Correspondence to Christof R. Hauck: christof.hauck{at}mail.uni-wuerzburg.de
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Introduction |
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Despite host cell exfoliation, N. gonorrhoeae is highly successful in establishing itself on epithelial surfaces of the human body. Gonococci express an array of adhesive factors that together allow for multiple, independent host cell interactions (Dehio et al., 2000). Although the exquisite host adaptation of N. gonorrhoeae has restricted in vivo experimentation, a limited number of volunteer studies and extensive in vitro experiments have suggested an orchestrated sequence of events upon infection. In particular, these investigations have pointed to type IV pili, which are filamentous surface appendages of microorganisms, to establish initial bacterial contact with the mucosa (Dehio et al., 2000). Furthermore, the expression of another group of outer membrane proteins, the so-called colony opacity-associated (Opa) proteins, appears to mediate intimate binding of bacteria to host cells. Although Opa protein expression is subject to phase variation, the opaque phenotype dominates in vivo because mainly Opa-expressing variants are reisolated from volunteers even when nonopaque gonococci are used for infection (Swanson et al., 1988; Jerse et al., 1994).
Work over the last decade has identified cellular receptors that are targeted by Opa proteins from N. gonorrhoeae and its close relative Neisseria meningitidis, which is a major cause of bacterial meningitis and is a common colonizer of nasopharyngeal mucosa (for review see Hauck and Meyer, 2003). Besides a few Opa proteins that recognize heparansulfate proteoglycans (OpaHSPG), most gonococcal and meningococcal Opa proteins that have been characterized so far recognize members of the human carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family (OpaCEA). OpaCEACEACAM association involves two of the four extracellular loops of outer membraneembedded Opa proteins and the unglycosylated face of the NH2-terminal Ig variable-like domain of CEACAM1, CEACAM3, CEA (the product of the CEACAM5 gene), or CEACAM6 (Bos et al., 1999, 2002; Virji et al., 1999).
Epithelial cells express the transmembrane receptor CEACAM1 as well as glycosylphosphatidylinositol-linked CEA and CEACAM6, which usually localize to the apical membrane domain (Hammarstrom, 1999). The in vivo function of CEACAMs on epithelia is not clear, though some family members can mediate cellcell adhesion in vitro (Benchimol et al., 1989). It is important to note that only a few CEACAM homologues are present in rodents, whereas the CEACAM family has expanded considerably during primate evolution and is still diversifying (Hammarstrom and Baranov, 2001; Zhou et al., 2001). As these glycoproteins are recognized by a variety of gram-negative bacteria that are found in association with human mucosa (Leusch et al., 1991; Sauter et al., 1993; Virji et al., 1996, 2000; Toleman et al., 2001; Hill and Virji, 2003), it has been speculated that CEACAMs regulate bacterial colonization; however, direct evidence for this hypothesis is lacking (Hammarstrom and Baranov, 2001).
In this study, we demonstrate that CEACAM engagement by bacterial pathogens triggers enhanced host cell adhesion to the ECM, thereby abrogating cell detachment, which is an essential component of the exfoliation response. Enhanced cell adhesion depends on de novo expression of CD105, a TGF-ß1 receptor. Moreover, bacteria-triggered CD105 surface expression is necessary and sufficient to prevent detachment of infected cells by modulating integrin affinity. These results not only provide, for the first time, molecular insight into microbial strategies that counteract epithelial exfoliation but, in addition, help to explain the prevalence of CEACAM recognition amongst human pathogens.
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Results |
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CD105 requires the presence of ß1 integrins to enhance cell adhesion
Because the enhanced cell adhesion that was triggered by CD105 was most pronounced on surfaces coated with ligands for ß1 integrins, we wondered whether this process involved integrins. Cell adhesion assays that were performed in the presence of monoclonal antibodies directed against either CD105, integrin ß1, or the hyaluronate receptor CD44 demonstrated that interference with both CD105 or integrin ß1 abolished enhanced cell adhesion after bacterial infection or CD105 expression (Fig. 7 A). In contrast, anti-CD44 antibodies or control mouse IgG had no influence on cell adhesion to collagen (Fig. 7 A). Importantly, CD105 expression did not lead to enhanced cell adhesion in cells that were genetically deficient for integrin ß1, whereas reexpression of human integrin ß1 restored the ability of these cells to display enhanced cell adhesion upon CD105 expression, demonstrating that integrin ß1 is essential for this process (Fig. 7 B). Flow cytometry revealed that the levels of integrin ß1 are not modulated by the presence of CD105 (Fig. 7 C). Furthermore, CD105 expression did not alter the production of ECM proteins, such as collagen or fibronectin, by 293T cells (Fig. 7 D), suggesting that CD105 exerts its effect by altering the ligand-binding properties of integrin ß1.
