Calprotectin S100A9 Calcium-binding Loops I and II Are Essential for Keratinocyte Resistance to Bacterial Invasion
2009; Elsevier BV; Volume: 284; Issue: 11 Linguagem: Inglês
10.1074/jbc.m806605200
ISSN1083-351X
AutoresChantrakorn Champaiboon, Kaia J. Sappington, Brian Guenther, Karen F. Ross, Mark C. Herzberg,
Tópico(s)Heat shock proteins research
ResumoEpithelial cells expressing calprotectin, a heterodimer of S100A8 and S100A9 proteins, are more resistant to bacterial invasion. To determine structural motifs that affect resistance to bacterial invasion, mutations were constructed in S100A9 targeting the calcium-binding loops I and II (E36Q, E78Q, E36Q,E78Q) and the C terminus (S100A91–99 and S100A91–112), which contains putative antimicrobial zinc-binding and phosphorylation sites. The S100A8 and mutated S100A9 encoding plasmids were transfected into calprotectin-negative KB carcinoma cells. All transfected cells (except KB-sham) expressed 27E10-reactive heterodimers. In bacterial invasion assays with Listeria monocytogenes and Salmonella enterica serovar Typhimurium (Salmonella typhimurium), cell lines expressing S100A8 in complex with S100A9E36Q, S100A9E78Q, S100A91–99, or S100A91–112 mutants or the S100A91–114 (full-length) calprotectin resisted bacterial invasion better than KB-sham. When compared with KB-S100A8/A91–114, cells expressing truncated S100A91–99 or S100A91–112 with S100A8 also showed increased resistance to bacterial invasion. In contrast, glutamic acid residues 36 and 78 in calcium-binding loops I and II promote resistance in epithelial cells, because cells expressing S100A9E36Q,E78Q with S100A8 were unable to resist bacterial invasion. Mutations in S100A9 E36Q, E78Q were predicted to cause loss of the calcium-induced positive face in calprotectin, reducing interactions with microtubules and appearing to be crucial for keratinocyte resistance to bacterial invasion. Epithelial cells expressing calprotectin, a heterodimer of S100A8 and S100A9 proteins, are more resistant to bacterial invasion. To determine structural motifs that affect resistance to bacterial invasion, mutations were constructed in S100A9 targeting the calcium-binding loops I and II (E36Q, E78Q, E36Q,E78Q) and the C terminus (S100A91–99 and S100A91–112), which contains putative antimicrobial zinc-binding and phosphorylation sites. The S100A8 and mutated S100A9 encoding plasmids were transfected into calprotectin-negative KB carcinoma cells. All transfected cells (except KB-sham) expressed 27E10-reactive heterodimers. In bacterial invasion assays with Listeria monocytogenes and Salmonella enterica serovar Typhimurium (Salmonella typhimurium), cell lines expressing S100A8 in complex with S100A9E36Q, S100A9E78Q, S100A91–99, or S100A91–112 mutants or the S100A91–114 (full-length) calprotectin resisted bacterial invasion better than KB-sham. When compared with KB-S100A8/A91–114, cells expressing truncated S100A91–99 or S100A91–112 with S100A8 also showed increased resistance to bacterial invasion. In contrast, glutamic acid residues 36 and 78 in calcium-binding loops I and II promote resistance in epithelial cells, because cells expressing S100A9E36Q,E78Q with S100A8 were unable to resist bacterial invasion. Mutations in S100A9 E36Q, E78Q were predicted to cause loss of the calcium-induced positive face in calprotectin, reducing interactions with microtubules and appearing to be crucial for keratinocyte resistance to bacterial invasion. Mucosal keratinocytes continuously confront endogenous and exogenous invading microorganisms. Consequently the superficial keratinocytes of the oral mucosa contain a variety of indigenous bacteria (1Rudney J.D. Chen R. Arch. Oral. Biol. 2006; 51: 291-298Crossref PubMed Scopus (39) Google Scholar). Yet the keratinocytes appear to resist large scale invasion and intracellular infection. Expressed in the cytoplasm of squamous mucosal keratinocytes, calprotectin (S100A8 and S100A9, MRP8 and MRP14, calgranulin A and B, L1, cystic fibrosis antigen, and 27E10 antigen) is a heterodimeric complex of polypeptides of 10.8 and 13.2 kDa, respectively (2Ross K.F. Herzberg M.C. Infect. Immun. 