ARTICLE |
Correspondence to: Sophie de Bentzmann, INSERM U514, IFR 53, CHU Maison-Blanche, 45 rue Cognacq-Jay, 51092 Reims Cedex, France.
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Summary |
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Staphylococcus aureus is a common human pathogen involved in non-bronchial diseases and in genetic and acquired bronchial diseases. In this study, we applied an immunolabeling approach for in vivo and in vitro detection of S. aureus, based on the affinity of staphylococcal protein A (SpA) for the Fc region of immunoglobulins, especially IgG. Most strains of S. aureus, including clinical strains, can be detected with this labeling technique. The approach can be used for detection and localization with transmission electron microscopy or light-fluorescence microscopy of S. aureus in infected tissues such as human bronchial tissue from cystic fibrosis (CF) patients. The methodology can also be applied to cell culture models with the aim of characterizing bacterial adherence to epithelial cells in backscattered electron imaging with scanning electron microscopy. Application to the study of S. aureus adherence to airway epithelium showed that the bacteria did not adhere in vivo to intact airway epithelium. In contrast, bacteria adhered to the basolateral plasma membrane of columnar cells, to basal cells, to the basement membrane and were identified beneath the lamina propria when the epithelium was injured and remodeled, or in vitro when the epithelial cells were dedifferentiated. (J Histochem Cytochem 48:523533, 2000)
Key Words: Staphylococcus aureus, immunodetection, protein A, bacterial adherence, human airway epithelium, cystic fibrosis
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Introduction |
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Staphylococcus aureus is a common human pathogen associated with various infections, particularly those involving the respiratory tract. S. aureus is the first pathogen to appear in cystic fibrosis (CF) respiratory infection and is also involved in acquired respiratory diseases (e.g., chronic bronchitis and nosocomial infections). S. aureus binds to many host matrix, plasma, and tissue proteins, such as fibronectin (
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Materials and Methods |
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Human Airway Tissues
Chronically infected lungs were obtained from six CF patients (five males and one female; four homozygous for the F508 mutation, two with unknown genotypes; mean age 23.5 ± 10.3 years) before surgical lung transplantation. Sputum bacteriology showed that these CF patients were chronically colonized by S. aureus or P. aeruginosa isolated as a unique infecting pathogen. Tissue fragments were taken at lobar and segmental levels and were cryofixed in optimal cutting temperature (OCT) medium (Tissue Tek; Miles, Elkhart, IN).
Human Airway Epithelial Cell Culture
Nasal polyps from non-CF patients were used as human airway tissue for the cell culture study in 24-well plates. The polyps were cut into small explants (2 mm2) and seeded onto a Type I collagen matrix in a defined RPMI 1640 culture medium (Seromed Polylabo; Strasbourg, France) supplemented with insulin (1 µg/ml; Sigma Chemical, St Louis, MO), apo-transferrin (1 µg/ml; Serva, Heidelberg, Germany), epidermal growth factor (10 ng/ml; Serva), hydrocortisone (0.5 µg/ml; Sigma), retinoic acid (10 ng/ml; Sigma), penicillin (100 U/ml) and streptomycin (100 µg/ml; Sigma) at 37C in air with 5% CO2 (
Bacteria
The Cowan III strain (ATCC, Rockville, MD; #12600) and the protein A (SpA)-deficient strain of S. aureus (DU5875 strain; a generous gift from Pr. T.J. Foster, Department of Microbiology, Trinity College, Dublin, Ireland) were maintained at -20C in trypticase soy broth (TSB; Institut Pasteur, Paris, France) containing 20% v/v glycerol. For adherence assays, bacteria were cultured overnight in TSB at 37C, harvested by centrifugation, and resuspended in RPMI 1640 culture medium containing 20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid). The bacterial suspension was adjusted to a final concentration of 107 colony forming units (CFU)/ml. Bacterial self-aggregation was limited by extruding ex tempo the bacterial suspension through a fine needle just before contact with the airway epithelial cell cultures.
