Galactosylated wall teichoic acid,but not lipoteichoic acid,retains InlB on the surface of serovar 4b Listeria monocytogenes

Listeria monocytogenes is a Gram-positive, intracellular pathogen harboring the surface-associated virulence factor InlB, which enables entry into certain host cells. Structurally diverse wall teichoic acids (WTAs), which can also be differentially glycosylated, determine the antigenic basis of the various Listeria serovars. WTAs have many physiological functions; they can serve as receptors for bacteriophages, and provide a substrate for binding of surface proteins such as InlB. In contrast, the membrane-anchored lipoteichoic acids (LTAs) do not show significant variation and do not contribute to serovar determination. It was previously demonstrated that surface-associated InlB non-covalently adheres to both WTA and LTA, mediating its retention on the cell wall. Here, we demonstrate that in a highly virulent serovar 4b strain, two genes gtlB and gttB are responsible for galactosylation of LTA and WTA respectively. We evaluated the InlB surface retention in mutants lacking each of these two genes, and found that only galactosylated WTA is required for InlB surface presentation and function, cellular invasiveness and phage adsorption, while galactosylated LTA plays no role thereof. Our findings demonstrate that a simple pathogen-defining serovar antigen, that mediates bacteriophage susceptibility, is necessary and sufficient to sustain the function of an important virulence factor.     

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.     

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.     

The carboxyl-terminal SH3 domain of the mammalian adaptor CrkII promotes internalization of Listeria monocytogenes through activation of host phosphoinositide 3-kinase

The intracellular bacterial pathogen Listeria monocytogenes causes food-borne illnesses leading to gastroenteritis, meningitis or abortion. Listeria induces its internalization into some mammalian cells through binding of the bacterial surface protein InlB to its host receptor, the Met Receptor Tyrosine Kinase. InlB-induced activation of Met stimulates host signal transduction pathways that culminate in cell surface changes driving pathogen engulfment. One mammalian protein with the potential to couple Met to downstream signalling is the adaptor CrkII. CrkII contains an unusual carboxyl-terminal SH3 domain (SH3C) that promotes entry of Listeria. However, binding partners or downstream effectors of SH3C remain unknown. Here, we use RNA interference and overexpression studies to demonstrate that SH3C affects bacterial uptake, at least in part, through stimulation of host phosphatidylinositide (PI) 3-kinase. Experiments with latex beads coated with InlB protein indicated that one potential role of SH3C and PI 3 kinase is to promote changes in the F-actin cytoskeleton necessary for particle engulfment. Taken together, our results indicate that the CrkII SH3C domain engages a cellular ligand that regulates PI 3 kinase activity and host cell surface rearrangements.     

Aromatic amino acids at the surface of InlB are essential for host cell invasion by Listeria monocytogenes

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

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.     

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.     

An amino-terminal domain of Enterococcus faecalis aggregation substance is required for aggregation, bacterial internalization by epithelial cells and binding to lipoteichoic acid

Aggregation substance (AS), a plasmid-encoded surface protein of Enterococcus faecalis, plays important roles in virulence and antibiotic resistance transfer. Previous studies have suggested that AS-mediated aggregation of enterococcal cells could involve the binding of this protein to cell wall lipoteichoic acid (LTA). Here, a method to purify an undegraded form of Asc10, the AS of the plasmid pCF10, is described. Using this purified protein, direct binding of Asc10 to purified E. faecalis LTA was demonstrated. Equivalent binding of Asc10 to LTA purified from INY3000, an E. faecalis strain that is incapable of aggregation, was also observed. Surprisingly, mutations in a previously identified aggregation domain from amino acids 473 to 683 that abolished aggregation had no effect on LTA binding. In frame deletion analysis of Asc10 was used to identify a second aggregation domain located in the N-terminus of the protein from amino acids 156 to 358. A purified Asc10 mutant protein lacking this domain showed reduced LTA binding, while a purified N-terminal fragment from amino acids 44-331 had high LTA binding. Like the previously described aggregation domain, the newly identified Asc10((156-358)) aggregation domain was also required for efficient internalization of E. faecalis into HT-29 enterocytes. Thus, Asc10 possess two distinct domains required for aggregation and eukaryotic cell internalization: an N-terminal domain that promotes binding to LTA and a second domain located near the middle of the protein.     

The Listeria monocytogenes protein InlB is an agonist of mammalian phosphoinositide 3-kinase.