2001; 69: 3248-3254Crossref PubMed Scopus (74) Google Scholar, 3Striz I. Trebichavsky I. Physiol. Res. 2004; 53: 245-253Crossref PubMed Google Scholar, 4Korndorfer I.P. Brueckner F. Skerra A. J. Mol. Biol. 2007; 370: 887-898Crossref PubMed Scopus (213) Google Scholar). These two subunits are members of the S100 protein family, which are involved in cell cycle progression, cell differentiation, and cytoskeleton-membrane interaction (5Kligman D. Hilt D.C. Trends Biochem. Sci. 1988; 13: 437-443Abstract Full Text PDF PubMed Scopus (480) Google Scholar, 6Marenholz I. Heizmann C.W. Fritz G. Biochem. Biophys. Res. Commun. 2004; 322: 1111-1122Crossref PubMed Scopus (685) Google Scholar, 7Zimmer D.B. Wright Sadosky P. Weber D.J. Microsc. Res. Tech. 2003; 60: 552-559Crossref PubMed Scopus (128) Google Scholar). Calprotectin is the most abundant protein found in the cytoplasm of neutrophils (8Dale I. Fagerhol M.K. Naesgaard I. Eur. J. Biochem. 1983; 134: 1-6Crossref PubMed Scopus (195) Google Scholar, 9Hessian P.A. Edgeworth J. Hogg N. J. Leukocyte Biol. 1993; 53: 197-204Crossref PubMed Scopus (353) Google Scholar) and is also found in monocytes (10Dale I. Brandtzaeg P. Fagerhol M.K. Scott H. Am. J. Clin. Pathol. 1985; 84: 24-34Crossref PubMed Scopus (154) Google Scholar), macrophages (11Odink K. Cerletti N. Bruggen J. Clerc R.G. Tarcsay L. Zwadlo G. Gerhards G. Schlegel R. Sorg C. Nature. 1987; 330: 80-82Crossref PubMed Scopus (561) Google Scholar), and human gingival keratinocytes (2Ross K.F. Herzberg M.C. Infect. Immun. 2001; 69: 3248-3254Crossref PubMed Scopus (74) Google Scholar). Elevated levels of calprotectin have been observed in body fluids such as plasma, saliva, gingival crevicular fluid, stools, and synovial fluid during infections and inflammatory conditions (12Johne B. Fagerhol M.K. Lyberg T. Prydz H. Brandtzaeg P. Naess-Andresen C.F. Dale I. Mol. Pathol. 1997; 50: 113-123Crossref PubMed Scopus (301) Google Scholar). Consequently, calprotectin is broadly used as a marker for inflammatory bowel diseases (13Roseth A.G. Schmidt P.N. Fagerhol M.K. Scand. J. Gastroenterol. 1999; 34: 50-54Crossref PubMed Scopus (367) Google Scholar), reactive arthritis (14Hammer H.B. Kvien T.K. Glennas A. Melby K. Clin. Exp. Rheumatol. 1995; 13: 59-64PubMed Google Scholar), and Sjogren syndrome (15Cuida M. Halse A.K. Johannessen A.C. Tynning T. Jonsson R. Eur. J. Oral. Sci. 1997; 105: 228-233Crossref PubMed Scopus (41) Google Scholar). Functioning as an antimicrobial protein (complex), calprotectin shows broad spectrum activities against microorganisms, including Capnocytophaga sputigena (16Miyasaki K.T. Bodeau A.L. Murthy A.R. Lehrer R.I. J. Dent. Res. 1993; 72: 517-523Crossref PubMed Scopus (62) Google Scholar), Candida albicans (17Murthy A.R. Lehrer R.I. Harwig S.S. Miyasaki K.T. J. Immunol. 1993; 151: 6291-6301PubMed Google Scholar), Escherichia coli, Staphylococcus aureus, Staphylococcus epidermis (18Sohnle P.G. Collins-Lech C. Wiessner J.H. J. Infect. Dis. 1991; 163: 187-192Crossref PubMed Scopus (120) Google Scholar), and Borrrelia burgdorferi (19Lusitani D. Malawista S.E. Montgomery R.R. Infect. Immun. 2003; 71: 4711-4716Crossref PubMed Scopus (80) Google Scholar). Calprotectin also inhibits bacterial invasion of epithelial cells by Listeria monocytogenes, S. typhimurium, and Porphyromonas gingivalis (20Nisapakultorn K. Ross K.F. Herzberg M.C. Infect. Immun. 