Bacterial Adherence
On Day 3 or 4, antibiotics were removed from primary human airway epithelial cell cultures by repeated rinsing with RPMI 1640 culture medium. A 200-µl aliquot of bacterial suspension was added to the cultures for 1 hr at 37C. To remove non-adherent bacteria, cultures were rinsed three times with 0.5 ml phosphate-buffered saline (PBS; 0.1 M, pH 7.2) and were prepared for immunofluorescence microscopy or for scanning or transmission electron microscopy.
Immunodetection of S. aureus
The immunodetection of S. aureus was based on the high affinity of SpA, a cell wall-associated protein of S. aureus, for Type G immunoglobulins (IgG) through binding to the Fc fragment (
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Immunofluorescent detection of S. aureus. For immunofluorescence, cultures and tissues were embedded in OCT, immersed in liquid nitrogen, and stored at -80C until cryosectioning. Five µm-thick cryosections deposited on gelatin-coated slides were fixed in pre-cooled methanol (-20C) for 10 min, rinsed in PBS, and preincubated with 3% PBSbovine serum albumin (BSA). Sections were first exposed to biotinylated donkey anti-rabbit (DAR) IgG (1:50 in 1% PBSBSA; Amersham, Les Ulis, France) for 60 min. After incubation with 3% PBSBSA, SpA-biotinylated IgG complexes were detected by streptavidinTexas Red (1:50 in 1% PBSBSA for 60 min; Amersham).
When a double immunolabeling (detection of both bacteria and epithelial antigens) protocol was performed after the labeling of bacteria as described above, sections were further incubated for 60 min with pure nonimmune goat serum (NGS; Sigma) reconstituted in distilled water according to the manufacturer's recommendations. This step saturated SpA molecules present at the surface of the bacteria before the second immunolabeling step. Sections were then incubated with primary mouse monoclonal antibody (MAb) against cytokeratin-13 (CK13; 1:600 in 1% PBSBSA for 60 min; Sigma), a typical airway epithelial basal cell marker ( fractions (Sigma). To control the specificity of S. aureus labeling, CF bronchial tissues from a patient infected only with P. aeruginosa were processed as followed. Sections were first incubated with biotinylated sheep anti-mouse IgG (1:50 in 1% PBSBSA for 1 hr; Boehringer Mannheim), with streptavidinTexas Red and with NGS as described above. Sections were secondly treated with a mixture of P. aeruginosa rabbit anti-sera (PMA, PMC, PME, and PMF; 1:10 in PBSBSA 1% for 1 hr; Sanofi Diagnostics Pasteur, Marnes-La Coquette, France), with antirabbit F(ab')2 fractions coupled with digoxigenin (1:50 in PBSBSA 1% for 1 hr; Boehringer Mannheim), and then with anti-digoxigenin Fab fraction coupled with FITC (1:50 in PBSBSA 1% for 30 min).
The sections were finally counterstained with Harris hematoxylin, mounted in Citifluor antifading solution (Agar Scientific; Stansted, UK), and observed with an Axiophot microscope (Zeiss; Oberkochen, Germany) with epifluorescence and Nomarski differential interference illumination.
Immunoelectron Microscopic Detection of S. aureus.
For scanning electron microscopy (SEM) or transmission electron microscopy (TEM), cell cultures were fixed in 4% PBSparaformaldehyde for 60 min at 4C. The cultures were then rinsed twice in PBS and in 1% PBSBSA. Specimens were first incubated with biotinylated DAR IgG (1:50 in 1% PBSBSA for 60 min) and then, to saturate SpA molecules present at the surface of bacteria, with pure NGS (for 60 min). Specimens were finally exposed to goat anti-biotin antibody (1:200 in 1% PBSBSA for 60 min; Sigma) and to SpA15-nm or SpA40-nm gold particle complexes (for 60 min) prepared as previously described by
Negative controls were performed by first incubating specimens with pure NGS (for 60 min) or with non-biotinylated DAR IgG before incubation with goat anti-biotin antibody and SpA15-nm or 40-nm gold particle complexes. Free SpA (Sigma; ref. P3838) used for gold particle complexes had a binding capacity of 714 mg of human IgG per mg solid.