The Gram-positive pathogen Listeria monocytogenes induces its own internalization into some non-phagocytic mammalian cells by stimulating host tyrosine phosphorylation, phosphoinositide (PI) 3-kinase activity, and rearrangements in the actin cytoskeleton. Entry into many cultured cell lines is mediated by the bacterial protein InlB. Here we investigate the role of InlB in regulating mammalian signal transduction and cytoskeletal structure. Treatment of Vero cells with purified InlB caused rapid and transient increases in the lipid products of the PI 3-kinase p85-p110, tyrosine phosphorylation of the mammalian adaptor proteins Gab1, Cbl, and Shc, and association of these proteins with p85. InlB also stimulated large scale changes in the actin cytoskeleton (membrane ruffling), which were PI 3-kinase-dependent. These results identify InlB as the first reported non-mammalian agonist of PI 3-kinase and demonstrate similarities in the signal transduction events elicited by this bacterial protein and known agonists such as epidermal growth factor.     

Involvement of CD44v6 in InlB-dependent Listeria invasion

Listeria monocytogenes , a Gram-positive bacterium, is the causative agent for the disease called listeriosis. This pathogen utilizes host cell surface proteins such as E-cadherin or c-Met in order to invade eukaryotic cells. The invasion via c-Met depends on the bacterial protein InlB that activates c-Met phosphorylation and internalization mimicking in many regards HGF, the authentic c-Met ligand. In this paper, we demonstrate that the activation of c-Met induced by InlB is dependent on CD44v6, a member of the CD44 family of transmembrane glycoproteins. Inhibiting CD44v6 by means of a blocking peptide, a CD44v6 antibody or CD44v6-specific siRNA prevents the activation of c-Met induced by InlB. Subsequently, signalling, scattering and the entry of InlB-coated beads into host cells are also impaired by CD44v6 blocking reagents. For the entry process, ezrin, a protein that links the CD44v6 cytoplasmic domain to the cytoskeleton, is required as well. Most importantly, this collaboration between c-Met and CD44v6 contributes to the invasion of L. monocytogenes into target cells as demonstrated by a drastic decrease in bacterial invasion in the presence of blocking agents such as the CD44v6 peptide or antibody.     

Docking interactions of the JNK scaffold protein WDR62

JNK (c-Jun N-terminal kinase) is part of a MAPK (mitogen-activated protein kinase) signalling cascade. Scaffold proteins simultaneously associate with various components of the MAPK signalling pathway and play a crucial role in signal transmission and MAPK regulation. WDR62 (WD repeat domain 62) is a JNK scaffold protein. Recessive mutations within WDR62 result in severe cerebral cortical malformation. In the present study we demonstrate the association of WDR62 with endogenous and overexpressed proteins of both JNK2 and the JNK2-activating kinase MKK7 (MAPK kinase 7). Association of WDR62 with JNK2 and MKK7 occurs via direct protein-protein interactions. We mapped the docking domain of WDR62 responsible for the association with JNK. WDR62 interacts with all JNK isoforms through a D domain motif located at the C-terminus. A WDR62 mutant lacking the putative JNK-binding domain fails to activate and recruit JNK to cellular granules. Furthermore, a synthetic peptide composed of the WDR62 docking domain inhibits JNK2 activity in vitro. WDR62 association with JNK2 requires both the JNK CD and ED domains, and the binding requisite is distinct from that of the previously described JNK2 association with JIP1 (JNK-interacting protein 1). Next, we characterized the association between WDR62 and MKK7. WDR62 associates directly with the MKK7β1 isoform independently of JNK binding, but fails to interact with MKK7α1. Furthermore, MKK7β1 recruits a protein phosphatase that dephosphorylates WDR62. Interestingly, a premature termination mutation in WDR62 that results in severe brain developmental defects does not abrogate WDR62 association with either JNK or MKK7. Therefore such mutations represent a loss of WDR62 function independent of JNK signalling.     

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.     

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.     

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.     

Formation of D-alanyl-lipoteichoic acid is required for adhesion and virulence of Listeria monocytogenes

The dlt operon of Gram-positive bacteria comprises four genes (dltA, dltB, dltC and dltD) that catalyse the incorporation of D-alanine residues into the cell wall-associated lipoteichoic acids (LTAs). In this work, we characterized the dlt operon of Listeria monocytogenes and constructed a D-Ala-deficient LTA mutant by inactivating the first gene (dltA) of this operon. The DltA- mutant did not show any morphological alterations and its growth rate was similar to that of the wild-type strain. However, it exhibited an increased susceptibility to the cationic peptides colistin, nisin and polymyxin B. The virulence of the DltA- mutant was severely impaired in a mouse infection model (4 log increase in the LD50) and, in vitro, the adherence of the mutant to various cell lines (murine bone marrow-derived macrophages and hepatocytes and a human epithelial cell line) was strongly restricted, although the amounts of surface proteins implicated in virulence (ActA, InlA and InlB) remains unaffected. We suggest that the decreased adherence of the DltA- mutant to non-phagocytic and phagocytic cells might be as a result of the increased electronegativity of its charge surface and/or the presence at the bacterial surface of adhesins possessing altered binding activities. These results show that the D-alanylation of the LTAs contributes to the virulence of the intracellular pathogen L. monocytogenes.     

