Distinct mechanisms of internalization of Neisseria gonorrhoeae by members of the CEACAM receptor family involving Rac1- and Cdc42-dependent and -independent pathways
2002; Springer Nature; Volume: 21; Issue: 4 Linguagem: Inglês
10.1093/emboj/21.4.560
ISSN1460-2075
AutoresOliver Billker, Andreas Popp, Volker Brinkmann, Gerald Wenig, Jutta Schneider, Emmanuelle Caron, Thomas F. Meyer,
Tópico(s)Peptidase Inhibition and Analysis
ResumoArticle15 February 2002free access Distinct mechanisms of internalization of Neisseria gonorrhoeae by members of the CEACAM receptor family involving Rac1- and Cdc42-dependent and -independent pathways Oliver Billker Oliver Billker Max-Planck-Institut für Infektionsbiologie, Abteilung Molekulare Biologie, Schumannstraße 21/22, D-10117 Berlin, Germany Search for more papers by this author Andreas Popp Andreas Popp Max-Planck-Institut für Infektionsbiologie, Abteilung Molekulare Biologie, Schumannstraße 21/22, D-10117 Berlin, Germany Search for more papers by this author Volker Brinkmann Volker Brinkmann Max-Planck-Institut für Infektionsbiologie, Abteilung Molekulare Biologie, Schumannstraße 21/22, D-10117 Berlin, Germany Search for more papers by this author Gerald Wenig Gerald Wenig Max-Planck-Institut für Infektionsbiologie, Abteilung Molekulare Biologie, Schumannstraße 21/22, D-10117 Berlin, Germany Search for more papers by this author Jutta Schneider Jutta Schneider Universität Freiburg, Institut für Immunbiologie, Stefan-Meier-Straße 8, D-79104 Freiburg, Germany Present address: Institut für Molekulare Medizin und Zellforschung, Breisacher Straße 66, D-79106 Freiburg, Germany Search for more papers by this author Emmanuelle Caron Emmanuelle Caron Medical Research Council Laboratory for Molecular Cell Biology, University College London, Gower Street, London, WC1E 6BT GB Search for more papers by this author Thomas F. Meyer Corresponding Author Thomas F. Meyer Max-Planck-Institut für Infektionsbiologie, Abteilung Molekulare Biologie, Schumannstraße 21/22, D-10117 Berlin, Germany Search for more papers by this author Oliver Billker Oliver Billker Max-Planck-Institut für Infektionsbiologie, Abteilung Molekulare Biologie, Schumannstraße 21/22, D-10117 Berlin, Germany Search for more papers by this author Andreas Popp Andreas Popp Max-Planck-Institut für Infektionsbiologie, Abteilung Molekulare Biologie, Schumannstraße 21/22, D-10117 Berlin, Germany Search for more papers by this author Volker Brinkmann Volker Brinkmann Max-Planck-Institut für Infektionsbiologie, Abteilung Molekulare Biologie, Schumannstraße 21/22, D-10117 Berlin, Germany Search for more papers by this author Gerald Wenig Gerald Wenig Max-Planck-Institut für Infektionsbiologie, Abteilung Molekulare Biologie, Schumannstraße 21/22, D-10117 Berlin, Germany Search for more papers by this author Jutta Schneider Jutta Schneider Universität Freiburg, Institut für Immunbiologie, Stefan-Meier-Straße 8, D-79104 Freiburg, Germany Present address: Institut für Molekulare Medizin und Zellforschung, Breisacher Straße 66, D-79106 Freiburg, Germany Search for more papers by this author Emmanuelle Caron Emmanuelle Caron Medical Research Council Laboratory for Molecular Cell Biology, University College London, Gower Street, London, WC1E 6BT GB Search for more papers by this author Thomas F. Meyer Corresponding Author Thomas F. Meyer Max-Planck-Institut für Infektionsbiologie, Abteilung Molekulare Biologie, Schumannstraße 21/22, D-10117 Berlin, Germany Search for more papers by this author Author Information Oliver Billker1, Andreas Popp1, Volker Brinkmann1, Gerald Wenig1, Jutta Schneider2,4, Emmanuelle Caron3 and Thomas F. Meyer 1 1Max-Planck-Institut für Infektionsbiologie, Abteilung Molekulare Biologie, Schumannstraße 21/22, D-10117 Berlin, Germany 2Universität Freiburg, Institut für Immunbiologie, Stefan-Meier-Straße 8, D-79104 Freiburg, Germany 3Medical Research Council Laboratory for Molecular Cell Biology, University College London, Gower Street, London, WC1E 6BT GB 4Present address: Institut für Molekulare Medizin und Zellforschung, Breisacher Straße 66, D-79106 Freiburg, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:560-571https://doi.org/10.1093/emboj/21.4.560 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Opa