sufficient to promote entry into mammalian cells

InlB is one of the two Listeria monocytogenes invasion proteins required for bacterial entry into mammalian cells. Entry into human epithelial cells such as Caco-2 requires InlA, whereas InlB is needed for entry into cultured hepatocytes and some epithelial or fibroblast cell lines such as Vero, HEp-2 and HeLa cells. InlB-mediated entry requires tyrosine phosphorylation, cytoskeletal rearrangements and activation of the host protein phosphoinositide (PI) 3-kinase, probably in response to engagement of a receptor. In this study, we demonstrate for the first time that InlB is sufficient to promote internalization. Indeed, coating of normally non-invasive bacteria or inert latex beads with InlB leads to internalization into mammalian cells. In addition, a soluble form of InlB also appears to promote uptake of non-invasive bacteria, albeit at a very low level. Similar to entry of L. monocytogenes , uptake of InlB-coated beads required tyrosine phosphorylation in the host cell, PI 3-kinase activity and cytoskeletal reorganization. Taken together, these data indicate that InlB is sufficient for entry of L. monocytogenes into host cells and suggest that this protein is an effector of host cell signalling pathways.     

Synergy between the N- and C-terminal domains of InlB for efficient invasion of non-phagocytic cells by Listeria monocytogenes

InlB is a Listeria monocytogenes protein promoting entry in non-phagocytic cells, and has been shown recently to activate the hepatocyte growth factor receptor (HGFR or Met). The N-terminal domain of InlB (LRRs) binds and activates Met, whereas the C-terminal domain of InlB (GW modules) mediates loose attachment of InlB to the listerial surface. As HGF activation of Met is tightly controlled by glycosaminoglycans (GAGs), we tested if GAGs also modulate the Met-InlB interactions. We show that InlB-dependent invasion of non-phagocytic cells decreases up to 10 times in the absence of GAGs, and that soluble heparin releases InlB from the bacterial surface and promotes its clustering. Furthermore, we demonstrate that InlB binds cellular GAGs by its GW modules, and that this interaction is required for efficient InlB-mediated invasion. Therefore, GW modules have an unsuspected dual function: they attach InlB to the bacterial surface and enhance entry triggered by the LRRs domain. Our results thus provide the first evidence for a synergy between two host factor-binding domains of a bacterial invasion protein, and reinforce similarities between InlB and mammalian growth factors.     

A FRET analysis to unravel the role of cholesterol in Rac1 and PI 3-kinase activation in the InlB/Met signalling pathway

The signalling pathway for the hepatocyte growth factor receptor, Met/HGF-R, is hijacked by the bacterial surface protein InlB to induce Listeria monocytogenes entry into non-phagocytic cells. We previously showed that Listeria invades host cells by interacting with specialized microdomains of the host plasma membrane called lipid rafts. In this study, we analysed in living cells signalling events that are crucial for Listeria entry using a fluorescence resonance energy transfer-based microscopic method. Phosphoinositide (PI) 3-kinase activity and Rac1 signalling induced by Listeria interacting with epithelial cells were monitored as well as signalling induced by soluble InlB and the Met natural ligand HGF. We found that InlB and HGF induced similar kinetics of PI 3-kinase and Rac1 activation. PI 3-kinase activation was upstream and independent of Rac1 activation. Cholesterol-depletion experiments were performed to address the role of lipid rafts in Met signalling. The amount of 3'-phosphoinositides produced by PI 3-kinase was not affected by cholesterol depletion, while their membrane dynamic was cholesterol-dependent. Rac1 activation, downstream from PI 3-kinase, was cholesterol-dependent suggesting that the spatial distribution of 3'-phosphoinositides within membrane microdomains is critical for Rac1 activation and consequently for F-actin assembly at bacterial entry site.     

InIB-dependent internalization of Listeria is mediated by the Met receptor tyrosine kinase

The Listeria monocytogenes surface protein InlB promotes bacterial entry into mammalian cells. Here, we identify a cellular surface receptor required for InlB-mediated entry. Treatment of mammalian cells with InlB protein or infection with L. monocytogenes induces rapid tyrosine phosphorylation of Met, a receptor tyrosine kinase (RTK) for which the only known ligand is Hepatocyte Growth Factor (HGF). Like HGF, InlB binds to the extracellular domain of Met and induces "scattering" of epithelial cells. Experiments with Met-positive and Met-deficient cell lines demonstrate that Met is required for InlB-dependent entry of L. monocytogenes. InlB is a novel Met agonist that induces bacterial entry through exploitation of a host RTK pathway.     

