Role of Staphylococcus Aureus Surface-Associated Proteins in the Attachment to Cultured HaCaT Keratinocytes in a New Adhesion Assay
1998; Elsevier BV; Volume: 111; Issue: 3 Linguagem: Inglês
10.1046/j.1523-1747.1998.00293.x
ISSN1523-1747
AutoresMartin Mempel, Tanja Schmidt, Stephan Weidinger, Christina Schnopp, Johannes Ring, Dietrich Abeck, Timothy J. Foster,
Tópico(s)Nail Diseases and Treatments
ResumoColonization of human skin with Staphylococcus aureus is a common feature in a variety of dermatologic diseases. In order to reproducibly investigate the adherence of Staphylococcus aureus to human epidermal cells, an in vitro assay was established using the biotin/streptavidine labeling system and the HaCaT cell line. This assay was used to define the role of several Staphylococcus aureus surface proteins with regard to their function in the staphylococcal adhesion process. Our studies included the standard laboratory strain Newman as well as its genetically constructed mutants DU5873, DU5852, DU5854, and DU5886 generated by allele replacement or transposon mutagenesis, which are deficient in the elaboration of staphylococcal protein A (spa), clumping factor (clfA), coagulase (coa), and the fibronectin-binding proteins A and B (fnbA/B), respectively. In comparison with strain Newman all mutants showed remarkably reduced adherence to the HaCaT keratinocyte cell line in our assay, yielding only between 43% and 60% of the adherence capacity of strain Newman after 60 min. Bacterial adherence could be re-established by introducing the cloned wild-type genes for the surface proteins on shuttle plasmids into the chromosomally defective mutants, thus suggesting a pathogenetic role of these proteins in the attachment of Staphylococcus aureus to human keratinocytes. Bacterial adherence was additionally enhanced by alkaline pH-values that are characteristic for skin conditions with epidermal barrier dysfunction. The use of Staphylococcus aureus mutant strains, deficient in the elaboration of defined proteins, allows specific investigation of colonization and virulence factors of this dermatologic relevant microorganism. Key word: surface proteins. J Invest Dermatol 111:452–456 1998 Colonization of human skin with Staphylococcus aureus is a common feature in a variety of dermatologic diseases. In order to reproducibly investigate the adherence of Staphylococcus aureus to human epidermal cells, an in vitro assay was established using the biotin/streptavidine labeling system and the HaCaT cell line. This assay was used to define the role of several Staphylococcus aureus surface proteins with regard to their function in the staphylococcal adhesion process. Our studies included the standard laboratory strain Newman as well as its genetically constructed mutants DU5873, DU5852, DU5854, and DU5886 generated by allele replacement or transposon mutagenesis, which are deficient in the elaboration of staphylococcal protein A (spa), clumping factor (clfA), coagulase (coa), and the fibronectin-binding proteins A and B (fnbA/B), respectively. In comparison with strain Newman all mutants showed remarkably reduced adherence to the HaCaT keratinocyte cell line in our assay, yielding only between 43% and 60% of the adherence capacity of strain Newman after 60 min. Bacterial adherence could be re-established by introducing the cloned wild-type genes for the surface proteins on shuttle plasmids into the chromosomally defective mutants, thus suggesting a pathogenetic role of these proteins in the attachment of Staphylococcus aureus to human keratinocytes. Bacterial adherence was additionally enhanced by alkaline pH-values that are characteristic for skin conditions with epidermal barrier dysfunction. The use of Staphylococcus aureus mutant strains, deficient in the elaboration of defined proteins, allows specific investigation of colonization and virulence factors of this dermatologic relevant microorganism. Key word: surface proteins. J Invest Dermatol 111:452–456 1998 colony forming unit Colonization of the skin with Staphylococcus aureus plays an important role in the pathogenesis of a variety of dermatologic diseases such as atopic dermatitis, psoriasis, and cutaneous T cell lymphoma (Aly et al., 1977Aly R. Maibach H.J. Shinefield H.R. Microbial flora of atopic dermatitis.Arch Dermatol. 