In1C

Figure 8.2. Schematic representation of different types of internalins involved in invasion of target cells by L. monocytogenes. InlA is an LPXTG-anchored membrane protein that promotes bacterial entry into polarized epithelial cells by interacting with the adhesion molecule E-cadherin through its leucine-rich repeats. InlB is loosely anchored to the bacterial cell wall through its GW domains and can be detached from the L. monocytogenes surface; soluble InlB interacts with host cell glycosaminoglycans (GAGs) and the globular receptor for the complement gC1 molecule (gC1q-R) through its GW domains, while its leucine-rich repeats induce invasion of a large panel of target cell by stimulation of the hepatocyte growth factor receptor Met. InlC is a soluble internalin that potentiates the InlA-mediated entry pathway in the presence of InlB by interacting with a still unknown ligand.

regions are sufficient to induce the entry of inert latex beads or of the noninvasive L. innocua species into target cells (Lecuit et al., 1997). Recent in vivo studies have highlighted the critical role played by InlA during the traversal of the human intestinal and materno-fetal barriers (Lecuit et al., 2001, 2004) (see below). The importance of InlA has also been estimated at the population level: although certain L. monocytogenes strains carry a truncated form of InlA that can be released from the bacterial cell wall (Jonquieres et al., 1998), an epidemiological study carried out in France demonstrated that 96% of clinical L. monocytogenes isolates present a full-length protein; moreover, 100% of isolates from placental infections present the full-length form of InlA, confirming the critical role of InlA in the listeriosis pathogenesis, as well as its potential value as an indicator of virulence in food-safety assessment programs (Jacquet et al., 2004).

8.2.1.2. E-cadherin: the InlA Receptor

By affinity chromatography on an InlA-column, E-cadherin was identified as the cellular receptor for InlA (Mengaud et al., 1996). E-cadherin belongs to the cadherin superfamily of calcium-dependent cell adhesion molecules, which includes other classical cadherins such as the N-, P-, or H-cadherins, but also includes more than 80 protocadherins (the largest subgroup in the cadherin super-family) involved in the morphogenesis and formation of neuronal circuits, and in modulation of synaptic transmission (Junghans et al., 2005). Classical cadherins in neighboring cells establish homophilic interactions between their extracellular domains, while their cytoplasmic tails interact with several molecules, including proteins of the Armadillo repeat family such as P-catenin and p120 catenin, which mediate association of cell adhesion complexes to the cytoskeleton, and regulate cadherin stability/retention at the plasma membrane (Nelson and Nusse, 2004). E-cadherin, in particular, is involved in the formation of adherens junctions in polarized epithelial cells of different tissues such as the intestine or the feto-placental barrier and is regarded as the main organizer of the epithelial phenotype; indeed, E-cadherin dysfunction or down-regulation is closely linked to cancer (Gumbiner, 2005). Distribution of E-cadherin in polarized epithelial layers is normally restricted to basolateral membranes where E-cadherin is not accessible to the lumen; however, within an epithelial barrier, sites of senescent cell extrusion exist in which E-cadherin is transiently exposed to the luminal surface, and these sites have been shown to be used by L. monocytogenes to access epithelial junctions, promoting cellular invasion (Pentecost et al., 2006).

The initial interaction between bacterial InlA and human E-cadherin is mediated by the LLRs present on InlA and the first extracellular domain of E-cadherin. Interestingly, this interaction is species-specific: a proline at position 16 on E-cadherin (such as in humans and guinea pigs) is necessary for InlA binding, and mutation of this proline to glutamic acid (such as in the mouse or rat) not only inhibits adhesion of L. monocytogenes to E-cadherin-expressing cells but also inhibits invasion (Lecuit et al., 1999). The crystal structure of the InlA LRRs in complex with the first extracellular domain of human E-cadherin illustrates the exquisite fine interaction between these two molecules: an hydrophobic pocket between the LRR 5 and the LRR 7 (due to the absence of one amino acid on LLR 6) accommodates precisely the proline at position 16 (Figure 8.3.) (Schubert et al., 2002). The generation of transgenic mice expressing the human E-cadherin at the intestinal level revealed that only the interaction of L. monocytogenes with the transgenic human InlA-binding E-cadherin (and not with the endogenous mouse E-cadherin) allowed bacterial translocation across the intestinal barrier, highlighting the crucial role of this interaction for the initial steps of the disease (Lecuit et al., 2001). E-cadherin is also present in syncytiotrophoblasts and villious cytotrophoblasts of the placenta, and it has been recently shown that the InlA/E-cadherin interaction is as well required for traversal of the human materno-fetal barrier (Lecuit et al., 2004). E-cadherin is also present at epithelial cells in contact with the encephalorachidean liquid, and it is suspected that InlA plays a role during bacterial translocation through the blood-brain barrier.