Investigation of the mechanism of binding between internalin B and heparin using surface plasmon resonance

Listeria monocytogenes, a food-borne pathogen that infects immunocompromised patients, enters and proliferates within mammalian cells by taking advantage of host cell machinery. While entry into macrophages and other phagocytic cells occurs constitutively, intracellular invasion of nonphagocytic cells, such as epithelial and endothelial cells, occurs through induced phagocytosis. Invasion of these nonphagocytic cell types is under the control of the secreted L. monocytogenes protein internalin B (InlB), which directly associates with and activates the receptor tyrosine kinase Met. Activation of Met by InlB has previously been shown to be potentiated by binding of glycosaminoglycans to the GW domains of this protein. We studied the interaction between heparin and full-length InlB as well as a truncated, functional form of InlB to understand the mode of interaction between these two molecules. InlB preferred long-chain (>or=dp14) heparin oligosaccharides, and the interaction with heparin fit a complicated binding model with a dissociation constant in the nanomolar range. While there are various explanations for this complicated binding model, one supported by our data involves binding and rebinding of InlB to multiple binding sites on heparin in a positive and weakly cooperative manner. This mode is consistent with enhancement of interaction of InlB with glycosaminoglycans for activation of Met.     

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

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

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.     

Role of lipid rafts in E-cadherin-- and HGF-R/Met--mediated entry of Listeria monocytogenes into host cells

Listeria monocytogenes uptake by nonphagocytic cells is promoted by the bacterial invasion proteins internalin and InlB, which bind to their host receptors E-cadherin and hepatocyte growth factor receptor (HGF-R)/Met, respectively. Here, we present evidence that plasma membrane organization in lipid domains is critical for Listeria uptake. Cholesterol depletion by methyl-beta-cyclodextrin reversibly inhibited Listeria entry. Lipid raft markers, such as glycosylphosphatidylinositol-linked proteins, a myristoylated and palmitoylated peptide and the ganglioside GM1 were recruited at the bacterial entry site. We analyzed which molecular events require membrane cholesterol and found that the presence of E-cadherin in lipid domains was necessary for initial interaction with internalin to promote bacterial entry. In contrast, the initial interaction of InlB with HGF-R did not require membrane cholesterol, whereas downstream signaling leading to F-actin polymerization was cholesterol dependent. Our work, in addition to documenting for the first time the role of lipid rafts in Listeria entry, provides the first evidence that E-cadherin and HGF-R require lipid domain integrity for their full activity.     

GW domains of the Listeria monocytogenes invasion protein InlB are SH3-like and mediate binding to host ligands

InlB, a surface-localized protein of Listeria monocytogenes, induces phagocytosis in non-phagocytic mammalian cells by activating Met, a receptor tyrosine kinase. InlB also binds glycosaminoglycans and the protein gC1q-R, two additional host ligands implicated in invasion. We present the structure of InlB, revealing a highly elongated molecule with leucine-rich repeats that bind Met at one end, and GW domains that dissociably bind the bacterial surface at the other. Surprisingly, the GW domains are seen to resemble SH3 domains. Despite this, GW domains are unlikely to act as functional mimics of SH3 domains since their potential proline-binding sites are blocked or destroyed. However, we do show that the GW domains, in addition to binding glycosaminoglycans, bind gC1q-R specifically, and that this binding requires release of InlB from the bacterial surface. Dissociable attachment to the bacterial surface via the GW domains may be responsible for restricting Met activation to a small, localized area of the host cell and for coupling InlB-induced host membrane dynamics with bacterial proximity during invasion.     

