InlBMediated Pathway InlB Structure and Function

InlB is a 630-amino acids protein from the internalin family encoded in the same operon as InlA (Gaillard et al., 1991). InlB presents an amino-terminal signal peptide followed by the short conserved internalin cap and seven LRRs; the IR region that follows the LRR region has a minimal immunoglobulin (Ig)-like fold that, together with the LRRs and with the cap (which presents itself a truncated EF-hand fold structure), constitutes aunique domain with a continuous hydrophobic core and an extended ß-sheet (Figure 8.3.) (Schubert et al., 2001). This amino-terminal domain is followed by a central B repeat, while the carboxy-terminal domain is constituted by three C repeats of approximately 80 amino acids, each of them starting with the residues glycine/tryptophane (GW) and hence known as the GW region (Braun et al., 1997). The GW domains mediate loose attachment of InlB with the lipoteichoic acids of the L. monocytogenes cell wall (Jonquieres et al., 1999).

As in the case of InlA, InlB has been shown to be required for invasion of human epithelial cells; however, InlB allows entry within a larger panel of target cells than InlA (Braun et al., 1998; Dramsi et al., 1995). The LRR region of InlB has been shown to be sufficient for inducing entry into cells of latex beads covalently bound to this domain (Braun et al., 1999). Exogenously added InlB can also favor entry of noninvasive L. innocua or Streptococcus carnosus (Braun et al., 1998). Interestingly, InlB can be detached from the bacterial cell wall, and it has been shown that the GW domains of its carboxy terminal can also interact with cellular targets (Jonquieres et al., 2001), indicating that both the amino-and carboxy-terminal domains of InlB cooperate to promote efficient invasion of nonphagocytic cells (see below). Met, GAGs, and gC1qR: InlB Receptors

The main signaling receptor for InlB is the hepatocyte growth factor (HGF) receptor, also known as Met (Shen et al., 2000). Met belongs to the family of receptor tyrosine kinases (RTK), one of the largest and most important families of transmembrane signaling receptors (Hubbard and Till, 2000). Upon binding to their cognate ligands, RTKs can transduce extracellular signals into the cytoplasm by autophosphorylation and by phosphorylation of downstream molecules, activating multiple pathways involved in cell proliferation, migration, and/or differentiation. Met, in particular, plays an important role during development, controlling epithelial cell migration and growth during embryogenesis; Met also controls growth, invasion and metastasis in cancer cells, and activating Met mutations predispose to human cancer (Birchmeier et al., 2003).

The crystal structure of the InlB LRRs reveals that this region adopts an elongated curved conformation (Figure 8.3.) (Marino et al., 1999), and it has been recently shown that four aromatic residues within the concave surface of the LRR domain are crucial for binding to Met (Machner et al., 2003). The InlB-Met interaction is necessary for L. monocytogenes entry into target cells (Shen et al., 2000). As is the case for the interaction between the HGF and the Met, interaction between InlB and Met is potentiated by glycosaminoglycans (GAGs) of the extracellular matrix (Jonquieres et al., 2001). It is relevant to note that InlB is able to bind GAGs directly through its GW domains (Banerjee et al., 2004; Jonquieres et al., 2001). The GW repeats of InlB actually present similarity to eukaryotic Src-homologytype 3 (SH3)-like domains; however, the potential proline-binding sites typical from functional SH3 domains are absent from the GW repeats (Marino et al., 2002). The GW domains of InlB also interact with the receptor for the globular head of the complement C1q molecule (known as gC1q-R) (Braun et al., 2000; Marino et al., 2002); however, the functional relevance of this binding during interaction of L. monocytogenes with target cells has not been established yet. Signaling Downstream of InlB-Met Interaction