1977; 113: 780-782Crossref PubMed Scopus (221) Google Scholar;Abeck and Ruzicka, 1992Abeck C. Ruzicka T. Bacteria and atopic eczema: merely association or etiologic factor?.in: Ruzicka T Ring J Przybilla B Handbook of Atopic Eczema. Springer-Verlag, Berlin1992: 212-220Google Scholar;Nickoloff et al., 1993Nickoloff B.J. Mitra R.J. Green J. Shimizu Y. Thompson C. Turka L.A. Activated keratinocytes present bacterial-derived superantigens to T-lymphocytes: relevance to psoriasis.J Dermatol Sci. 1993; 6: 127-133Abstract Full Text PDF PubMed Scopus (25) Google Scholar;Cooper, 1994Cooper K.D. Atopic dermatitis: recent trends in pathogenesis and therapy.J Invest Dermatol. 1994; 102: 128-137Abstract Full Text PDF PubMed Google Scholar;Tokura et al., 1995Tokura Y. Yagi H. Ohshima A. et al.Cutaneous colonization with staphylococci influences the disease activity of Sézary syndrome: a potential role for bacterial superantigens.Br J Dermatol. 1995; 133: 6-12Crossref PubMed Scopus (69) Google Scholar). Well-established virulence factors of these bacteria include their encymatic repertoire (Kornblum et al., 1990Kornblum J. Kreiswirth B. Projan S.J. Ross H. Novick R.P. Agr: a polycistronic locus regulating exoprotein synthesis in Staphylococcus aureus, p.in: Novick RP Molecular Biology of the Staphylococci. VCH Publishers, New York1990: 373-402Google Scholar), their ability to trigger chronic inflammatory processes via the elaboration of superantigens (Tokura et al., 1994Tokura Y. Yagi J. O’malley M. Lewis J.M. Takigawa M. Edelson R.L. Tigelaar R.E. Superantigenetic staphylococcal exotoxins induce T cell proliferation in the presence of Langerhans cells or class II-bearing keratinocytes and stimulate keratinocytes to produce T cell-activating cytokines.J Invest Dermatol. 1994; 102: 31-38Abstract Full Text PDF PubMed Google Scholar;Leung et al., 1995Leung D.Y.M. Travers J.B. Norris D.A. The role of superantigens in skin disease.J Invest Dermatol. 1995; 105: 37s-42sAbstract Full Text PDF PubMed Scopus (87) Google Scholar), and their widespread resistance to commonly used anti-microbial agents (Chambers and Hackbarth, 1989Chambers H.F. Hackbarth C.J. Methicillin-resistant Staphylococci: genetics and mechanisms of resistance.Antimicrob Agents Chemother. 1989; 33: 991-994Crossref PubMed Scopus (75) Google Scholar). Although numerous reports dealt with their pathogenetic properties, little is known about colonization factors of S. aureus to human skin. Staphylococcus aureus surface proteins have been supposed to play a major role in the adhesion process and a previous report established the influence of Protein A on the bacterial adherence to isolated human corneocytes (Cole and Silverberg, 1986Cole G.W. Silverberg N.L. The adherence of Staphylococcus aureus to human corneocytes.Arch Dermatol. 1986; 122: 166-169Crossref PubMed Scopus (92) Google Scholar). Further investigations gave evidence for an important role of the fibronectin receptor in the colonizing process of low granular and fully keratinized cells (Bibel et al., 1983Bibel D.J. Aly R. Shinefield H.R. Maibach H.I. The Staphylococcus aureus receptor for fibronectin.J Invest Dermatol. 1983; 80: 494-496Abstract Full Text PDF PubMed Scopus (29) Google Scholar). In the meantime, additional S. aureus structures have been identified enabling this microorganism to gain access to different types of human tissue (Lopes et al., 1985Lopes J.D. dos Reis M. Brentani R.R. Presence of laminin receptors in Staphylococcus aureus..Science. 1985; 229: 275-276Crossref PubMed Scopus (174) Google Scholar;Jönsson et al., 1991Jönsson K. Signäs C. Müller H-P Lindberg M. Two different genes encode fibronectin binding-proteins in Staphylococcus aureus..Eur J Biochem. 