2001; 69: 4242-4247Crossref PubMed Scopus (115) Google Scholar, 21Nisapakultorn K. Ross K.F. Herzberg M.C. Infect. Immun. 2001; 69: 3692-3696Crossref PubMed Scopus (71) Google Scholar). By promoting resistance to bacterial invasion, calprotectin-expressing cells, including squamous oral keratinocytes, are likely to contribute to mucosal innate immunity. We have been studying the structural basis of calprotectin-mediated, cell-associated antimicrobial resistance. Unlike S100A8 and other members of the S100 family, S100A9 has a extended C-terminal region, which has an amino acid sequence (residues 89–108) that is identical to the N-terminal region of neutrophil immobilizing factor (22Edgeworth J. Freemont P. Hogg N. Nature. 1989; 342: 189-192Crossref PubMed Scopus (87) Google Scholar, 23Watt K.W. Brightman I.L. Goetzl E.J. Immunology. 1983; 48: 79-86PubMed Google Scholar) and homologous to domain 5 of high molecular weight kininogen (24Hessian P.A. Fisher L. Eur. J. Biochem. 2001; 268: 353-363Crossref PubMed Scopus (38) Google Scholar). Domain 5 of high molecular weight kininogen has antimicrobial activity against E. coli, Pseudomonas aeruginosa, and Enterococcus faecalis (25Nordahl E.A. Rydengard V. Morgelin M. Schmidtchen A. J. Biol. Chem. 2005; 280: 34832-34839Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). In addition, S100A9 C-terminal residues 103–105 form a polyhistidine motif (HHH), which may be involved in zinc binding (26Sohnle P.G. Collins-Lech C. Wiessner J.H. J. Infect. Dis. 1991; 164: 137-142Crossref PubMed Scopus (123) Google Scholar, 27Loomans H.J. Hahn B.L. Li Q.Q. Phadnis S.H. Sohnle P.G. J. Infect. Dis. 1998; 177: 812-814Crossref PubMed Scopus (96) Google Scholar). Also suggested to be zinc-binding domains, the HXXXH motifs in S100A8 and S100A9 are commonly found in S100 proteins (4Korndorfer I.P. Brueckner F. Skerra A. J. Mol. Biol. 2007; 370: 887-898Crossref PubMed Scopus (213) Google Scholar, 27Loomans H.J. Hahn B.L. Li Q.Q. Phadnis S.H. Sohnle P.G. J. Infect. Dis. 1998; 177: 812-814Crossref PubMed Scopus (96) Google Scholar, 28Clohessy P.A. Golden B.E. J. Leukocyte Biol. 1996; 60: 674Crossref PubMed Scopus (19) Google Scholar). Because zinc is required for bacterial growth, either the polyhistidine or HXXXH motifs have been suggested to bind and sequester zinc from microorganisms and inhibit bacterial growth (4Korndorfer I.P. Brueckner F. Skerra A. J. Mol. Biol. 2007; 370: 887-898Crossref PubMed Scopus (213) Google Scholar, 27Loomans H.J. Hahn B.L. Li Q.Q. Phadnis S.H. Sohnle P.G. J. Infect. Dis. 1998; 177: 812-814Crossref PubMed Scopus (96) Google Scholar, 28Clohessy P.A. Golden B.E. J. Leukocyte Biol. 1996; 60: 674Crossref PubMed Scopus (19) Google Scholar, 29Sohnle P.G. Hahn B.L. Antimicrob. Agents Chemother. 2000; 44: 139-142Crossref PubMed Scopus (13) Google Scholar). In addition to zinc, calprotectin chelates other metal ions, including Mn2+, which inhibits growth of S. aureus in tissue abscesses (30Corbin B.D. Seeley E.H. Raab A. Feldmann J. Miller M.R. Torres V.J. Anderson K.L. Dattilo B.M. Dunman P.M. Gerads R. Caprioli R.M. Nacken W. Chazin W.J. Skaar E.P. Science. 2008; 319: 962-965Crossref PubMed Scopus (671) Google Scholar). Independent of direct antimicrobial activity, epithelial resistance to invasion may also reflect the ability of bacteria to bind and internalize. Bacterial binding and internalization could be regulated by calprotectin as an interacting partner with the cytoskeleton, although distinguishing from antimicrobial activity may not always be clear. For example, S100A8/A9 translocates across the plasma membrane and is released from the cell in a tubulin-dependent manner (31Rammes A. Roth J. Goebeler M. Klempt M. Hartmann M. Sorg C. J. Biol. Chem. 1997; 