adhesins of pathogenic Neisseria species target four members of the human carcinoembryonic antigen-related cellular adhesion molecule (CEACAM) family. CEACAM receptors mediate opsonization-independent phagocytosis of Neisseria gonorrhoeae by human granulocytes and each receptor individually can mediate gonococcal invasion of epithelial cells. We show here that gonococcal internalization occurs by distinct mechanisms depending on the CEACAM receptor expressed. For the invasion of epithelial cell lines via CEACAM1 and CEACAM6, a pathogen-directed reorganization of the actin cytoskeleton is not required. In marked contrast, ligation of CEACAM3 triggers a dramatic but localized reorganization of the host cell surface leading to highly efficient engulfment of bacteria in a process regulated by the small GTPases Rac1 and Cdc42, but not Rho. Two tyrosine residues of a cytoplasmic immune receptor tyrosine-based activating motif of CEACAM3 are essential for the induction of phagocytic actin structures and subsequent gonococcal internalization. The granulocyte-specific CEACAM3 receptor has properties of a single chain phagocytic receptor and may thus contribute to innate immunity by the elimination of Neisseria and other CEACAM-binding pathogens that colonize human mucosal surfaces. Introduction Neisseria gonorrhoeae is a human-specific, Gram-negative pathogen that colonizes mucosal surfaces of the urogenital tract, but also infects the rectum, nasopharynx and the conjunctiva of the eye. For the colonization of such diverse human mucosal epithelia, gonococci rely on a combinatorial strategy that involves the phase-variable expresssion of a large panel of adhesive functions, including type IV pili with the PilC adhesin, colony opacity-associated (Opa) proteins, PorB and specific lipooligosaccharides (Dehio et al., 2000; Merz and So, 2000). Molecular mechanisms of invasion are only partly understood but appear to vary with the complement of adhesins expressed and with the host cell receptors involved in the interaction. The Opa proteins comprise a family of antigenically diverse outer membrane proteins of Neisseria that function as adhesins and invasins (Dehio et al., 1998). Up to 11 unlinked chromosomal alleles encoding distinct Opa variants (Kupsch et al., 1993) are regulated independently by phase variation, resulting in a heterogeneous population of bacteria expressing none, one or several Opa variants (Stern et al., 1986). An important role for the Opa adhesins during infection is suggested by the observation that mostly Opa+ bacteria are recovered during natural infection and following inoculation of human volunteers with Opa− bacteria (Swanson et al., 1988; Jerse et al., 1994). Some Opa proteins (e.g. Opa30 of strain MS11) bind cell surface-associated heparan sulfate proteoglycans (HSPGs), but most Opa variants characterized to date interact with the family of human carcinoembryonic antigen-related cellular adhesion molecules (CEACAMs; for a review of Opa receptors see Dehio et al., 1998). The CEACAM family belongs to the immunoglobulin (Ig) superfamily of adhesion molecules (Öbrink, 1997). It comprises seven members, four of which are receptors for Opa proteins: CEA (carcinoembryonic antigen; CD66e), CEACAM1 (biliary glycoprotein; BGP; CD66a), CEACAM3 (CEA gene family member 1; CGM1; CD66d) and CEACAM6 (non-specific cross-reacting antigen; NCA; CD66c). All CEACAM molecules share a conserved N-terminal Ig variable (Igv)-like domain that is followed by 0–6 Ig constant (Igc)-like domains. The Opa-binding CEACAM receptors are characterized by a particularly conserved Igv-like domain that contains the CD66 epitopes and is engaged in a protein–protein interaction by the Opa proteins (Bos et al., 1999; Popp et al., 1999; Virji et al., 1999). CEACAM1 is the Opa receptor with the widest tissue distribution. It is expressed by endothelial and epithelial cells of a wide range of human tissues and also on leukocytes, including granulocytes, activated T cells, B cells and CD16−/CD56+ natural killer cells (Grunert et al., 1998). Differential splicing of CEACAM1 generates at least eight transmembrane isoforms with different numbers of extracellular domains and either a long or a truncated cytoplasmic