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Discussion |
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Microorganisms have elaborated an array of adhesive factors, allowing them to firmly attach to host surfaces (Finlay and Falkow, 1997; Abraham et al., 1998). However, stratified epithelial tissues can rapidly respond to bacterial attachment with accelerated renewal, including detachment of superficial cell layers as well as differentiation of basal and intermediate cells (Mulvey et al., 2000; Mysorekar et al., 2002). Infection of the mouse bladder with uropathogenic Escherichia coli has demonstrated that within 6 h after bacterial inoculation, detachment of bacteria-laden superficial epithelial cells can be detected in vivo (Mulvey et al., 1998). In the same time frame, urothelial gene expression is altered to reflect the microbe-induced renewal of the epithelial strata (Mysorekar et al., 2002). It is interesting to note that CD105 up-regulation and increased adhesion of epithelial cells after CEACAM stimulation can be observed within 38 h upon infection. This is consistent with the idea that the impaired detachment of epithelial cells colonized via CEACAMs could operate to effectively counteract the exfoliation response.
CD105 is a nonsignaling member of the TGF-ß1 receptor family (type III receptor) that is known to modulate cellular responses to this growth factor (Lebrin et al., 2004). Although the role of CD105 in TGF-ß1 signaling is far from understood (Fonsatti et al., 2001), CD105 has an essential function in establishing a well-differentiated vasculature (Li et al., 1999; Sorensen et al., 2003). Accordingly, mutations in CD105 are found in hereditary hemorrhagic telangiectasia type 1, which is a human disease characterized by vascular malformations (McAllister et al., 1994). In addition, an antiapoptotic function has been reported for CD105 in endothelial cells, where CD105 is induced upon hypoxia (Li et al., 2003; Zhu et al., 2003). However, we did not observe differences in apoptosis upon infection of CEACAM1-expressing cells with OpaCEA-expressing gonococci compared with cells that were infected with nonopaque bacteria (unpublished data). Clearly, de novo CD105 expression in human epithelial cells has a positive influence on cell adhesion to the ECM in the absence of bacterial infection, further suggesting that the ability of CD105 to counteract the detachment of infected cells is not caused by an antiapoptotic effect.
Interestingly, the adhesion-promoting activity of CD105 mapped to the last 35 residues of the 47amino acid-long cytoplasmic domain. These results are in accordance with recent investigations that have revealed a novel function for CD105 in modulating the actin cytoskeleton and cell migration (Liu et al., 2002; Conley et al., 2004; Sanz-Rodriguez et al., 2004). In particular, two recent studies have demonstrated that the cytoplasmic tail of CD105 interacts with the LIM (Lin-11, Isl-1, and Mec-3) domains of zyxin and ZRP-1 (zyxin-related protein 1), respectively, and the presence of CD105 redistributes zyxin and ZRP-1 from their regular localization at focal adhesion sites (Conley et al., 2004; Sanz-Rodriguez et al., 2004). Zyxin and ZRP-1 bind to members of the p130CAS family of adaptor molecules that, together with their binding partners FAK, c-Src, and c-Crk, play important roles in cell motility and are involved in the turnover of focal adhesion structures (Honda et al., 1998; Klemke et al., 1998; O'Neill et al., 2000; Yi et al., 2002). Importantly, CD105 expression not only disturbs the focal adhesion association of zyxin and ZRP-1 but also disturbs the subcellular distribution of p130CAS and c-Crk (Conley et al., 2004). These molecular interactions might allow CD105 to influence focal adhesion composition and, thereby, impact cell adhesion.
It is important to note that CD105-triggered adhesion was most pronounced on substrates encompassing integrin ß1 ligands such as collagen type I or IV and laminin (the major constituents of matrigel; Hynes, 2002). Whereas CD105 expression had no influence on the expression of matrix proteins or on the surface expression levels of integrin ß1, enhanced adhesion could be blocked by antibodies directed against this integrin. In addition, cells that were genetically deficient for integrin ß1 showed no increase in cell adhesion upon CD105 expression. The affinity of integrins for their substrate can be altered by signals from within the cell (integrin activation) that modulate the association of focal adhesion proteins such as talin with integrin ß1 cytoplasmic domains (Tadokoro et al., 2003). As CD105 expression did not affect the total amount of integrin ß1 but affected the ligand-binding capabilities of integrins, we suggest that CD105 positively influences integrin activity by modulating the protein composition of integrin-dependent cell attachment sites, thereby influencing integrin activity by inside-out signaling.