For SEM, cultures were dehydrated through graded ethanol concentrations, critical point-dried with CO2, affixed onto stubs, coated with carbon (20 nm), and observed with a Philips XL30 scanning electron microscope operating at 10 kV with secondary electron imaging (SEI). In addition, immunogold-stained bacteria were identified with backscattered electron imaging (BEI) at 25 kV. Secondary and backscattered electron signals could also be mixed.
For TEM, the cultures were dehydrated through graded concentrations of ethanol and embedded in agar resin 100 (Agar Scientific; Orsay, France). Ultrathin sections were stained with uranyl acetate and lead citrate and then observed with a Hitachi 300 transmission electron microscope operating at 75 kV.
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Results |
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S. aureus Immunodetection with Light Microscopy
S. aureus that had adhered to the apex of airway epithelial cells or was present at the contact of basolateral membranes of airway epithelial cells could be easily detected in cell cultures (Fig 2A). Labeling of S. aureus, in parallel with the detection of basal cells using anti-CK13 antibody, allowed specific detection of all bacteria in the absence of any fluorescent background (Fig 2B). Negative control slides for S. aureus detection, using non-biotinylated donkey anti-rabbit IgG (Fig 2C and Fig 2D), as well as double control experiments negative for S. aureus (preincubation with NGS) and epithelial cell marker immunodetection, showed no immunolabeling (Fig 2E and Fig 2F). No Texas Red labeling was observed when immunodetection of S. aureus was performed on cell cultures exposed to the SpA-deficient strain (Fig 2G and Fig 2H).
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In all infected bronchial tissues from five different S. aureus-infected CF patients (Fig 3), bacteria could be detected in the sputum covering pseudostratified bronchial epithelial areas (Fig 3A and Fig 3B). When the epithelium was pseudostratified (Fig 3C and Fig 3D), S. aureus was detected on the basal side of the CK13 basal epithelial cells, beneath the basement membrane, and in the lamina propria. This shows that the bacteria had gained access to these sites of adherence after epithelial barrier integrity breakdown. In patchy areas of injury, bacteria were found adhered to CK13-positive airway epithelial cells, identified as basal cells, and beneath the basement membrane in the lamina propria (Fig 3E and Fig 3F). No FITC or Texas Red labeling was observed in negative controls for S. aureus or for CK13 basal cell immunodetection (not shown). In P. aeruginosa-infected CF bronchi, the specificity of S. aureus labeling was assessed by double labeling for P. aeruginosa (in green with FITC) and S. aureus (in red with Texas Red). Using double exposure (Fig 3G and Fig 3H), only P. aeruginosa was detected.
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S. aureus Immunodetection with Electron Microscopy
In TEM, pre-embedding immunogold labeling of S. aureus was found over the entire surface of the bacteria (Fig 4). Many 15-nm colloidal particles were present at the surface of the bacteria that adhered to airway epithelial cells (Fig 4A) and to the collagen matrix on which cells had grown (Fig 4B). In contrast, bacteria were sparsely labeled with SpA40-nm gold particle complexes (Fig 4C). Very few 40-nm gold particles were present at the surface of the bacteria, and some bacteria were not labeled (Fig 4D). In control experiments performed with preincubation with NGS before incubation with goat anti-biotin antibody and SpA15-nm gold complex, no gold particles could be detected on bacteria (Fig 4E). Similarly, when immunodetection of S. aureus was performed in cell cultures exposed to the SpA-deficient strain, no bacteria were labeled with SpA15-nm colloidal gold particles (Fig 4F). This pre-embedding labeling did not generate any background, inclusive of the collagen matrix or in contact with cellular membranes.