The development of rapid fluorescence-based immunoassays, using quantum dot-labelled antibodies for the detection of Listeria monocytogenes cell surface proteins

Listeria monocytogenes is an important food-borne pathogen with an extremely high mortality rate (approximately 30%). Therefore, a highly sensitive, reproducible and rapid assay for its detection is vital. L. monocytogenes cells employ two surface bound proteins, Internalin A (InlA) and Internalin B (InlB) to promote invasion into host cells. Recombinant forms of both proteins were previously cloned and expressed in Escherichia coli. In this paper we describe how the InlB protein was sub-divided into three shorter overlapping peptide fragments yielding truncated functional protein of M(R) 23, 35 and 45 kDa, respectively. Purification of the InlB fragments by immobilised metal affinity chromatography (IMAC) was optimised and confirmed by electrophoresis and Western blotting. Identification of the antibody binding regions was achieved by probing the expressed polypeptide domains with a panel of antibodies and antibody fragments. The cloned peptide fragments were also used to develop novel fluorescence-based immunoassays incorporating quantum dots. The application of quantum dot-labelled anti-InlA monoclonal antibodies for immunostaining L. monocytogenes was also demonstrated.     

CD44 binds to the Shigella IpaB protein and participates in bacterial invasion of epithelial cells

Shigella entry into epithelial cells is characterized by a transient reorganization of the host cell cytoskeleton at the site of bacterial interaction with the cell membrane, which leads to bacterial engulfment in a macropinocytic process. Using affinity chromatography on HeLa cell extracts, we show here that the hyaluronan receptor CD44 associates with IpaB, a Shigella protein that is secreted upon cell contact. Overlay and solid-phase assays indicated that IpaB binds directly to the extracellular domain of CD44; binding is saturable and inhibitable, with a half- maximal inhibitory concentration of 175 nM. Immunoprecipitation experiments showed that IpaB associates with CD44 during Shigella entry. CD44 is recruited at bacterial entry sites and localizes at the plasma membrane of cellular extensions induced by Shigella . Pretreatment of cells with an anti-CD44 monoclonal antibody resulted in inhibition of Shigella -induced cytoskeletal reorganization, as well as inhibition of bacterial entry, whereas transfection of CD44 in cells that are deficient for CD44 results in increased bacterial binding to cells and internalization. The IpaB–CD44 interaction appears to be required for Shigella invasion by initiating the early steps of the entry process.     

c-Met recruits ICAM-1 as a coreceptor to compensate for the loss of CD44 in Cd44 null mice

CD44 isoforms act as coreceptors for the receptor tyrosine kinases c-Met and VEGFR-2. However, Cd44 knockout mice do not show overt phenotypes, in contrast to Met and Vegfr-2 knockout mice. We hypothesized that CD44 is being compensated for by another factor in Cd44 null mice. Using RNAi technology and blocking experiments with antibodies, peptides, and purified ectodomains, as well as overexpression studies, we identified intercellular adhesion molecule-1 (ICAM-1) as a new coreceptor for c-Met in CD44-negative tumor cells and in primary hepatocytes obtained from Cd44 null mice. Most strikingly, after partial hepatectomy, CD44v6-specific antibodies inhibited liver cell proliferation and c-Met activation in wild-type mice, whereas ICAM-1-specific antibodies interfered with liver cell proliferation and c-Met activation in Cd44 knockout mice. These data show that ICAM-1 compensates for CD44v6 as a coreceptor for c-Met in Cd44 null mice. Compensation of proteins by members of the same family has been widely proposed to explain the lack of phenotype of several knockout mice. Our experiments demonstrate the functional substitution of a protein by a heterologous one in a knockout mouse.     

Listeria InlB takes a different route to met

InlB, a surface protein of the human bacterial pathogen Listeria monocytogenes, interacts with the receptor tyrosine kinase Met on host cells to enable bacterial invasion. In this issue, Niemann et al. (2007) provide the first structural evidence that InlB does not compete for the same interaction site on Met as the natural ligand HGF.     