272: 9496-9502Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar). Release from the cell is controlled by the penultimate threonine (Thr-113) residue in the C terminus of S100A9, a substrate for protein kinase C (31Rammes A. Roth J. Goebeler M. Klempt M. Hartmann M. Sorg C. J. Biol. Chem. 1997; 272: 9496-9502Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar). Although tubulin-dependent interactions may bring calprotectin in proximity to surface bacteria, these interactions could regulate cytoskeleton-dependent internalization (32Steele-Mortimer O. Curr. Opin. Microbiol. 2008; 11: 38-45Crossref PubMed Scopus (210) Google Scholar). In epithelial cells, calprotectin exists primarily as a heterodimeric complex of S100A8 and S100A9 and the individual subunits are not readily found (2Ross K.F. Herzberg M.C. Infect. Immun. 2001; 69: 3248-3254Crossref PubMed Scopus (74) Google Scholar). S100A9 integrity is critical to the formation of complexes with S100A8 (33van den Bos C. Roth J. Koch H.G. Hartmann M. Sorg C. J. Immunol. 1996; 156: 1247-1254PubMed Google Scholar) and the calcium-binding loops within the EF-hands contribute to intermolecular stability (4Korndorfer I.P. Brueckner F. Skerra A. J. Mol. Biol. 2007; 370: 887-898Crossref PubMed Scopus (213) Google Scholar). The calcium-binding loops of S100 proteins also modulate intracellular calcium signaling, which affects cell differentiation, and cell cycle and cytoskeletal interactions (5Kligman D. Hilt D.C. Trends Biochem. Sci. 1988; 13: 437-443Abstract Full Text PDF PubMed Scopus (480) Google Scholar). Integrity of the S100A9 calcium-binding loops may also be critical to resistance against bacterial invasion. We considered that keratinocyte resistance to invasion reflected the ability of the cells to bind, internalize, and host viable invaders within the cell. In this study, we hypothesized that specific structural motifs of S100A9 in the calprotectin complex regulate epithelial cell resistance to bacterial invasion. To test this hypothesis, we designed five different S100A9 mutant constructs either in the calcium-binding or C-terminal domains using in vitro site-directed mutagenesis and deletion mutagenesis, respectively. Each mutated S100A9 was then expressed in KB cells with S100A8. As we reported previously (20Nisapakultorn K. Ross K.F. Herzberg M.C. Infect. Immun. 2001; 69: 4242-4247Crossref PubMed Scopus (115) Google Scholar), calprotectin (S100A8/A9) increased the resistance of epithelial cells to bacterial invasion. In the presence of S100A8, truncation of the C-terminal domain of S100A9 made the cells more resistant to invasion than with full-length S100A9. In contrast, mutations of S100A9 calcium-binding loops resulted in complete loss of resistance to bacterial invasion. Therefore, the central core polypeptide domain of S100A9 in the calprotectin complex plays a crucial role in epithelial resistance to bacterial invasion. Cells-Wild-type calprotectin-negative KB cells (American Type Culture Collection, ATCC CCL-17) were maintained in modified Eagle's media (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (Mediatech) in 5% CO2 at 37 °C. Transfected KB cells were maintained in modified Eagle's media supplemented with 10% fetal bovine serum and 700 μg/ml G418 sulfate (Mediatech). To test the effect of calprotectin expression on viable bacteria, mutants and controls were maintained in medium without G418 sulfate for 4 days before the experiments were performed. Bacteria-L. monocytogenes ATCC 10403S (provided by Dr. Daniel Portnoy, University of California, Berkley) and S. enterica serovar Typhimurium (S. typhimurium) ATCC 14028 (provided by Dr. Roy Curtiss III, Washington University, St. Louis) were grown in brain heart infusion medium (Difco) and