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For the SEM experiments, the efficiency of labeling, characterized by the superimposition of the SEI and BEI views of bacteria, was analyzed as a function of the diameter of gold particles. Whereas the entire surface of the bacteria observed with SEI was labeled with SpA15-nm colloidal gold particles (Fig 5A and Fig 5B), labeling with BEI was heterogeneous, of low intensity, and with a speckled distribution of SpA40-nm colloidal gold particles (Fig 5C and Fig 5D). Apart from the gold particle diameter, optimal dilutions of IgG and anti-biotin antibody were adjusted to generate the best signal:noise ratio, which was characterized by the superimposition of bacteria observed with SEI with the respective labeled surface observed with BEI.
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S. aureus Adherence to Airway Epithelial Cells
We further investigated, using SEM, the adherence of S. aureus to airway epithelial cells in vitro as a function of their state of differentiation. This could be easily reproduced by using the primary explantoutgrowth culture model (
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Imaging of bacteria adhered to airway epithelial cells in cell cultures was performed with the SEI and BEI configurations of the SEM. Under standard observation with SEI, the segregation of bacteria from mucous granules or microdebris present in the preparations was difficult, especially for isolated bacteria (Fig 6C). In contrast, labeled isolated bacteria were easily distinguished from mucous secretory granules and from other objects (Fig 6D).
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Discussion |
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In the present work, an immunolabeling methodology was applied to the detection of S. aureus. The immunolabeling methodology involved is applicable to in vivo and in vitro study of the interaction of S. aureus with various tissues and particularly with airway epithelium. This method permits the immunodetection of the bacteria with optical and electron microscopic techniques in infected human bronchial tissues and in primary cell cultures.
The labeling approach is based on the detection of the most characteristic protein produced by S. aureus, i.e., protein A (SpA). SpA is a cell wall-associated protein that binds in a nonantigenic but specific way to the Fc region of IgG from several species (
Because of the specific interaction of SpA with IgG, the signal detection reported here is highly specific to S. aureus. Moreover, clinical strains devoid of protein A have been shown to be uncommon, because it has been reported that among 190 clinical strains of S. aureus, only one strain was lacking the spa gene (
Because SpA (and, to a lesser extent, Sbi) binds not only IgG but also IgA, IgE, and IgM molecules, the difficulty in the present experiments was to saturate all SpA molecules before detection of other antigens in the preparations. This was overcome by incubating specimens first with biotinylated IgG and second with nonimmune goat serum. This procedure ensures that vacant SpA molecules present at the surface of bacteria, and possibly incompletely saturated during the immunodetection of S. aureus, do not trap immunoglobulins that will be further used for the immunolabeling of epithelial cell markers in the double labeling procedure. The labeling methodology applied in the present study is different from that used by
Using TEM, it could be shown that colloidal gold complexes were localized over the entire bacterial membrane surface, without any background signal arising in the collagen matrix or the epithelial cell membrane. To confirm that immunolabeling of S. aureus via SpA is a specific process, we also used an SpA-deficient strain of S. aureus. No labeling of the SpA-deficient strain was observed by light microscopy or electron microscopy. One could have expected a weak labeling of bacteria due to expression of the Sbi protein. However, the parent strain 8325-4 used to obtain this SpA-deficient strain has been reported to have undetectable levels of Sbi even though the sbi gene could be detected by Southern blot analysis (