The pore-forming toxin listeriolysin O mediates a novel entry pathway of L. monocytogenes into human hepatocytes

Intracellular pathogens have evolved diverse strategies to invade and survive within host cells. Among the most studied facultative intracellular pathogens, Listeria monocytogenes is known to express two invasins-InlA and InlB-that induce bacterial internalization into nonphagocytic cells. The pore-forming toxin listeriolysin O (LLO) facilitates bacterial escape from the internalization vesicle into the cytoplasm, where bacteria divide and undergo cell-to-cell spreading via actin-based motility. In the present study we demonstrate that in addition to InlA and InlB, LLO is required for efficient internalization of L. monocytogenes into human hepatocytes (HepG2). Surprisingly, LLO is an invasion factor sufficient to induce the internalization of noninvasive Listeria innocua or polystyrene beads into host cells in a dose-dependent fashion and at the concentrations produced by L. monocytogenes. To elucidate the mechanisms underlying LLO-induced bacterial entry, we constructed novel LLO derivatives locked at different stages of the toxin assembly on host membranes. We found that LLO-induced bacterial or bead entry only occurs upon LLO pore formation. Scanning electron and fluorescence microscopy studies show that LLO-coated beads stimulate the formation of membrane extensions that ingest the beads into an early endosomal compartment. This LLO-induced internalization pathway is dynamin-and F-actin-dependent, and clathrin-independent. Interestingly, further linking pore formation to bacteria/bead uptake, LLO induces F-actin polymerization in a tyrosine kinase-and pore-dependent fashion. In conclusion, we demonstrate for the first time that a bacterial pathogen perforates the host cell plasma membrane as a strategy to activate the endocytic machinery and gain entry into the host cell.     

CD44 stimulation by fragmented hyaluronic acid induces upregulation and tyrosine phosphorylation of c-Met receptor protein in human chondrosarcoma cells

Hepatocyte growth factor/scatter factor (HGF/SF) can induce proliferation and motility and promote invasion of tumor cells. Since HGF/SF receptor, c-Met, is expressed by tumor cells, and since stimulation of CD44, a transmembrane glycoprotein known to bind hyaluronic acid (HA) in its extracellular domain, is involved in activation of c-Met, we have studied the effects of CD44 stimulation by ligation with HA upon the expression and tyrosine phosphorylation of c-Met on human chondrosarcoma cell line HCS-2/8. The current study indicates that (a) CD44 stimulation by fragmented HA upregulates expression of c-Met proteins; (b) fragmented HA also induces tyrosine phosphorylation of c-Met protein within 30 min, an early event in this pathway as shown by the early time course of stimulation; (c) the effects of HA fragments are critically HA size-dependent. High molecular weight HA is inactive, but lower molecular weight fragments (M(r) 3.5 kDa) are active with maximal effect in the microg/ml range; (d) the standard form of CD44 (CD44s) is critical for the response because the effect on c-Met, both in terms of upregulation and phosphorylation, is inhibited by preincubation with an anti-CD44 monoclonal antibody; and (e) phosphorylation of c-Met induced by CD44 stimulation is inhibited by protein tyrosine kinase inhibitor, tyrphostin. Therefore, our study represents the first report that CD44 stimulation induced by fragmented HA enhances c-Met expression and tyrosine phosphorylation in human chondrosarcoma cells. Taken together, these studies establish a signal transduction cascade or cross-talk emanating from CD44 to c-Met.     

GW domains of the Listeria monocytogenes invasion protein InlB are required for potentiation of Met activation

The Listeria monocytogenes protein InlB promotes intracellular invasion by activating the receptor tyrosine kinase Met. Earlier studies have indicated that the LRR fragment of InlB is sufficient for Met activation, but we show that this is not the case unless the LRR fragment is artificially dimerized through a disulphide bond. In contrast, activation of Met proceeds through monomers of intact InlB and, at physiologically relevant concentrations, requires coordinated action in cis of both InlB N-terminal LRR region and C-terminal GW domains. The GW domains are shown to be crucial for potentiating Met activation and inducing intracellular invasion, with these effects depending on association between GW domains and glycosaminoglycans. Glycosaminoglycans do not alter the monomeric state of InlB, and are likely to enhance Met activation through a receptor-mediated mode, as opposed to the ligand-mediated mode observed for the LRR fragment. Surprisingly, we find that gC1q-R, a host protein implicated in InlB-mediated invasion, specifically antagonizes rather than enhances InlB signalling, and that interaction between InlB and gC1q-R is unnecessary for bacterial invasion. Lastly, we demonstrate that HGF, the endogenous ligand of Met, substitutes for InlB in promoting intracellular invasion, suggesting that no special properties are required of InlB in invasion besides its hormone-like mimicry of HGF.     