1991; 202: 1041-1048Crossref PubMed Scopus (291) Google Scholar;Liang et al., 1993Liang O.D. Maccarena M. Flock J-I Paulsson M. Preissner K.T. Waldström T. Multiple interactions between human vitronectin and Staphylococcus aureus.Biochim Biophys Acta. 1993; 1225: 57-63Crossref PubMed Scopus (34) Google Scholar;Foster and McDevitt, 1994Foster T.J. McDevitt D. Surface associated proteins of Staphylococcus aureus: their possible roles in virulence.FEMS Microbiol Lett. 1994; 118: 199-206Crossref PubMed Scopus (159) Google Scholar;Kanzaki et al., 1996Kanzaki H. Morishita Y. Akiyama H. Arata J. Adhesion of Staphylococcus aureus to horny layer: role of fibrinogen.J Dermatol Sci. 1996; 12: 132-139Abstract Full Text PDF PubMed Scopus (15) Google Scholar;Foster et al., 1997Foster T.J. Hartford O. O’connell D. Host-pathogen protein–protein interactions in staphylococcus.in: McCrae MA Saunders JR Smyth CJ Stow ND Molecular Aspects of Host–Pathogen Interaction. Cambridge University Press, Cambridge1997Google Scholar). Among these are coagulase and clumping factor (Phonimdaeng et al., 1990Phonimdaeng P. O’reilly M. Nowlan P. Bramkley A.J. Foster T.J. The coagulase of Staphylococcus aureus 8325–4: sequence analysis and virulence of site-specific coagulase-deficient mutants.Mol Microbiol. 1990; 4: 393-404Crossref PubMed Scopus (112) Google Scholar;McDevitt et al., 1992McDevitt D. Vaudaux P. Foster T.J. Genetic evidence that bound coagulase of Staphylococcus aureus is not clumping factor.Infect Immun. 1992; 60: 1514-1523Crossref PubMed Google Scholar, McDevitt et al., 1994McDevitt D. Francois P. Vaudaux P. Foster T.J. Molecular characterization of the clumping factor (fibrinogen receptor) of Staphylococcus aureus.Mol Microbiol. 1994; 11: 237-248Crossref PubMed Scopus (428) Google Scholar). Genetic manipulation of the well-characterized strain Newman allows the construction of stable mutants of this strain deficient in the expression of Protein A, coagulase, clumping factor, and fibronectin-binding protein A and B (Patel et al., 1987Patel A.H. Nowlan P. Weavers E.D. Foster T. Virulence of Protein A-deficient and alpha-toxin-deficient mutants of Staphylococcus aureus isolated by allele replacement.Infect Immun. 1987; 55: 3103-3110Crossref PubMed Google Scholar;Phonimdaeng et al., 1990Phonimdaeng P. O’reilly M. Nowlan P. Bramkley A.J. Foster T.J. The coagulase of Staphylococcus aureus 8325–4: sequence analysis and virulence of site-specific coagulase-deficient mutants.Mol Microbiol. 1990; 4: 393-404Crossref PubMed Scopus (112) Google Scholar;McDevitt et al., 1994McDevitt D. Francois P. Vaudaux P. Foster T.J. Molecular characterization of the clumping factor (fibrinogen receptor) of Staphylococcus aureus.Mol Microbiol. 1994; 11: 237-248Crossref PubMed Scopus (428) Google Scholar;Greene et al., 1995Greene C. McDevitt D. Francois P. Vaudaux P.E. Lew D.P. Foster T.J. Adhesion properties of mutants of Staphylococcus aureus in fibronectin-binding proteins and studies on the expression of the fnb genes.Mol Microbiol. 1995; 17: 1143-1152Crossref PubMed Scopus (232) Google Scholar). To investigate their capacity to adhere to human keratinocytes, we established an in vitro adherence assay that allows the quantitation of adherent bacteria using the biotin/streptavidine labeling system. In addition the influence of pH values and temperature on bacterial adherence was investigated. The HaCaT keratinocyte cell line that was generously provided by Prof. Fusenig (DKFZ, Heidelberg, Germany) was used throughout the study. Cells were grown in Dulbecco’s keratinocyte SF medium (Gibco, Grand Island, NY) with the addition of epidermal growth factor and bovine pituary extract (Gibco) at 37°C and 5% CO2. No fetal calf serum was added to the medium. Cells were grown in 96 well plates (Nunc, Roskilde, Denmark) until confluent monolayers were obtained. Repeated counts of the keratinocytes in a Neubauer counting chamber at that point revealed a concentration of 1.5 × 104 cells per well. In order to minimize unspecific adhesion the keratinocyte plates were blocked for 120 min with 2% bovine serum albumin (Boehringer, Mannheim, Germany) in phosphate-buffered saline (PBS) before starting the assay. Staphylococcus aureus strain Newman is a standard strain and has been used in previous studies (Cole and Silverberg, 1986Cole G.W. Silverberg N.L. The adherence of Staphylococcus aureus to human corneocytes.Arch Dermatol. 