on tryptic soy agar (Difco) at 37 °C. Listeria and Salmonella were harvested from log phase or stationary phase, respectively (absorbance of 0.4–0.6 at 620 nm), and used to infect KB cells. Construction of Calprotectin and S100A9 Mutant Expressing KB Cells-The structure of S100A8, S100A9, and mutant constructs in selected S100A9 functional domains are shown in Fig. 1, A and B. To construct S100A8 and S100A9 expression vectors, sequences were amplified using the following primers: S100A8, sense 5′-GGGCATCATGTTGACCGAGC-3′ and antisense 5′-GTAACTCAGCTACTCTTTGTGGCTT-3′; S100A9, sense 5′-CGATGACTTGCAAAATGTCGCAG-3′ and antisense 5′-GCCACTGTGGTCTTAGGGT-3′. To construct truncated S100A9 mutants (Fig. 1B), the sense primer was identical to S100A9 above, and the antisense primers were as follows: S100A91–112, 5′-TTAGCCCTCCCCGAGGGCTG-3′, and S100A91–99, 5′-TTACTCGTCACCCTCGTGCATCTTC-3′. S100A9 mutant sequences with point mutations in the calcium-binding loops, E36Q and E78Q (Fig. 1B), were constructed using the following oligonucleotides: S100A9E36Q, 5′-GCACCCTGAACCAGGGGCAATTCAAAGAGCTGGTGCG-3′, and 5′-CGCACCGCCTTGAATTGCCCCTGGTTCAGGGTG-3′, and S100A9E78Q, 5′-GCAGCTGAGTTCGACAGTTCATCATGCTGATGGCG-3′ and 5′-CGCCATCAGCATGATAATGCTCGAAGCCAGCTGC-3′, with the QuickChange® site-directed mutagenesis kit (Stratagene, Rockville, MD). S100A9E36Q,E78Q was constructed using all the oligonucleotides from above. PCR products were cloned and amplified using pPCR-Script® (Stratagene, La Jolla, CA). All mutants were verified by sequencing. S100A8 and mutated S100A9 sequences were then cloned into pIRES (BD Biosciences) and pKN-1 (pIRES-EGFP; BD Biosciences with the BamHI site at 1887 bp attenuated) plasmids and co-transfected into KB cells using Superfect (Qiagen, Valencia, CA). Transfectants were selected by resistance to 700 μg/ml G418 sulfate and sorted for enhanced green fluorescent protein expression using a FACSorter (BD Biosciences). Cells co-transfected with insertless pIRES and pKN-1 served as a sham-control transfectant (KB-sham). Plasmids containing S100A8 and unmodified S100A9 were co-transfected into KB cells and served as a positive calprotectin-expressing control (KB-S100A8/A91–114). Stable transfectants were confirmed by reverse transcription-PCR using PCR primers listed above. Immunofluorescence-Cells were grown on coverslips overnight, washed with PBS, 2The abbreviations used are: PBS, phosphate-buffered saline; mAb, monoclonal antibody; CFU, colony-forming unit; PDB, Protein Data Bank; m.o.i., multiplicity of infection; ELISA, enzyme-linked immunosorbent assay. and fixed with 4% paraformaldehyde for 10 min at room temperature. Monolayers were washed three times and permeabilized with 0.2% Triton X-100 for 2 min. After washing, monolayers were then incubated with murine monoclonal antibody against the calprotectin complex (mAb 27E10, diluted 1:50; Bachem, King of Prussia, PA) for 1 h at room temperature, followed by Alexa Fluor 568-conjugated goat anti-mouse IgG (diluted 1:500; Molecular Probes, Eugene, OR) for 1 h. Both antibodies were diluted in 3% (w/v) bovine serum albumin (Sigma) in PBS. The monolayers were washed and mounted with Fluoromount G (Southern Biotechnology, Birmingham, AL). Slides were examined using a Nikon Eclipse epifluorescence microscope and photographed using a Spot digital camera (Diagnostic Instruments Inc, Sterling Heights, MI). Sandwich ELISA-To detect calprotectin complex, cells were resuspended in Hanks' balanced salt solution (Invitrogen) and sonicated three times on ice at 50 watts for 15 s each (Sonifier Cell Disruptor W185, Heat Systems, Ultrasonics Inc., Plainview, MA). To obtain cell cytosol, sonicates were centrifuged at 10,000 × g for 20 min, and supernatants were collected, and total protein