domain. The long cytoplasmic domain associates with Src family tyrosine kinases (Brummer et al., 1995; Skubitz et al., 1995) and with the tyrosine phosphatases SHP-1 and SHP-2 (Huber et al., 1999), and contains a functional immune receptor tyrosine-based inhibitory motif (ITIM; Chen et al., 2001b). This domain is also responsible for the growth-inhibitory effects of CEACAM1 in epithelial carcinoma cells (Fournes et al., 2001) and localizes the adhesion molecule to cell–cell contacts between epithelial cells in a way that depends on association with the actin cytoskeleton and that is subject to regulation by Rho-family GTPases (Sadekova et al., 2000). CEA, a widely used tumour marker, and CEACAM6 can be co-expressed with CEACAM1 on epithelial cells, where they engage in homophilic and heterophilic interactions with CEACAM molecules of neighbouring cells (Öbrink, 1997). CEA and CEACAM6 are inserted into the membrane by a glycosylphosphatidylinositol (GPI) anchor. Granulocytes also express CEACAM1 and CEACAM6, together with CEACAM3 and CEACAM8, which are limited to this cell type. CEACAM3 has a cytoplasmic domain distinct from CEACAM1. It contains a functional immune receptor tyrosine-based activating motif (ITAM) that was shown recently to participate in gonococcal internalization when the recombinant receptor is expressed in a chicken B-cell line (Chen et al., 2001a). The specific cellular function of CEACAM3 is unknown, but all CEACAM family members of granulocytes have been implicated in cellular activation, resulting in degranulation, priming of oxidant production and increased β2 integrin-mediated adhesion (Skubitz et al., 1996; Stocks et al., 1996). Neisseria expressing CEACAM-binding Opa variants adhere to and invade human epithelial cell lines expressing recombinant or endogenous CEACAM molecules and primary endothelial cells expressing CEACAM1 (Virji et al., 1996; Chen et al., 1997; Gray-Owen et al., 1997b; Muenzner et al., 2000). In polarized T84 epithelial monolayers, CEA, CEACAM1 and CEACAM6 are transported apically, where they mediate invasion and subsequent transcytosis of Opa+ gonococci by an intracellular route (Wang et al., 1998). CEACAM-binding Opa variants are also responsible for efficient, opsonization-independent phagocytosis of Neisseria by human granulocytes (Chen and Gotschlich, 1996; Virji et al., 1996; Gray-Owen et al., 1997b). Studying an in vitro differentiated myelomonocytic cell line expressing CEACAM1 and CEACAM6, we found that phagocytosis of gonococci expressing the CEACAM-binding Opa52 requires activation of Src-family tyrosine kinases and the small GTPase Rac (Hauck et al., 1998). Whether similar mechanisms are involved in epithelial cell invasion is not known. In the present study, we use transfected epithelial cells for a comparative analysis of signalling through individual CEACAM receptors during neisserial invasion. This model circumvents complications arising from the expression of multiple, functionally diverse endogenous CEACAM family members and natural splice variants in many human tissues and cell lines. We provide evidence that ligation of CEACAM3 by gonococcal Opa proteins mediates bacterial internalization by a unique and highly efficient mechanism that is not induced by either CEACAM1 or CEACAM6. We show that upon receptor aggregation, tyrosine residues of the cytoplasmic ITAM motif of CEACAM3 are involved in triggering a localized reorganization of the host cell surface, leading to engulfment of adhering bacteria. We further identify key functions for the small GTP-binding proteins Rac1 and Cdc42 in regulating gonococcal internalization by this phagocytic mechanism and the underlying rearrangements of the cellular actin cytoskeleton. Our results suggest that the granulocyte-specific CEACAM3 receptor may have a phagocytic function in vivo. In contrast, epithelial cell invasion through CEACAM1 or CEACAM6 occurs by a less efficient mechanism that is insensitive to disruption of the F-actin cytoskeleton and does not require Rho-family GTPases. Results Internalization of Neisseria gonorrhoeae N313 (Opa57) by CEACAM3-expressing HeLa transfectants is a rapid process Previous studies consistently observed that Opa-mediated invasion of a HeLa cell