Invasion of mammalian cells by Listeria monocytogenes: functional mimicry to subvert cellular functions

The bacterium Listeria monocytogenes has the unusual capacity to enter and to multiply in nonphagocytic cells. Bacterially induced phagocytosis is triggered mainly by the two surface proteins internalin (also called InlA) and InlB, which interact with host cell receptors and either mimic or act in place of the normal cellular ligands. Internalin interacts specifically with human E-cadherin, whereas InlB activates the tyrosine kinase receptor Met and also interacts with the ubiquitous receptor gC1qR and proteoglycans. Signals induced by crosstalk between the bacterium and the host cell allow internalization, which is a prelude to intracellular multiplication, actin-based movement and spread of the bacterium from cell to cell. Manipulating the bacterial invasion proteins offers us an unprecedented tool with which to understand the complex phenomenon of phagocytosis.     

Heparan sulfate-modified CD44 promotes hepatocyte growth factor/scatter factor-induced signal transduction through the receptor tyrosine kinase c-Met

CD44 has been implicated in tumor progression and metastasis, but the mechanism(s) involved is as yet poorly understood. Recent studies have shown that CD44 isoforms containing the alternatively spliced exon v3 carry heparan sulfate side chains and are able to bind heparin-binding growth factors. In the present study, we have explored the possibility of a physical and functional interaction between CD44 and hepatocyte growth factor/scatter factor (HGF/SF), the ligand of the receptor tyrosine kinase c-Met. The HGF/SF-c-Met pathway mediates cell growth and motility and has been implicated in tumor invasion and metastasis. We demonstrate that a CD44v3 splice variant efficiently binds HGF/SF via its heparan sulfate side chain. To address the functional relevance of this interaction, Namalwa Burkitt's lymphoma cells were stably co-transfected with c-Met and either CD44v3 or the isoform CD44s, which lacks heparan sulfate. We show that, as compared with CD44s, CD44v3 promotes: (i) HGF/SF-induced phosphorylation of c-Met, (ii) phosphorylation of several downstream proteins, and (iii) activation of the MAP kinases ERK1 and -2. By heparitinase treatment and the use of a mutant HGF/SF with greatly decreased affinity for heparan sulfate, we show that the enhancement of c-Met signal transduction induced by CD44v3 was critically dependent on heparan sulfate moieties. Our results identify heparan sulfate-modified CD44 (CD44-HS) as a functional co-receptor for HGF/SF which promotes signaling through the receptor tyrosine kinase c-Met, presumably by concentrating and presenting HGF/SF. As both CD44-HS and c-Met are overexpressed on several types of tumors, we propose that the observed functional collaboration might be instrumental in promoting tumor growth and metastasis.     

Entry of the bacterial pathogen Listeria monocytogenes into mammalian cells

The bacterial pathogen Listeria monocytogenes causes food-borne illnesses leading to meningitis or abortion. Listeria provokes its internalization ('entry') into mammalian cells that are normally non-phagocytic, such as intestinal epithelial cells and hepatocytes. Entry provides access to a nutrient-rich cytosol and allows translocation across anatomical barriers. Here I discuss the two major internalization pathways used by Listeria. These pathways are initiated by binding of the bacterial surface proteins InlA or InlB to their respective host receptors, E-cadherin or Met. InlA mediates traversal of the intestinal barrier, whereas InlB promotes infection of the liver. At the cellular level, both InlA- and InlB-dependent entry require host signalling that promotes cytoskeletal rearrangements and pathogen engulfment. However, many of the specific signalling proteins in the two entry routes differ. InlA-mediated uptake uses components of adherens junctions that are coupled to F-actin and myosin, whereas InlB-dependent entry involves cytosolic adaptors that bridge Met to regulators of F-actin, including phosphoinositide 3-kinase and activators of the Arp2/3 complex. Unexpectedly, entry directed by InlB also involves endocytic components. Future work on InlA and InlB will lead to a better understanding of virulence, and may also provide novel insights into the normal biological functions of E-cadherin and Met.