in each sample was determined by BCA protein assay kit (Pierce). Cell cytosol (50 μg) was analyzed for calprotectin using an ELISA. Briefly, 96-well plates were coated overnight at 4 °C with mAb 27E10 (diluted 1:100; Bachem), washed three times with PBS, pH 7.2, and 0.1% Tween 20, blocked for 1 h at 37 °C with blocking buffer (PBS, 0.1% Tween 20 and 0.5 mm CaCl2), and washed three more times. Cell cytosol was added, incubated for 1 h at 37 °C, and washed three times. Biotinylated murine monoclonal antibody to S100A9 (S 36.48-biotin, diluted 1:200; Bachem) was then added and incubated for 1 h at 37 °C. Extravidin-horseradish peroxidase and 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma) were used for colorimetric detection, and the absorbance was measured at 405 nm. Co-immunoprecipitation, Gel Electrophoresis, Silver Staining, and Western Blotting-mAb 27E10 was used for immunoprecipitation. To demonstrate co-precipitation of S100A8 and mutant S100A9 proteins, products were analyzed on silver-stained gels and Western blots. In brief, cells were treated with lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, pH 8.0, 1 mm EGTA, 1% Triton X-100, 0.5% Nonidet P-40 with the proteinase inhibitors, 2 μg/ml phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin A, 1 μg/ml aprotinin, 2.5 μg/ml leupeptin). Lysate protein concentration was determined using the BCA protein assay kit (Pierce). In preparation for immunoprecipitation, cell lysates were pre-cleared (reduced nonspecific binding) using protein A/G beads (50 μl), which were previously equilibrated twice in 450 μl of cold lysis buffer and centrifuged at 7500 × g for 30–45 s. Cell lysates (1 mg of protein) were incubated with the equilibrated protein A/G beads at 4 °C for 1 h using constant mixing and then centrifuged at 7500 × g for 10 min. The supernatants (pre-cleared lysates) were collected and incubated with mAb 27E10 (5 μg) at 4 °C for 1 h with constant mixing. Next, equilibrated protein A/G beads (50 μl) were added into the mixture and incubated overnight at 4 °C with constant mixing, pelleted at 7500 × g for 30–45 s, and washed five times with lysis buffer. Immunoprecipitated protein associated with the beads was resuspended in 50 μl of 2× SDS-PAGE buffer (1.2 ml of 0.5 m Tris, pH 6.8, 2% SDS, 20% glycerol, 0.5 ml of β-mercaptoethanol, and 1.6 ml of 1% bromphenol blue) and boiled to dissociate the immunoprecipitated protein from the beads. Immunoprecipitates (30 μl) were analyzed on 15% SDS-polyacrylamide gels, which were stained with metachromatic silver following the manufacturer's instructions (Bio-Rad). For Western blotting, KB cell lysates or the immunoprecipitated samples were separated on 15% SDS-PAGE, transferred onto a 0.2-μm nitrocellulose membrane (Bio-Rad), using a semi-dry transfer apparatus (Bio-Rad), and blocked overnight with 5% nonfat milk in TBST buffer (0.5 m NaCl, 20 mm Tris, pH 7.5, and 0.1% Tween 20). The membranes were then incubated with mouse anti-human S100A8 monoclonal antibody (C-10, Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human S100A9 monoclonal antibody (S 36.48, Bachem), or rabbit anti-human S100A9 polyclonal antibody (H-90, Santa Cruz Biotechnology) (each diluted 1:500) for 1 h at room temperature, washed three times with TBST buffer, and then incubated with either horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG (diluted 1:1000; Santa Cruz Biotechnology), respectively, for 1 h at room temperature. After washing, immunoblots were developed using ECL Western blot detection reagents (Amersham Biosciences). Nonspecific isotype IgG was used as a negative control. Bacterial Invasion Assay-Bacterial invasion was determined by the antibiotic protection assay as we described previously (20Nisapakultorn K. Ross K.F. Herzberg M.C. Infect. Immun. 