Met is a heterodimer formed by an extracellular a- and a P-subunit that comprises extracellular, transmembrane and cytoplasmic domains. The cytoplasmic tail of the P-subunit contains several potential sites for tyrosine phosphorylation: tyrosines 1234 and 1235 are referred as the activation loop since they increase Met kinase activity (Birchmeier and Gherardi, 1998). The juxtamembrane tyrosine 1003 serves as a binding site for the ubiquitin ligase Cbl (Peschard et al., 2001), while tyrosines 1349 and 1356 are referred as the multi-docking site since they are involved in the recruitment of several molecules including the adaptor proteins Shc and Gab1, which in turn can be phospho-rylated in several tyrosine residues and bind other signaling proteins (Ponzetto et al., 1994). It has been reported that upon stimulation by InlB, phosphorylation of Cbl, Shc, and Gab1 occurs on target cells (Ireton et al., 1999; Shen et al., 2000). Of note, recruitment/activation of Gab1 by InlB can take place by two redundant pathways that require either phosphorylation of Met tyrosines 1349/1356 and Gab1-binding to the Met multidocking site, or formation of the phosphoinositide phosphatidylinositol-3,4-5-triphosphate (PI[3,4,5]P3) at the plasma membrane and Gab1-recruitment via its pleckstrin homology domain, able to directly bind PI(3,4,5)P3 (Basar et al., 2005). Gab1, Shc, and Cbl are involved in the recruitment of the phosphatidylinositol 3-kinase (PI3K) that precisely promotes PI[3,4,5]P3 production at the site of L. monocytogenes entry

(Ireton et al., 1996, 1999) and favors cytoskeletal rearrangements required for bacterial engulfment (Figure 8.4.).

Regulation of actin modification downstream of PI3K during InlB stimulation is complex and varies in different cell lines. In the green monkey hepatocyte cell line Vero, it has been demonstrated that the small GTPase Racl is involved in the WAVE2-dependent activation of the Arp2/3 complex (Bierne et al., 2001, 2005), which nucleates and polymerizes actin filaments in branched networks (Pollard and Beltzner, 2002; Stradal and Scita, 2006). Proteins of the Ena/VASP family are required for elongation of actin filaments (Bierne et al., 2005). Formation and disruption of the phagocytic cup during L. monocy-togenes entry require a fine-tuning of the activity of the actin depolymerizing factor cofilin, its activity in turn being regulated by the LIM kinase (Bierne et al., 2001). In HeLa cells, activation of the Arp2/3 complex by InlB requires Racl upstream of WAVE2 and WAVE1, and also Cdc42 upstream of N-WASP (Bierne et al. 2005). Clathrin-Mediated Endocytosis of Met and Invasion

As mentioned above, the ubiquitin ligase Cbl is recruited to Met upon cellular stimulation by InlB (Ireton et al., 1999). It has been recently determined that during this stimulation, Cbl ubiquitinates Met and triggers activation of the endocytosis machinery, favoring the clathrin-dependent internalization of Met (Li et al., 2005) and of L. monocytogenes to produce infection (Li et al., 2005; Veiga and Cossart, 2005). This result is highly surprising since it was thought that the endocytic machinery supported internalization of vesicles presenting a size inferior only to 100 nm (Gao et al., 2005). However, it seems that the potential use of the endocytic machinery for phagocytosis is a broad phenomenon also observed with other bacterial pathogens (E. Veiga, 2007, personal communication). Effect of Rafts on the Downstream Signaling of Met upon InlB Stimulation

As in the case of the InlA-dependent pathway, the contribution of lipid microdomains in the context of the InlB-dependent entry of L. monocytogenes has been analyzed. As opposed to the InlA-E-cadherin interaction, which requires the presence of intact rafts to take place, interaction between InlB and Met can occur in cells which have been depleted of cholesterol (Seveau et al., 2004). However, despite recruitment of Met to the bacterial entry site, downstream signaling leading to actin polymerization was disrupted in M^CD-treated cells. Activation of PI3K is not affected in these cells, but activation of Rac1 is compromised, suggesting that it is the recruitment of PI(3,4,5)P3-binding protein(s) involved in Rac1 activation that requires integrity of plasma membrane microdomains (S. Séveau and J. Swanson, 2006, personal communication).

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