The intriguing molecular cross talk that is revealed in this study sheds light onto an unexplored process in the establishment of infectious disease; namely, how bacterial pathogens overcome the exfoliation response of epithelial cells. A surprising, yet simple, solution to this challenge might be the pathogen-triggered modulation of host cell adhesion, as examplified in this study in the case of diverse CEACAM-binding human-specific microorganisms. In vivo, bacteria-triggered up-regulation of CD105 via CEACAM engagement and the resulting suppression of epithelial cell detachment could provide CEACAM-binding microorganisms with a selective advantage during initial colonization of the human mucosa. It is interesting to speculate that such an advantage could be the major driving force behind the convergent evolution of distinct CEACAM-binding adhesins in several gram-negative species of the genera Neisseria, Haemophilus, and Moraxella (Hill et al., 2001; Hauck and Meyer, 2003; Hill and Virji, 2003). Aside from bacterial colonization, the functional link between CEACAMs, CD105, and integrin activation could also play important roles in physiological settings such as angiogenesis (Ergun et al., 2000; Lebrin et al., 2004) and warrants further investigation.
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Materials and methods |
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Bacteria and infection
The following strains were provided by T. Meyer (Max-Planck Institut für Infektionsbiologie, Berlin, Germany): Opa52-expressing (OpaCEA) nonpiliated N. gonorrhoeae MS11-B2.1 (strain N309); Opa50-expressing (OpaHSPG) nonpiliated N. gonorrhoeae MS11-B2.1 (strain N303); nonopaque, piliated gonococci (strain N280); nonpiliated, nonopaque gonococci (strain N302); and commensal N. cinerea. Opa-expressing nonencapsulated N. meningitidis (SiaD mutant of strain MC58) was obtained from M. Frosch (Institut für Hygiene und Mikrobiologie, Universität Würzburg, Germany). M. catarrhalis strain 11994 was obtained from DSMZ. M. catarrhalis and all Neisseriae species were grown on GC agar plates (Difco BRL) supplemented with vitamins at 5% CO2 and 37°C and were subcultured daily. The unencapsulated variant of H. influenzae strain RD was obtained from A. Reidl (Zentrum für Infektionsforschung, Universität Würzburg, Germany). H. influenzae was grown on brainheart infusion agar at 5% CO2 37°C. For infection, bacteria were suspended in DME, the OD of the suspension was used to estimate the number of microorganisms, and bacteria were added to cells at the indicated multiplicity of infection (MOI).
Recombinant DNA constructs
Mammalian expression plasmids encoding cDNAs of human CEACAM1-4L (CEACAM1) and CEACAM6 were provided by W. Zimmermann (Universitätsklinikum Grosshadern, München, Germany). CEACAM1 CT was constructed by PCR amplification with primers CEACAM1 sense (5'-GGGAAGCTTGCCATGGGGCACCTCTCAGCCCCACTTCAC-3') and CEA1-HA-
CT antisense (5'-GGGGACGTCATAGGGATATTTCCCGAAATGCAGAAAACATGCCAGGGC-3') and were cloned into the HindIII-AatIIdigested plasmid pBluescript CEACAM3-HA (Schmitter et al., 2004) before further subcloning via HindIII-NotI into pcDNA3.1 Hygro (Invitrogen). Plasmid pDS red encoding RFP was from BD Biosciences. A cDNA clone encoding human CD105 (endoglin) was obtained from Deutsches Ressourcenzentrum für Genomforschung GmbH (RZPD; clone ID IMAGp958J07537QZ). Amplification of full-length CD105 was performed by using the primer pair EndoIF sense (5'-GAAGTTATCAGTCGATACCATGGACCGCGGCACGCTCCCTCTGGC-3') and EndoIF antisense (5'-ATGGTCTAGAAAGCTTCCTGCCATGCTGCTGGTGGAGCAGGGGGTGC-3'). CD105 variants with deletions in the cytoplasmic domain were generated by amplification from the same template using the primer EndoIF sense combined with either EndoIF
CT antisense (5'-ATGGTCTAGAAAGCTTCCCCAGAGTGCAGCAGTGAGCAGGGC-3') or EndoIF
35 antisense (5'-ATGGTCTAGAAAGCTTTCCCGCTTGCTGGGGGAACGCGTGTGCG-3') to result in CD105
CT or CD105
35, respectively. The resulting PCR fragments were cloned into pDNR-Dual using the In-Fusion PCR Cloning Kit (BD Biosciences) and were transferred by Cre-mediated recombination into pLPS-3' EGFP (BD Biosciences), resulting in GFP fused to the COOH terminus of the expressed proteins.