1986; 122: 166-169Crossref PubMed Scopus (92) Google Scholar;Moreillon et al., 1995Moreillon P. Entenza J.M. Francioli P. McDevitt D. Foster T.J. Francois P. Vaudaux P. Role of Staphylococcus aureus coagulase and clumping factor in pathogenesis of experimental endocarditis.Infect Immun. 1995; 63: 4738-4743Crossref PubMed Google Scholar). The following genetically manipulated strains were used for the determination of staphylococcal adhesion structures: strain DU5852 is a clumping factor negative mutant that was constructed by transposon Tn917 mutagenesis of strain Newman, forming clfA1::Tn917 (McDevitt et al., 1994McDevitt D. Francois P. Vaudaux P. Foster T.J. Molecular characterization of the clumping factor (fibrinogen receptor) of Staphylococcus aureus.Mol Microbiol. 1994; 11: 237-248Crossref PubMed Scopus (428) Google Scholar). Strain DU5854 is a coagulase-defective mutant of strain Newman with a deletion in the coa gene substituted by a fragment encoding tetracycline resistance (Δcoa::TcR) (McDevitt et al., 1992McDevitt D. Vaudaux P. Foster T.J. Genetic evidence that bound coagulase of Staphylococcus aureus is not clumping factor.Infect Immun. 1992; 60: 1514-1523Crossref PubMed Google Scholar). Strain DU5886 is strain Newman carrying mutations in the fibronectin-binding proteins A + B, marked by a tetracycline resistance in the fibronectin-binding protein A (fnbA::TcR) and a erythromycin resistance in the fibronectin-binding protein B (fnbB::EmR) (Greene et al., 1995Greene C. McDevitt D. Francois P. Vaudaux P.E. Lew D.P. Foster T.J. Adhesion properties of mutants of Staphylococcus aureus in fibronectin-binding proteins and studies on the expression of the fnb genes.Mol Microbiol. 1995; 17: 1143-1152Crossref PubMed Scopus (232) Google Scholar). Strain DU5873 is a Protein A-negative Newman mutant with a tetracycline resistance in the spa gene (Δspa::TcR) (Patel et al., 1987Patel A.H. Nowlan P. Weavers E.D. Foster T. Virulence of Protein A-deficient and alpha-toxin-deficient mutants of Staphylococcus aureus isolated by allele replacement.Infect Immun. 1987; 55: 3103-3110Crossref PubMed Google Scholar). In order to verify the role of the relevant surface-protein genes the wild-type gene was restored in all mutants using staphylococcal shuttle plasmids in accordance to previously described reports (Greene et al., 1995Greene C. McDevitt D. Francois P. Vaudaux P.E. Lew D.P. Foster T.J. Adhesion properties of mutants of Staphylococcus aureus in fibronectin-binding proteins and studies on the expression of the fnb genes.Mol Microbiol. 1995; 17: 1143-1152Crossref PubMed Scopus (232) Google Scholar). Table 1 gives a summary of the strains and their relevant phenotype.Table IBacterial strains used in the studyS. aureus strainRelevant genotypeClumpingIgG-binding(OD)CoagulasetitresFibronectin binding (OD)Newmancoa+, clf +, spa+, fnbA/B++2.341>1:1282.147Parent strainDU5854Δcoa::Tcr+2.316>1:82.052Coagulase-deficientDU5852clfA1::Tn917–2.472>1:1281.786Clumping factor-deficientDU5873Δspa::TcR+0.232>1:1282.011Protein A-deficientDU5886fnbA::TcR; fnbB::EmR+2.186>1:1280.566Fibronectin-binding protein A/B-deficientDU5854/pcoaΔcoa::TcR/pcoa++2.250>1:1282.002Coagulase-restoredDU5852/pclfclfA1::Tn917/pclf++2.017>1:1282.201Clumping factor-restoredDU5873/pspaΔspa::TcR/pspa++1.989>1:1281.989Protein A-restoredDU5886/pfnbAfnbA::TcR; fnbB::EmR/pfnbA++2.004>1:1281.967Fibronectin-binding protein A-restoredDU5886/pfnbBΔfnbA::TcR;ΔfnbB::EmR/pfnbB++2.183>1:1282.111Fibronectin-binding protein B-restored Open table in a new tab For the detection of staphylococcal protein A we used a method described byvan Belkum et al., 1997van Belkum A. Riewarts Eriksen N.H. Sijmons M. et al.Coagulase and protein A polymorphisms do not contribute to persistence of nasal colonisation by.Staphylococcus Aureus. J Med Microbiol. 