The identification of S. aureus with the aid of SEM is difficult because of morphological and size similarities of S. aureus with mucous granules or microdebris. The immunodetection of S. aureus with BEI coupled with SEI observations represents a useful tool for studying the mechanisms involved in S. aureus adherence to airway epithelial cells. This could be particularly helpful in the identification of bacterial adhesins and epithelial receptors using quantitative methods with computer-aided on-line image analysis techniques. We observed that immunodetection of S. aureus with BEI was optimal for SpA15-nm gold compared to SpA40-nm colloidal gold particles, the SpA5-nm colloidal gold particles giving no more benefit for S. aureus detection with BEI observation (personal data). These results agree with previous reports showing that for the 15-nm gold particle size, 60 molecules of SpA are present at the surface of each gold particle (
This cytochemical methodology based on staphylococcal protein A can be applied either in light microscopy or in electron microscopy, but also for immunodetection of the bacteria in many staphylococcal pathologies including bronchial diseases. In the present study, the application of this methodology to the detection of S. aureus in infected tissues was validated with the study of S. aureus interaction with airway epithelium. Mechanisms of colonization of airway epithelium by S. aureus remain unknown. S. aureus is capable of adhering to purified components of epithelial cell membranes, such as the binding sequence Gal-Nacß1-4Gal present in the glycolipid asialo GM1 (
In the present study, we have applied an immunolabeling methodology based on the affinity of SpA for IgG, which is applicable to either immunofluorescence observation using light microscopy or immunogold detection with electron microscopic techniques for immunodetection of the bacteria in many staphylococcal pathologies, including bronchial diseases. The study of S. aureus adherence to airway epithelium, which is the first step involved in the pathogenesis of staphylococcal lung diseases, implies the identification of bacterial adhesins and epithelial receptors. This will be further achieved by using human primary airway cell culture models, with immuofluorescence and quantitative methods as well as computer-aided on-line image analysis techniques coupled with BEI and SEI in SEM.
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Acknowledgments |
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Supported by a grant from DRET/DGA no. 96.34.053.00. 470 and partially funded by the Association Française de Lutte contre la Mucoviscidose (AFLM). E. Mongodin is a doctoral fellow of the Ministère de la Défense - DRET/DGA.
We thank Pr T.J. Foster for providing us with the SpA-deficient strain of S. aureus. We are grateful to Pr Klossek (CHU J. Bernard; Poitiers, France) for providing human nasal polyp tissues and to Pr Couetil (Hopital Broussais; Paris, France) for CF lung tissues.
Received for publication June 3, 1999; accepted November 17, 1999.
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Baltimore RS, Christie CD, Smith GJ (1989) Immunohistopathologic localization of Pseudomonas aeruginosa in lungs from patients with cystic fibrosis. Implications for the pathogenesis of progressive lung deterioration. Am Rev Respir Dis 140:1650-1661[Medline]
Brézillon S, Hamm H, Heilmann M, Schäfers HJ, Hinnrasky J, Friedrich Wagner TO, Puchelle E, Tümmler B (1997) Decreased expression of the cystic fibrosis transmembrane conductance regulator protein in remodelled airway epithelium from lung transplanted patients. Hum Pathol 28:944-952[Medline]
Cheung AL, Fischetti VA (1990) The role of fibrinogen in staphylococcal adherence to catheters in vitro. J Infect Dis 161:1177-1186[Medline]
Chevillard M, Hinnrasky J, Zahm JM, Plotkowski MC, Puchelle E (1991) Proliferation, differentiation and ciliary beating of human respiratory ciliated cells in different conditions of primary cultures. Cell Tissue Res 264:49-55[Medline]