EM
ME-180 cells were seeded at 2.5 x 105 cells/well in 24-well plates on acid-washed glass coverslips that were coated with 25 µg/ml collagen. The next day, medium was replaced with DME and 0.5% CS for 8 h. Then, cells were infected for 14 h at an MOI of 20 or were left uninfected. Samples were fixed in situ with 2% glutaraldehyde/3% formaldehyde in 0.1 M cacodylate, 0.09 M sucrose, 0.01 M CaCl2, and 0.01 M MgCl2, pH 6.9, for at least 1 h at 4°C. The samples were washed with 20 mM Tris and 1 mM EDTA, pH 7.0, and were dehydrated in a graded series of aceton on ice. After critical point drying from liquid CO2, samples were sputter coated with 10-nm gold and examined at 5 kV of accelerating voltage in a field emission scanning electron microscope (model DSM982 Gemini; Carl Zeiss MicroImaging, Inc.) using Everhardt Thornley and inlense secondary electron detectors (Carl Zeiss MicroImaging, Inc.) in a 1:1 ratio. Images were digitally recorded and processed in Adobe Photoshop 6.
Detachment assay
ME-180 cells were seeded at 2 x 104 cells/well onto collagen-coated (16 h at 4°C) wells of 96-well plates. The next day, growth medium was replaced with DME containing 0.5% CS, and after 8 h, the confluent monolayers were infected or left uninfected for 14 h with the indicated bacteria at an MOI of 20. In some cases, aliquots of sample that were infected for 14 h were used for the determination of viable bacteria by dilution plating onto GC agar. After infection, plates were either fixed in situ or inverted and centrifuged for 5 min at 2,500 rpm to remove detached cells and medium before fixation with 4% PFA (20 min at RT). Samples were stained with 0.1% crystal violet in 0.1 M borate, pH 9.0 (CV stain). The staining intensity was measured in a microplate reader (Bio-Rad Laboratories) at 570 nm. The percentage of detached cells was determined by comparing samples fixed before and after centrifugation. 293T cells were seeded at 105 cells/well in a 24-well plate coated with 25 µg/ml collagen type I (in PBS for 16 h at 4°C) and infected with an MOI of 20 for 14 h. Cells were washed with PBS, fixed, stained with CV stain, and photographed.
Cell adhesion assay
The wells of 96-well plates were coated with 100 µl PBS containing the indicated concentrations of collagen (collagen type 1 from calf skin [ICN Biomedicals], gelatine [Merck], matrigel [BD Biosciences], and bovine fibronectin [ICN Biomedicals]) or 0.2% BSA, respectively, for 24 h at 4°C. Serum-starved cells were infected or left uninfected with the indicated bacteria at an MOI of 30 for 8 h in the absence or presence of cycloheximide or staurosporine (Calbiochem). Then, the cells were detached by limited trypsin/EDTA digestion that was stopped by the addition of 0.5 mg/ml of soybean trypsin inhibitor in DME. Detached cells were kept in suspension medium (DME and 0.2% BSA) for 1 h at 37°C and were replated at 2 x 104 cells/well onto protein-coated wells in replicates of five. Where indicated, 1 µg/ml mAbs against CD105 (clone P4A4; Developmental Studies Hybridoma Bank [DSHB], under the auspices of the National Institutes of Child Health and Human Development), integrin ß1 (clone P5D2; DSHB), CD44 (clone H4C4; DSHB), or mouse IgG were present during replating. After 90 min at 37°C, nonadherent cells were removed by washing with PBS, adherent cells were fixed and stained with CV stain, and the staining intensity was measured.