Folding and stability of the leucine-rich repeat domain of internalin B from Listeri monocytogenes

Internalin B (InlB), a surface protein of the human pathogen Listeria monocytogenes, promotes invasion into various host cell types by inducing phagocytosis of the entire bacterium. The N-terminal half of InlB (residues 36-321, InlB321), which is sufficient for this process, contains a central leucine-rich repeat (LRR) domain that is flanked by a small alpha-helical cap and an immunoglobulin (Ig)-like domain. Here we investigated the spectroscopic properties, stability and folding of InlB321 and of a shorter variant lacking the Ig-like domain (InlB248). The circular dichroism spectra of both protein variants in the far ultraviolet region are very similar, with a characteristic minimum found at approximately 200 nm, possibly resulting from the high 3(10)-helical content in the LRR domain. Upon addition of chemical denaturants, both variants unfold in single transitions with unusually high cooperativity that are fully reversible and best described by two-state equilibria. The free energies of GdmCl-induced unfolding determined from transitions at 20 degrees C are 9.9(+/-0.8)kcal/mol for InlB321 and 5.4(+/-0.4)kcal/mol for InlB248. InlB321 is also more stable against thermal denaturation, as observed by scanning calorimetry. This suggests, that the Ig-like domain, which presumably does not directly interact with the host cell receptor during bacterial invasion, plays a critical role for the in vivo stability of InlB.     

Ligand-Mediated Dimerization of the Met Receptor Tyrosine Kinase by the Bacterial Invasion Protein InlB

The Listeria monocytogenes surface protein InlB mediates bacterial invasion into host cells by activating the human receptor tyrosine kinase Met. So far, it is unknown how InlB or the physiological Met ligand hepatocyte growth factor/scatter factor causes Met dimerization, which is considered a prerequisite for receptor activation. We determined two new structures of InlB, revealing a recurring, antiparallel, dimeric arrangement, in which the two protomers interact through the convex face of the leucine-rich repeat domain. The same contact is found in one structure of the InlB-Met complex. Mutations disrupting the interprotomeric contact of InlB reduced its ability to activate Met and downstream signaling. Conversely, stabilization of this crystal contact by two intermolecular disulfide bonds generates a constitutively dimeric InlB variant with exceptionally high signaling activity, which can stimulate cell motility and cell division. These data demonstrate that the signaling-competent InlB-Met complex assembles with 2:2 stoichiometry around a back-to-back InlB dimer, enabling the direct contact between the stalk region of two Met molecules.     

Structure of the human receptor tyrosine kinase met in complex with the Listeria invasion protein InlB

The tyrosine kinase Met, the product of the c-met proto-oncogene and the receptor for hepatocyte growth factor/scatter factor (HGF/SF), mediates signals critical for cell survival and migration. The human pathogen Listeria monocytogenes exploits Met signaling for invasion of host cells via its surface protein InlB. We present the crystal structure of the complex between a large fragment of the human Met ectodomain and the Met-binding domain of InlB. The concave face of the InlB leucine-rich repeat region interacts tightly with the first immunoglobulin-like domain of the Met stalk, a domain which does not bind HGF/SF. A second contact between InlB and the Met Sema domain locks the otherwise flexible receptor in a rigid, signaling competent conformation. Full Met activation requires the additional C-terminal domains of InlB which induce heparin-mediated receptor clustering and potent signaling. Thus, although it elicits a similar cellular response, InlB is not a structural mimic of HGF/SF.     

Multiple regions of internalin B contribute to its ability to turn on the Ras-mitogen-activated protein kinase pathway

Internalin B (InlB) is a protein present on the surface of Listeria monocytogenes that mediates bacterial entry into mammalian cells. It is thought that InlB acts by binding directly to the hepatocyte growth factor (HGF) receptor, present on the surface of host cells. Binding of InlB to the HGF receptor results in mitogen-activated protein (MAP) kinase and phosphoinositide 3-kinase activation, followed by changes in the organization of the actin cytoskeleton. Here we have compared signaling by HGF and InlB. Whereas stimulation with equivalent concentrations of HGF and InlB elicits similar activation of the HGF receptor, we observed striking differences in downstream activation of MAP kinase. InlB leads to a greater activation of the Ras-MAP kinase pathway than does HGF. The leucine-rich repeat region, which was previously shown to be sufficient for binding and activation of the HGF receptor, lacks the ability to super-activate the Ras-MAP kinase pathway. Analysis of a series of deletion mutants suggests that it is the B repeat region between the leucine-rich repeat and GW domains that endows InlB with an increased ability to turn on the Ras-MAP kinase pathway. These unexpected observations suggest that HGF and InlB use alternative mechanisms to turn on cellular signaling pathways.