1997; 46: 222-232Crossref PubMed Scopus (67) Google Scholar quantifying this protein by its capacity to bind to purified IgG. For this test, overnight cultures of the strains were grown in tryptic soy broth (Gibco) and diluted to an OD570 of 0.1. One hundred microliters of this suspension were used for coating Nunc Immunoplates (Nunc). After 1 h at 20°C the plates were washed three times with PBS. Remaining binding sites were blocked with 2% bovine serum albumin (Boehringer) in PBS. After two washes with PBS, 50 μl of alkaline-phosphatase coupled rabbit anti-mouse immunoglobulin (Sigma, Munich, Germany) diluted in PBS were applied to the wells and incubated for 1 h at 20°C. After three washes with PBS, alkaline phosphatase substrate (Sigma) was added to the wells and allowed to interact for 30 min. The obtained optical density was read at 492 nm. All strains were tested in quadruplicate. Coagulase titres were measured by adding 0.5 ml of dilutions of staphylococcal culture supernatans to 0.5 ml of human plasma diluted 1:3 in PBS. The titer was the reciprocal of the highest dilution showing evidence for clotting after 24 h incubation at 37°C (Anderson et al., 1982Anderson J.C. Adlam C. Knights J.M. The effect of staphylocoagulase in the mammary gland of the mouse.Br J Exptl Pathol. 1982; 63: 336-340PubMed Google Scholar). Fibronectin binding was tested by screening their capacity to adhere to bovine fibronectin-coated 96 well culture plates (Becton-Dickinson, Heidelberg, Germany). Therefore the strains were grown overnight in tryptic soy broth (Gibco) at 37°C. Biotin-NHS (Sigma) was added at a concentration of 5 mM to the growth medium and was allowed to interact with the bacteria for 90 min at 37°C. Bacteria were harvested by 10 min of centrifugation at 3000 r.p.m. and resuspended in PBS. In order to remove unbound biotin from the suspension this washing procedure was repeated three times. The pelleted bacteria were diluted in PBS to an optical density of 0.1 at 570 nm. Two hundred microliters of this dilution were applied to each well of the fibronectin coated plates and were incubated at 37°C for 1 h. After three washes with PBS, 50 μl alkaline phosphatase coupled streptavidine at a concentration of 250 ng per ml (Sigma) was added and incubated for 30 min, followed by another three washes. Thereafter 50 μl of alkaline phosphatase substrate (Sigma) was added to the wells and the reaction was stopped with 3 M NaOH after 30 min. Extinction was read at 405 nm. Strains were tested in triplicate. Measurement of cell clumping was detected using purified fibrinogen (Sigma) in the slide agglutination test. Bacteria were grown in tryptic soy broth (Gibco) into early logarithmic growth phase (≈4 h) at 37°C with gentle shaking. Labeling of the bacteria with biotin was carried out in modification of a previously described method (Tompkins et al., 1990Tompkins C.T. Hatcher V.B. Patel D. Orr G.A. Higgins L.L. Lowy F.D. A human endothelial cell membrane protein that binds Staphylococcus aureus in vitro.J Clin Invest. 1990; 85: 1248-1254Crossref PubMed Scopus (42) Google Scholar;von Boxberg et al., 1990von Boxberg Y. Wütz R. Schwarz U. Use of the biotin-avidin system for labelling, isolation and characterization of neural cell-surface proteins.Eur J Biochem. 