2001; 69: 4242-4247Crossref PubMed Scopus (115) Google Scholar). In brief, KB transfectants (1.2 × 105 cells) were seeded overnight in 24-well plates. Cells were then incubated with L. monocytogenes or S. typhimurium at a multiplicity of infection (m.o.i.) of 100:1 and 1:1, respectively. After 2 h of incubation, the monolayers were washed with Dulbecco's PBS (Sigma) and incubated in medium containing 100 μg/ml gentamicin (Sigma) for 1.5 h to eliminate extracellular bacteria. The monolayers were then washed and incubated with sterile distilled water for 15 min to release intracellular bacteria. Released bacteria were diluted, plated with a spiral plater (Spiral Biotech, Bethesda, MD), and incubated overnight at 37 °C, and the numbers of colony-forming units (CFUs) of internalized bacteria were enumerated on a New Brunswick C-110 colony counter (New Brunswick, NJ). The invasion assay was performed in triplicate and repeated at least three times. Immunofluorescence Analysis of Intracellular and Extracellular Listeria-Cells (1.2 × 105) were seeded on glass coverslips and grown overnight. As described previously (21Nisapakultorn K. Ross K.F. Herzberg M.C. Infect. Immun. 2001; 69: 3692-3696Crossref PubMed Scopus (71) Google Scholar), the monolayers were infected with L. monocytogenes for 2 h at an m.o.i. of 100:1, washed twice with Dulbecco's PBS, and fixed with 4% paraformaldehyde. Extracellular Listeria were stained using rabbit anti-Listeria serum (diluted 1:3000; Biodesign, Kennebunk, ME) for 1 h, washed with PBS, and then incubated with Alexa Fluor 568-conjugated goat anti-rabbit IgG (diluted 1:500; Molecular Probes) for another hour. All antibodies were diluted in 3% bovine serum albumin in PBS. Cells were then permeabilized with 0.2% Triton X-100 for 2 min and then stained for both intracellular and extracellular Listeria. Permeabilized monolayers were washed, incubated with rabbit anti-Listeria serum for 1 h, washed three times, and then incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:500; Molecular Probes) for 1 h. Nuclei were stained using 4′,6′-diamidino-2-phenylindole (diluted 1:3000; Molecular Probes). To verify antibody specificity, primary antibodies were replaced by rabbit serum. To determine nonspecific binding, secondary antibodies were added without primary antibody. Cells were observed using a Nikon Eclipse fluorescence microscope at ×400 magnification, and images from 20 random fields were captured with a Spot digital camera (Diagnostic Instruments Inc.). In each field, total Listeria (Alexa 488) and extracellular Listeria (Alexa 568) were counted. The number of intracellular Listeria was determined by subtracting the number of extracellular Listeria from the total count. Bacterial Binding Assay-Binding of Listeria to KB cells was performed as described previously (21Nisapakultorn K. Ross K.F. Herzberg M.C. Infect. Immun. 2001; 69: 3692-3696Crossref PubMed Scopus (71) Google Scholar). Cells (1.2 × 105) were seeded on glass coverslips and grown overnight. Monolayers were then incubated with L. monocytogenes at an m.o.i. of 100:1 for up to 60 min at 37 °C, washed, and fixed using 4% paraformaldehyde for 10 min. Adherent Listeria were labeled for 1 h with rabbit anti-Listeria serum (diluted 1:3000; Biodesign), washed, and incubated for 1 h with Alexa Fluor 568-conjugated goat anti-rabbit IgG. Separate coverslips were incubated with rabbit serum or secondary antibody as controls. At each time point, images from 10 random microscopic fields at ×200 magnification were captured with a Spot digital camera, and adherent bacteria were enumerated by visual counting. Structural Analysis of Calcium-free and Calcium-bound Calprotectin-Because the structure of