InlA

InlB

cap cap

Figure 8.3. Structure of the N-terminal domains of InlA and InlB. A Leucine-rich repeats (LRRs) from InlA are composed of 16 P-sheets that accommodate the first extracellular domain of E-cadherin; an hydrophobic pocket created between LRR 5 and 7 (due to the absence of one amino acid in LRR 6) accommodates E-cadherin proline 16, which is critical for InlA-E-cadherin interaction. B 90°C rotation of the figure depicted in A. C View toward the concave surface of the InlB LRRs. Reprinted from Cell, Vol. 111, W.D. Schubert et al., Structure of internalin, a major invasion protein of Listeria monocytogenes, in complex with its human receptor E-cadherin, pp. 825-836, copyright 2001, and JMolBiol, Vol. 312, W.D. Schubert et al., Internalins of the human pathogen L. monocytogenes combine three distinct folds into a contiguous internalin domain, pp. 783-794, copyright 2002, with permission from Elsevier. (A color version of this figure appears between pages 196 and 197.)

Inter Repeat

LRRs

Figure 8.3. Structure of the N-terminal domains of InlA and InlB. A Leucine-rich repeats (LRRs) from InlA are composed of 16 P-sheets that accommodate the first extracellular domain of E-cadherin; an hydrophobic pocket created between LRR 5 and 7 (due to the absence of one amino acid in LRR 6) accommodates E-cadherin proline 16, which is critical for InlA-E-cadherin interaction. B 90°C rotation of the figure depicted in A. C View toward the concave surface of the InlB LRRs. Reprinted from Cell, Vol. 111, W.D. Schubert et al., Structure of internalin, a major invasion protein of Listeria monocytogenes, in complex with its human receptor E-cadherin, pp. 825-836, copyright 2001, and JMolBiol, Vol. 312, W.D. Schubert et al., Internalins of the human pathogen L. monocytogenes combine three distinct folds into a contiguous internalin domain, pp. 783-794, copyright 2002, with permission from Elsevier. (A color version of this figure appears between pages 196 and 197.)

8.2.1.3. Adherens Junctions and the Molecular Machinery Involved in the InlA-Entry Pathway

As stated above, the cytoplasmic tail of E-cadherin is able to interact with several proteins of the catenin family that link the adherens junction complex to the actin cytoskeleton. In particular, P-catenin is able to bind to the last 35 amino acids of the E-cadherin cytoplasmic domain and also interacts with a-catenin, which in turn is able to directly bind actin (Jamora and Fuchs, 2002). Taking into account this model of interaction in which a-catenin provides a stable link between E-cadherin and the cytoskeleton, a chimera was constructed which contained the E-cadherin ecto-domain fused to the actin-binding site of a-catenin: this fusion molecule allowed similar levels of bacterial infection as wild-type cells (as opposed to E-cadherin molecules that lack the P-catenin-binding site and which are nonpermisive for infection), suggesting that L. monocytogenes exploits the same molecular scaffold used for adherens junction formation to induce entry into target cells (Figure 8.4.) (Lecuit et al., 2000). Recently, the model depicting the static binding of E-cadherin to the actin cytoskeleton via a-catenin has been challenged; in fact, it has been shown that the interaction of a-catenin with either E-cadherin or P-catenin is exclusive (Drees et al., 2005; Yamada et al., 2005). These authors have suggested that the direct connection between adherens junctions and the actin cytoskeleton could be mediated by