1990; 190: 249-256Crossref PubMed Scopus (34) Google Scholar). In brief, biotin-NHS (Sigma) was added at a concentration of 5 mM to the growth medium and was allowed to interact with the bacteria for 90 min at 37°C. Bacteria were harvested by 10 min of centrifugation at 3000 r.p.m. and resuspended in PBS. In order to remove unbound biotin from the suspension this washing procedure was repeated three times. The pelleted bacteria were diluted in PBS and the optical density (OD) was controlled in a spectrophotometer at 570 nm. One hundred microliters of the bacterial suspension was added to each confluent keratinocyte well and the plates were incubated at 37°C. After various time points the plates were washed three times with PBS and 100 μl of streptavidine coupled with alkaline phosphatase (Sigma) at a concentration of 250 ng per ml was added to each well. After 30 min of incubation at 37°C 100 μl alkaline phosphatase substrat (Sigma) was added and was allowed to interact with the enzyme for 30 min before the reaction was stopped with the addition of 50 μl 3 M NaOH. Optical density (OD) was measured at a wavelength of 405 nm. All strains were tested in quadruplicate. Internal controls were used as follows: to rule out unspecific binding of the bacteria to the culture plates, appropriate dilutions of the bacterial suspension were incubated on an empty row of the plates and treated in the same manner as the keratinocyte-coated rows. The obtained OD was considered unspecific and was substracted from the experiment results as background value. To control for comparable labeling of the different strains with biotin, the labeled strains were diluted (OD570 0.1) in PBS and 100 μl of the dilution were exposed to 100 μl of several concentrations (25 ng per ml, 250 ng per ml, and 2.5 μg per ml) of the alkaline phosphatase/streptavidine complex for 15 min at 37°C and were then allowed to interact with 50 μl of the alkaline phosphatase substrat for 30 min. Strains were only considered equally labeled when the photometric evaluation (OD405) of this dilutions yielded comparable results. The possibility of endogenous phosphatase activity of the tested strains influencing the results of the experiment was ruled out by incubating 4 × 108 bacteria, diluted in PBS (OD570 0.5) with the alkaline phosphatase substrate for 60 min. This control showed negative results for all tested strains. Binding of streptavidine to the keratinocytes was separately tested and revealed no positive results. The obtained results of the adhesion assay in the dose defining experiment were verified with the colony forming units (CFU) count. For this procedure the bacteria/keratinocytes complex was lifted with trypsin-ethylenediamine tetraacetic acid (Sigma) from the plates and was diluted in distilled water to rupture the keratinocytes. Dilutions of the bacteria were plated on Columbia agar from BioMerieux (France) containing 5% sheep blood. After 24 h at 37°C the CFU were counted. Cells were grown on sterile glass slides in cell culture dishes in SFB medium until confluency was obtained. Slides were fixed in acetone and immunohistochemistry was carried out following standard protocols using the APAAP-technique (Dako, Hamburg, Germany). The anti-fibronectin antibody was obtained from Sigma and used in a 1:500 dilution in PBS. Keratinocytes were counterstained in Mayer’s Hematoxylin (Merck, Darmstadt, Germany) and staphylococci were stained with gentian violet in a modified Gram stain. The S. aureus mutant strains were controled for lack of elaboration of the deleted gene products by a battery of tests. Table 1 gives a summary of the specific results. Interestingly, despite the deletion of the fibronectin-binding protein as well as the Protein A encoding gene sequences, we still obtained a rest of binding to the fibronectin and IgG-coated wells, respectively. This result may represent a nonreceptor/ligand-specific attachment to these molecules. In order to define an adequate keratinocyte/bacteria ratio, the parent strain Newman was used in several dilutions. As shown in Figure 1 an OD570nm of 0.2 turned out to allow a good evaluation of S. aureus adherence. Bacterial counts at that OD revealed a number of 5 × 106 CFU per milliliter. This dilution was used throughout the following experiments. Counts for the adherent bacteria yielded results between 0.7 × 103 CFU per ml at OD570 0.05 and 0.9 × 108 CFU at OD570 1.0 on Columbia agar. Changes of the skin surface pH are known to influence the microbial colonization