calcium-free calprotectin has not been determined, we generated the homology modeled structure using the program MODELLER (34Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10636) Google Scholar). This program was chosen because of its ability to handle the alignment of a heterodimer target sequence (S100A8 and S100A9) with a homodimer structural template. Calcium-free S100A1, S100A4, and S100B structures (PDB codes 1K2H, 1M31, and 2PRU, respectively) were chosen as templates for homology modeling because these proteins have over 30% sequence identity with both S100A8 and S100A9 (35Rustandi R.R. Baldisseri D.M. Inman K.G. Nizner P. Hamilton S.M. Landar A. Landar A. Zimmer D.B. Weber D.J. Biochemistry. 2002; 41: 788-796Crossref PubMed Scopus (62) Google Scholar, 36Vallely K.M. Rustandi R.R. Ellis K.C. Varlamova O. Bresnick A.R. Weber D.J. Biochemistry. 2002; 41: 12670-12680Crossref PubMed Scopus (67) Google Scholar, 37Malik S. Revington M. Smith S.P. Shaw G.S. Proteins. 2008; 73: 28-42Crossref PubMed Scopus (20) Google Scholar). The length and level of sequence identity are important factors in accurate homology model generation. These three structures were used as templates both individually and combined into a single composite template for homology model generation. There is little overall difference in the final models, but the structure used in Fig. 8 is based on the composite template to remove the bias of any individual starting structure. Additionally, the residues in the homology model generated structure were trimmed to match those described in PDB code 1XK4 (S100A8 Met-1 to His-87, S100A9 Lys-4 to Glu-92) to most closely compare changes in the molecular surface upon calcium binding. Furthermore, the absence of electron density for the C-terminal 22 residues in S100A9 in PDB code 1XK4 implies that these residues occupy multiple conformations and that there is no experimental support to favor a single prediction in our models for this tail region. The calcium-bound form of calprotectin has been experimentally determined by Skerra and co-workers, PDB code 1XK4 (4Korndorfer I.P. Brueckner F. Skerra A. J. Mol. Biol. 2007; 370: 887-898Crossref PubMed Scopus (213) Google Scholar), and this information was used in our analysis. The program Swiss-PdbViewer was used to generate the ribbon diagrams, molecular surface, and the calculation of electrostatic potential with the same settings throughout Fig. 8 (38Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9641) Google Scholar). The model structure for calcium-bound S100A8 in complex with calcium-free S100A9 was created by structural superimposition of the N- and C-terminal helices of the calcium-free calprotectin (composite model structure) onto calcium-bound calprotectin (PDB code 1XK4). Both structures were written out as one file followed by removal of the information for calcium-free S100A8 and calcium-bound S100A9. Statistical Analyses-Data are presented as the means ± S.E. Significant differences between control (KB-sham) and S100A9 mutants were determined using a two-sample Student's t test. p < 0.05 was considered to be statistically significant. Formation of S100A8 and Mutant S100A9 Heterodimers-As shown schematically in Fig. 1, KB cells were transfected to express calprotectin (S100A8/S100A9; Fig. 2B) and S100A8 in the presence of S100A9 C-terminal deletion mutants (Fig. 2, C and D) or point mutations in the calcium-binding loops (Fig. 2, E–G). Using complex-specific mAb 27E10, S100A8 in the presence of all mutant S100A9 variants appeared to form calprotectin complexes as suggested by immunofluorescence microscopy; KB-sham, the sham-transfected control cells, was negative (Fig. 2A). Antigen(s) precip
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