Listeria Monocytogenes Inla Cadherin
In1B entry pathway

Figure 8.4. Signaling pathways triggered by InlA and InlB. InlA entry pathway: InlA interacts with E-cadherin, recruiting to the bacterial entry site the adherens junction proteins and a-catenin, promoting rearrangements in the actin cytoskeleton required for invasion. The protein ARHGAP10 is a GTPase activating protein for RhoA and Cdc42 that interacts with a-catenin and is required for entry as well as adherens junction formation. The unconventional myosin VIIa is also required for entry, providing probably the

(continued)

Figure 8.4. Signaling pathways triggered by InlA and InlB. InlA entry pathway: InlA interacts with E-cadherin, recruiting to the bacterial entry site the adherens junction proteins and a-catenin, promoting rearrangements in the actin cytoskeleton required for invasion. The protein ARHGAP10 is a GTPase activating protein for RhoA and Cdc42 that interacts with a-catenin and is required for entry as well as adherens junction formation. The unconventional myosin VIIa is also required for entry, providing probably the

(continued)

other junction proteins such as nectin or afadin; alternatively, several weak transient and cumulative interactions between actin-binding proteins such as vinculin, afadin, or spectrin with the junction components could be the basis of the cytoskeleton connection to the adherens junctions. In the case of the L. monocytogenes entry, it has been determined that other proteins besides catenins also participate in the entry process which could also regulate the adherensjunction-cytoskeleton interaction: through a two-hybrid screen using a-catenin as a bait, the molecule ARHGAP10—which is a GTPase-activating protein (GAP) for the small Rho GTPases RhoA and Cdc42—has been found to be required for efficient bacterial invasion as well as for adherens junction formation (Sousa et al., 2005). The unconventional myosin VIIA and its ligand, the membrane-associated protein vezatin, are also required for entry, probably generating the contractile force necessary to promote bacterial internalization (Sousa et al., 2004). Polymerization of actin downstream of the InlA/E-cadherin interaction is mediated by the small Rho GTPase Rac, by the Src substrate cortactin and by the actin nucleating Arp2/3 complex (a cortactin substrate itself), but how these molecules are activated to favor cytoskeletal rearrangements during invasion is not known yet.

8.2.1.4. Effect of Rafts on the InlA-E-cadherin Interaction

It is important to note that a functional interaction between InlA and E-cadherin leading to bacterial entry cannot take place if the host membrane organization in lipid domains is altered. Indeed, formation of lipid membrane micro-domains or rafts provides a mechanism for the segregation of molecular effectors into functional subunits for efficient signaling and sorting processes (Simons and Toomre, 2000). The drug methyl-^-cyclodextrin (M^CD) that sequesters the major membrane lipid cholesterol and disrupts lipid rafts has been used to study the function of lipid micro-domains in the L. monocytogenes infection process; it has been demonstrated that M^CD does not alter the amounts of E-cadherin present at the plasma membrane of cholesterol-depleted cells, but affects the

Figure 8.4. (Continued) contractile force that drives bacterial internalization. The protein Vezatin is also localized at the bacterial entry site, but its function in the L. monocytogenes entry process is still unknown. InlB entry pathway: InlB stimulates the hepatocyte growth factor Met, inducing its phosphorylation and the recruitment to the bacterial entry site of the molecular adaptors Cbl, Shc, and Gabl, which in turn recruit the PI3K type I: this enzyme generates PI(3,4,5)P3, which is a potent second messenger upstream of Rac required for the induction of actin rearrangements associated with bacterial invasion. Cbl is also an ubiquitin ligase that targets the endocytic machinery to the bacterial entry site, favoring Met and L. monocytogenes internalization into target cells. Reprinted from Subversion of cellular functions by Listeria monocytogenes, J. Pizarro-Cerda and P. Cossart, J Pathol Vol 208, pp. 215-223, 2006, copyright Pathological Society of Great Britain and Ireland. Reproduced with permission. Permission granted by John Wiley & Sons Ltd on behalf of the Pathological Society.

efficient recruitment of E-cadherin complexes to the site of bacterial attachment (Seveau et al., 2004). Lipid raft markers such as glycosylphosphatidylinositol-conjugated proteins are also recruited at the bacterial entry site in an InlA-dependent manner. These results suggest that the function of E-cadherin, not only in the case of L. monocytogenes invasion but probably also in the context of native adherens junctions, requires the organization of the plasma membrane in intact lipid micro-domains that will favor the mobility and clustering of E-cadherin into functional units favoring downstream signaling.

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