line expressing CEACAM3 was more efficient than with other CEACAM receptors (Bos et al., 1997; Chen et al., 1997; Gray-Owen et al., 1997b), suggesting that different mechanisms of internalization may be involved. We first compared the time courses of neisserial invasion via CEACAM3 and CEACAM1. The gonococcal strain N313 expressing the recombinant CEACAM-binding Opa57 was used to infect HeLa cell lines stably transfected to express different CEACAM receptors. Bacterial adherence was determined in a plating assay and was found to be about twice as high with HeLa-CEACAM1 as compared with HeLa-CEACAM3 (Figure 1A). This difference reflects the different levels of CEACAM expression by the two cell lines, as shown by fluorescence-activated cell sorting (FACS) analysis using the cross-reactive monoclonal antibody D14HD11 (data not shown). An invasion assay, in which extacellular bacteria are killed selectively by treatment with gentamycin, showed that, in contrast to adherence, host cell invasion was increased in HeLa-CEACAM3 and occurred with markedly different kinetics as compared with HeLa-CEACAM1 (Figure 1B). In Figure 1C, we express numbers of viable intracellular bacteria as a percentage of total cell-associated bacteria to illustrate that the mechanism that mediates particularly efficient bacterial internalization via CEACAM3 may be largely limited to the first half hour after cell contact. Background adherence of N.gonorrhoeae N313 to a vector-transfected HeLa cell line was negligible and no invasion was observed (Gray-Owen et al., 1997a; data not shown). Figure 1.Different kinetics of internalization of N.gonorrhoeae N313 (Opa57) by HeLa cell lines expressing either CEACAM3 or CEACAM1. Cells were infected with a multiplicity of infection (m.o.i.) of 30 bacteria per cell. (A) Total cell-associated bacteria. (B) Gentamycin-resistant (i.e. intracellular, viable) bacteria. (C) Rate of internalization shown as the percentage of gentamycin-resistant bacteria calculated from data shown in (A) and (B). Data shown in (A) and (B) are mean results ± SD of triplicate samples for each time point. Data are representative of three independently performed experiments using either N313 (Opa57) or N309 (Opa52). Download figure Download PowerPoint N313 (Opa57) triggers marked cell surface rearrangements in CEACAM3-expressing HeLa cells that engulf adherent bacteria Having identified the first 30 min after infection as the critical time window for efficient uptake via CEACAM3, we were interested to study internalization at the ultrastructural level. Scanning electron microscopy (SEM) of HeLa-CEACAM3 cells infected for 30 min showed adhering diplococci in the centre of large, crown-like lamellipodial protrusions with long filopodial extensions emanating from the cell surface (Figure 2A and C). This marked reorganization of the cell surface was seen exclusively in association with individual gonococci or clusters of bacteria, suggesting that it was triggered by bacterial adherence. Transmission electron microscopy (TEM) of parallel cultures revealed corresponding structures extending up to 6 μm from the surface of the cells (Figure 2E). No such protrusions were triggered in HeLa lines expressing other CEACAM receptors. Instead, many bacteria adhering to HeLa-CEACAM1 or CEACAM6 were partially enclosed within modest pseudopods that enveloped bacteria without extending further from the cell (Figure 2B, D and F, and data not shown). TEM also confirmed that gonococci were capable of invading all HeLa cell lines in an Opa/CEACAM-dependent manner (data not shown) within 30 min after infection, albeit with different efficiencies depending on the CEACAM receptor expressed. Confocal laser scanning microscopy of phalloidin-stained infected cultures detected marked accumulations of F-actin around bacteria adhering to CEACAM3 (Figure 3) that corresponded well in shape and size to the lamellipodia-like cellular extensions seen by SEM. In many confocal sections, these F-actin-rich structures were highly reminiscent of 'phagocytic cups' engulfing adherent gonococci (arrow in Figure 3). These or similar structures were absent from vector-transfected control cells and from uninfected HeLa cells expressing CEACAM3 (data not shown). In