Microarray analysis
Total RNA was isolated from pcDNA, CEACAM1, or CEACAM6-transfected 293T cells 90 min after infection with OpaCEA-expressing N. gonorrhoeae or from the corresponding uninfected cells using the RNeasy kit (QIAGEN). 15 µg RNA was reverse transcribed into cDNA in the presence of fluorescently labeled nucleotides (Cy3 or Cy5) using the CyScribe First Strand cDNA kit (GE Healthcare). For each sample, reverse transcription and labeling was performed once in the presence of Cy3- and Cy5-labeled nucleotides. Labeled cDNA populations from an infected sample and the corresponding uninfected sample were hybridized in both dye configurations to custom-made cDNA microarrays using a Slide Pro station (Lucidea; GE Healthcare). The arrays contained PCR products with a mean size of 1,000 bp, which was derived from 4,500 mostly cancer-associated genes (generated from cDNA templates that were obtained from RZPD). A scheme of the different hybridizations is outlined in Fig. S2. After extensive washing, fluorescence of the Cy3- as well as the Cy5-labeled cDNA population that was hybridized to the slide was recorded with a ScanArray (model 4000; PerkinElmer), and 16-bit images for each fluorescence channel were generated. The resulting raw data were transformed into spot intensities by ScanAlyze 2 software (obtained from M. Eisen, University of California, Berkeley, Berkeley, CA). The data obtained for each hybridization experiment were normalized by using the whole set of measurements, and genes that were up-regulated >1.8-fold independently of the dye configuration in two repetitions of the experiment were regarded as induced by bacterial infection.
RT-PCR analysis
Total RNA was isolated from the indicated cells at different time points after infection by using the RNeasy kit and were further treated with RNase-free DNase-I (QIAGEN). 2 µg RNA were reverse transcribed into cDNA using SuperscriptIIRT and oligonucleotide (desoxythymidine) primers (Invitrogen). RNA that was used directly for PCR amplification did not yield any PCR product, indicating that signals were not caused by chromosomal DNA contamination. The amplification of a 640-bp fragment of human CD105 was performed using primers 5'-CGGTGGTAGGCTGCAGACCTCACC-3' and 5'-CCTATGGACTTCCTGGTCTTGAGACC-3' with 30 cycles of amplification and an annealing temperature of 58°C. Simultaneously, a 350-bp fragment of ß-actin (primers ß-actin sense, 5'-AGCGGGAAATCGTGCGTG-3'; and ß-actin antisense, 5'-GGGTACATGGTGGTGCCG-3') was coamplified and served as an internal control.
Flow cytometry and Western blotting
Analyses were performed as described previously (Schmitter et al., 2004) by using mAbs against CEACAM (clone D14HD11; Genovac), CD105 (clone P4A4; DSHB), fibronectin or ß-actin (clone F 3648 or AC-74; Sigma-Aldrich), and GFP (clone JL-8; BD Biosciences), pAbs against collagen IV (H-243; Santa Cruz Biotechnology, Inc.), and rabbit pAbs against N. gonorrhoeae (gift of T.F. Meyer, Max Planck Institut für Infektionsbiologie). Secondary antibodies were obtained from Jackson ImmunoResearch Laboratories.
Covalent coupling of proteins to microspheres and microsphere-binding assay
1.2 x 108 polybead carboxylate microspheres (1 µm in diameter; Polysciences) were sonicated and covalently coated with either 1 mg/ml collagen type 1, 100 µg/ml mAb integrin ß1 (P5D2; DSHB), or 4 mg/ml BSA for 2 h in coupling buffer (0.2 M NaHCO3 and 0.5 M NaCl, pH 8.6). Unreacted binding sites were saturated with 20 mg/ml BSA in coupling buffer for 1 h before extensive washing with PBS. To determine bead attachment to cells, transfected 293T cells were seeded at 2.5 x 105 cells/well in 24-well plates on acid-washed glass coverslips and were incubated with 2.5 x 107 coated microspheres. After 2 h at 37°C, nonadherent beads were removed by washing with PBS, and the cells were fixed and analyzed by microscopy to determine the number of beads that bound to transfected cells.
Online supplemental material
Fig. S1 shows that bacteria-triggered cell adhesion is blocked by the kinase inhibitor staurosporine. Fig. S2 presents the general layout of microarray analysis and provides original data of hybridized microarray slides. Fig. S3 provides evidence that OpaHS-expressing gonococci are unable to induce CD105 up-regulation, as analyzed by flow cytometry. Fig. S4 demonstrates the blockage of bacteria-triggered CD105 up-regulation by CD105-directed antisense oligonucleotides, as analyzed by flow cytometry. Fig. S5 shows that CD105 promotes cell adhesion to different ECM proteins and presents the expression of CD105 variants with COOH-terminal deletions in transfected 293T cells. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200412151/DC1.
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Acknowledgments |
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This study was supported by funds from the Bundesministerium für Bildung und Forschung and Deutschen Forschungsgemeinschaft (grant Ha2568/3-1) to C.R. Hauck.
Submitted: 23 December 2004
Accepted: 20 July 2005
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