de Bentzmann S, Mongodin E, Roger P, Puchelle E (in press). Bacterial adherence to airway epithelium. Eur Respir Rev
De Roe C, Courtoy PJ, Baudhuin P (1987) A model of proteincolloidal gold interactions. J Histochem Cytochem 35:1191-1198[Abstract]
Dupuit F, Kälin N, Brézillon S, Hinnrasky J, Tümmler B, Puchelle E (1995) CFTR and differentiation markers expression in non-CF and F508 homozygous CF nasal epithelium. J Clin Invest 96:1601-1611[Medline]
Forsgren A, Forsum U (1970) Role of protein A in non specific fluorescence of Staphylococcus aureus. Infect Immun 2:387-391
Forsgren A, Sjöquist J (1966) Protein A from S. aureus. I.-Pseudo-immune reaction with human -glogulin. J Immunol 97:822-827[Medline]
Foster TJ, McDevitt D (1994) Surface-associated proteins of Staphylococcus aureus: their possible roles in virulence. FEMS Microbiol Lett 118:199-206[Medline]
Frens G (1972) Particle size and sol stability in metal colloids. Kolloid Z Zellforsch Polymere 250:736
Frens G (1973) Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions. Nature 241:20-22. [Phys Sci]
Goding JW (1978) Use of staphylococcal protein A as an immunological reagent. J Immunol Methods 20:241-253[Medline]
Krivan HC, Roberts DD, Ginsburg V (1988) Many pulmonary pathogenic bacteria bind specifically to the carbohydrate sequence GalNAcß1-4Gal found in some glycolipids. Proc Natl Acad Sci USA 85:6157-6161[Abstract]
Kuypers JM, Proctor RA (1989) Reduced adherence to traumatized rat heart valves by a low-fibronectin binding mutant of Staphylococcus aureus. Infect Immun 57:2306-2312[Medline]
Lopes JD, Reis M, Brentani RR (1985) Presence of laminin receptors in Staphylococcus aureus. Science 229:275-277[Medline]
Park PW, Roberts DD, Grosso LE, Parks WC, Rosenbloom J, Abrams WR, Mecham RP (1991) Binding of elastin to Staphylococcus aureus. J Biol Chem 266:23399-23406
Patti JM, Allen BL, Mc Gavin MJ, Höök M (1994a) MSCRAMM-mediated adherence of microorganisms to host tissue. Annu Rev Microbiol 48:585-617[Medline]
Patti JM, Bremell T, KrajewskaPietrasik D, Abdelnour A, Tarkowski A, Ryden C, Höök M (1994b) The Staphylococcus aureus collagen adhesin is a virulence determinant in experimental septic arthritis. Infect Immun 62:152-161[Abstract]
Plotkowski MC, Chevillard M, Pierrot D, Altemayer D, Zahm JM, Colliot G, Puchelle E (1991) Differential adhesion of P. aeruginosa to human respiratory epithelial cell culture. J Clin Invest 87:2018-2028[Medline]
Sakurada J, Li Z, Seki K, Murai M, Usui A, Morihigashi M, Jitsukawa H, Seong HK, Mutou M, Masuda S (1994) Biochemical and genetic heterogeneity of staphylococcal protein A. FEMS Microbiol Lett 119:59-64[Medline]
Seki K, Sakurada J, Seong HK, Murai M, Tachi H, Ishii H, Masuda S (1998) Occurrence of coagulase serotype among Staphylococcus aureus strains isolated from healthy individualsspecial reference to correlation with size of protein A gene. Microbiol Immunol 42:407-409[Medline]
Switalski LM, Speziale P, Höök M (1989) Isolation and characterization of a putative collagen receptor from Staphylococcus aureus strain Cowan I. J Biol Chem 264:21080-21086
Uhlen M, Lindberg M, Philipson L (1984) The gene for staphylococcal protein A. Immunol Today 5:244-248
Ulrich M, Herbert S, Berger J, Bellon G, Louis D, Munker G, Doring G (1998) Localization of Staphylococcus aureus in infected airways of patients with cystic fibrosis and in a cell culture model of S. aureus adherence. Am J Respir Cell Mol Biol 19:83-91
Van Belkum A, Riewarts Eriksen NH, Sijmons M, Van Leeuwen W, Van den Bergh M, Kluytmans J, Espersen F, Verbrugh H (1997) Coagulase and protein A polymorphisms do not contribute to persistence of nasal colonization by Staphylococcus aureus. J Med Microbiol 46:222-232[Abstract]
Vercellotti GMN, Lussenhop D, Peterson PK, Furcht LT, MacCarthy JB, Jacob HS, Moldow CF (1984) Bacterial adherence to fibronectin and endothelial cells: a possible mechanism for bacterial tissue tropism. J Lab Clin Med 103:34-43[Medline]
Zhang L, Jacobsson K, Vasi J, Lindberg M, Frykberg L (1998) A second IgG-binding protein in Staphylococcus aureus. Microbiology 144:985-991[Abstract]