contrast, infected HeLa cells expressing CEACAM1 or CEACAM6 showed only fine rings of F-actin that marked the circumference of cell-associated gonococci either partially or completely (Figure 3) and that were similar in frequency and size to the pseudopods seen by SEM. Figure 2.Neisseria gonorrhoeae N313 (Opa57) triggers different ultrastructural modifications of the cell surface in HeLa lines, depending on the expression of either CEACAM3 or CEACAM1. Subconfluent monolayers were infected with an m.o.i. of 100 for 30 min at 37°C. (A–D) SEMs of infected cells showing large cellular protrusions extending from the cell surface of HeLa-CEACAM3 (A and C) and tightly fitting pseudopods enclosing bacteria that adhere to HeLa-CEACAM1 (B and D). (D) shows a detail of (B). Interactions between N313 and a CEACAM6-expressing HeLa cell line were indistinguishable at the ultrastructural level from those with HeLa-CEACAM1 (not shown). (E and F) TEMs of infected HeLa-CEACAM3 (E) and HeLa-CEACAM1 (F). Images are representative of three independent experiments. Download figure Download PowerPoint Figure 3.Confocal sections showing adherent gonococci and the distinct F-actin structures they trigger in different epithelial cell lines. HeLa lines expressing recombinant CEACAM receptors were infected with N.gonorrhoeae N313 (Opa57), whereas transfected COS-7 cells expressing human FcγRIIa were incubated with immunoglobulin-opsonized N302 (Opa−). After 20 min, cells were fixed and processed for indirect immunofluorescence staining of gonococci (green). F-actin was visualized by staining with TRITC–phalloidin (red). F-actin structures induced by the interaction of Opa-expressing bacteria with HeLa-CEACAM3 were highly reminiscent of the phagocytic actin structures triggered by the FcγRIIa receptor. This similarity extended to the formation of distinct phagocytic cups (arrow). In contrast, most Opa-expressing gonococci adhering to CEACAM1- and CEACAM6-expressing cells were only associated with fine rings of F-actin (arrowheads). Images are representative of three or more independent experiments. Download figure Download PowerPoint In time course experiments, the association of adherent gonococci with phagocytic actin structures in the HeLa-CEACAM3 cell line correlated well with gonococcal internalization as measured by the gentamycin protection assay. They were noticeable after 10 min of infection, became most prominent between 20 and 30 min, when internalization of bacteria was most efficient (Figure 1C), and had disappeared completely by 60 min of infection (data not shown). In marked contrast, in HeLa lines expressing CEACAM1 or CEACAM6, adherent bacteria remained associated with fine rings of F-actin throughout the 6 h study period and no other actin rearrangements were observed even after prolonged infection. We next determined the role of the actin cytoskeleton in mediating internalization via the different CEACAM receptors. A specific inhibitor of F-actin polymerization, cytochalasin D, blocked internalization of N313 Opa57 by the HeLa-CEACAM3 cell line, with half-maximal inhibition by <0.1 μM (Figure 4A). In contrast, invasion of HeLa-CEACAM1 was not inhibited at high (5 μM) concentrations of cytochalasin D (Figure 4B). Comparable results were obtained with a different F-actin-disrupting agent, latrunculin A (data not shown). Figure 4.Cytochalasin D differentially inhibits CEACAM3-mediated internalization. Following a 30 min pre-incubation period with either cytochalasin D or carrier, HeLa cell lines were infected with N313 (Opa57). After 30 min, total cell-associated bacteria and gentamycin-protected bacteria were quantitated by dilution plating. Black bars show efficient inhibition of gonococcal internalization by HeLa-CEACAM3 (A; black bars) but not by HeLa-CEACAM1 (B), whereas gonococcal adherence (grey bars) to either cell line was not affected by the inhibitor. Download figure Download PowerPoint Phagocytosis of Neisseria via CEACAM3 resembles Fc receptor-mediated phagocytosis and requires tyrosine residues of the ITAM motif in the cytoplasmic receptor domain The ability of CEACAM3 to trigger phagocytic actin rearrangements prompted us to compare the mechanism by which it mediates neisserial internalization to the human single chain phagocytic Fc receptor, FcγRIIa. Recombinant expression of human FcγRIIa in COS epithelial cells was found to render these phagocytic, and the COS model has since been used to study different aspects of phagocytic receptor signalling (Indik et al., 1995; Downey et al., 1999). In preliminary experiments, phagocytosis of gonococci by transfected COS-7 cells expressing recombinant FcγRIIa was found to require opsonization of bacteria by specific IgG but no Opa expression. Significantly, phagocytic F-actin structures triggered by the interaction between IgG-opsonized N.gonorrhoeae N302 (Opa−) with COS-7 cells expressing human FcγRIIa were indistinguishable from those induced by the Opa–CEACAM3 interaction in the HeLa line or COS-7 cells expressing recombinant CEACAM3 (Figure 3 and data not shown). The phagocytic activity of FcγRIIa in the epithelial cell model and in phagocytes critically depends on cytoplasmic tyrosine residues that form part of an ITAM signalling motif (Daëron, 1997). The cytoplasmic receptor domain of CEACAM3 lacks significant amino acid sequence similarity to FcγRIIa. However, it does contain two tyrosine residues arranged in an YxxLx(7)YxxM motif that resembles an ITAM motif, except for a methionine residue instead of leucine or isoleucine in the last position. To examine the importance of this motif for the phagocytic activity of CEACAM3, we constructed receptor mutants in which either one or both tyrosine residues were replaced by phenylalanine (Figure 5A). We then produced polyclonal COS-7 cell lines expressing either CEACAM3, the tyrosine mutants of CEACAM3 or CEACAM3-1C1, a short natural splice variant of the receptor in which most of the cytoplasmic domain is replaced by an unrelated amino acid sequence and which therefore lacks the ITAM sequence entirely (Figure 5A). Similar levels of receptor expression by the different COS-7 populations were confirmed by FACS analysis (data not shown) and were reflected by similar levels of adherence of the Opa57-expressing gonococcal strain N313 (Figure 5B). Interactions between N313 and transfected COS-7 cells were Opa and CEACAM dependent since only low levels of background adherence and no internalization were observed with vector-transfected control cells and with CEACAM3 transfectants infected with the Opa-negative isogenic strain N302 (Figure 5B). In contrast, COS-7 cells expressing the different CEACAM3 constructs were all able to internalize adhering N313, but with marked differences in efficiency. The phagocytic activities of CEACAM3 receptor mutants and splice variants determined in three to five independent experiments are summarized in Table I. CEACAM3 had the highest phagocytic activity in all experiments. Single tyrosine replacements (Y230F or Y241F) each had a marked impact on phagocytic activity (Table I). The effect was even more pronounced in the Y230/241F double mutant. The most dramatic reduction of phagocytic activity was observed with the short natural splice variant CEACAM3-1C1, which failed almost completely to internalize adherent gonococci. Figure 5.Critical role of tyrosine residues in the cytoplasmic receptor domain of CEACAM3 for phagocytic uptake of Opa-expressing gonococci. (A) Cytoplasmic domain mutants and natural splice variants of CEACAM3. (B) Gentamycin protection assay to determine the phagocytic activity of CEACAM3 receptor mutants and splice variants. COS-7 cells expressing the receptor variants shown in (A) or vector transfected control cells were infected with N.gonorrhoeae N313 (Opa57) or N302 (Opa−) for 30 min before quantitation of cell-associated and gentamycin-protected bacteria. Download figure Download PowerPoint Table 1. Phagocytic activity of CEACAM3 splice variants and mutants expressed in COS-7 cells Receptor Phagocytic activitya nb CEACAM3 100.00 ± 0.00 5 CEACAM3-Y230F 55.90 ± 36.19 3 CEACAM3-Y241F 37.95 ± 15.28 3 CEACAM3-Y230/241F 25.41 ± 17.51 5 CEACAM3-1C1 4.96 ± 2.69 5 pCEP4 (vector) 1.15 ± 2.09 5 a Following infection of cell lines with N.gonorrhoeae N313 (Opa57) for 30 min, the ratio between gentamycin-resistant (i.e. intracellular) and total cell-associated gonococci was determined and expressed as a percentage of the COS-7 cells expressing CEACAM3. Values are means ± SD. b Number of independent experiments. The strongly reduced ability of the Y230/241F double mutant to internalize adherent gonococci correlated with the inability to induce actin rearrangements, while receptor recruitment to the site of gonococcal adherence was preserved (Figure 6A). Receptor aggregation on its own was sufficient to trigger phagocytic actin rearrangements since magnetic beads coated with a monoclonal antibody against the N-terminal domain of CEACAM3 were capable of inducing F-actin accumulation in HeLa-CEACAM3 but not in HeLa cells expressing the Y230/241F double mutant (Figure 6B). These data show that aggregation of CEACAM3 by adherent gonococci triggers bacterial phagocytosis through actin rearrangements by a mechanism that critically depends on tyrosine residues in the cytoplasmic receptor domain of the long splice variant of CEACAM3 and that is independent of other neisserial factors. Figure 6.The formation of phagocytic F-actin structures by CEACAM3 is triggered by receptor aggregation and critically depends on the tyrosine residues of the cytoplasmic receptor domain. (A) Confocal sections through COS-7 cells transiently transfected to express either CEACAM3 or the double mutant Y230/241F and infected with N313 (Opa57) for 20 min. Phase contrast shows cells and adhering bacteria. Immunofluorescence labelling of CEACAM receptors (green) reveals strong aggregation of both receptors at sites of bacterial adherence. Phagocytic F-actin structures as visualized by TRITC–phalloidin (red) are present in cells expressing wild-type CEACAM3 (arrows) but absent from cells expressing the Y230/241F double mutant (arrowheads). (B) Magnetic beads coated with the D14HD11 mouse monoclonal antibody against CEACAM receptors were incubated with HeLa cell lines for 20 min at 37°C at a ratio of five beads per cell. Receptor aggregation by anti-CEACAM beads is sufficient to induce phagocytic actin rearrangements in cells expressing CEACAM3 (arrows) but not in cells producing the Y230/241F double mutant. Beads were visualized in fixed and permeabilized samples by a dichlorotriazinylamino fluorescein (DTAF)-conjugated secondary antibody against the coating mouse monoclonal antibody. Download figure Download PowerPoint Phagocytic activity of CEACAM3 is mediated by the GTPases Rac1 and Cdc42 Members of the Rho family of small GTP-binding proteins are key regulators of cellular F-actin structures (Mackay and Hall, 1998). Receptor-mediated phagocytosis of opsonized particles invariably requires rearrangements of the actin cytoskeleton but, depending on the receptor involved, these may be regulated by different Rho-family GTPases. While phagocytosis of IgG-opsonized particles through FcγRIIa has been shown to require Rac and Cdc42, phagocytic uptake of complement-opsonized particles by the complement receptor 3 depends on Rho, both in mouse macrophages and in the COS epithelial cell model (Caron and Hall, 1998). We were interested to determine which Rho-family GTPases regulate CEACAM3-mediated internalization of Opa-expressing Neisseria. First we examined the sensitivity of neisserial internalization to toxin B from Clostridium difficile, which inhibits all members of the Rho subfamily of small GTPases by glucosylation (Lerm et al., 2000). Pre-incubation of HeLa-CEACAM3 with different concentrations of toxin B inhibited internalization of N.gonorrhoeae N313 (Opa57) in a dose-dependent manner (Figure 7A). Inhibition correlated with rounding of the cells that is indicative of effective inactivation of Rho-family GTPases. In contrast, invasion of the CEACAM1-expressing HeLa line was not affected by toxin B over the same range of concentrations. Next we examined the effect of the C3 exoenzyme of Clostridium botulinum, which specifically inactivates Rho by ADP-ribosylation (Lerm et al., 2000). Pre-incubation for 24 h with up to 40 μg/ml toxin did not inhibit bacterial entry into either cell line, suggesting that Rho activity was not required for internalization via CEACAM3 or CEACAM1 (data not shown). Figure 7.Different roles for Rho-family GTPases in neisserial internalization via different CEACAM receptors. (A) Gentamycin pr
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