process. Therefore we screened the parent strain Newman for its adhesion properties at different pH values. This experiment showed optimal cell adherence at a slightly alkaline pH value between 7 and 8 (Figure 2). This pH was kept for testing the adherence of the mutant strains. Varying the incubation temperature resulted in highest adherence at 37°C (Figure 3), which was also used in the following experiments. Withdrawing the keratinocytes from epidermal growth factor for 24 h resulted in a clear decrease of adherence, possibly due to the diminished number of ligand structures on the keratinocye surface (data not shown). Testing the S. aureus mutant strains showed a decrease in the attachment to the HaCaT cells. As shown in Figure 4, the coagulase-negative mutant DU5854 showed only 53% of the adherence capacity of parent strain Newman, the clumping factor-deficient mutant DU5852 reached 50%, the fibronectin-binding-protein A and B-negative mutant DU5886 was reduced to 60% of the parent strain, and the Protein A-negative strain DU5873 reached 43%. Strains were read after 60 min. The results for DU5852 and DU5873 hit the criteria for statistical significance using the Student’s t test (p < 0.05), whereas the results for DU5854 and DU5886 remained sharply above the cut off for significance (p = 0.058 and p = 0.055, respectively). Restoring the wild-type gene of the knock-out mutants via the introduction of a shuttle plasmid carrying the responsible gene resulted in an overall regain of the adherence capacities of the strains, with the exception of the fibronectin-binding protein A and B negative double mutant that yielded statistically not significant lower adherence when complemented with either Fn-binding protein A or Fn-binding protein B. As shown in Figure 5, restoring the coagulase negative mutant resulted in 103%, restoring the clumping-factor-negative mutant resulted in 109%, and restoring the protein A-negative mutant resulted in 96% of the parental adherence. The fnb A and B double mutant yielded 85% when complemented with fnb A and 89% when complemented with fnb B. To demonstrate the expression of fibronectin as a matrix protein and well-known staphylococcal adhesion factor, we stained for fibronectin with a monoclonal antifibronectin antibody in the presence of strain Newman. Figure 6b shows adhesion of the staphylococci to the marked matrix protein and, although to a lesser extent, to additional keratinocyte structures. Attachment to and colonization of the human skin by S. aureus has previously been reported to play a crucial role in the pathogenesis and the perpetuation of several dermatologic diseases (Aly et al., 1977Aly R. Maibach H.J. Shinefield H.R. Microbial flora of atopic dermatitis.Arch Dermatol. 1977; 113: 780-782Crossref PubMed Scopus (221) Google Scholar;Abeck and Ruzicka, 1992Abeck C. Ruzicka T. Bacteria and atopic eczema: merely association or etiologic factor?.in: Ruzicka T Ring J Przybilla B Handbook of Atopic Eczema. Springer-Verlag, Berlin1992: 212-220Google Scholar;Nickoloff et al., 1993Nickoloff B.J. Mitra R.J. Green J. Shimizu Y. Thompson C. Turka L.A. Activated keratinocytes present bacterial-derived superantigens to T-lymphocytes: relevance to psoriasis.J Dermatol Sci. 1993; 6: 127-133Abstract Full Text PDF PubMed Scopus (25) Google Scholar;Cooper, 1994Cooper K.D. Atopic dermatitis: recent trends in pathogenesis and therapy.J Invest Dermatol. 1994; 102: 128-137Abstract Full Text PDF PubMed Google Scholar;Tokura et al., 1995Tokura Y. Yagi H. Ohshima A. et al.Cutaneous colonization with staphylococci influences the disease activity of Sézary syndrome: a potential role for bacterial superantigens.Br J Dermatol. 1995; 133: 6-12Crossref PubMed Scopus (69) Google Scholar). Quantitation of this process in recent studies was mainly based on counting the bacteria adherent to the single epidermal cell types microscopically (Cole and Silverberg, 1986Cole G.W. Silverberg N.L. The adherence of Staphylococcus aureus to human corneocytes.Arch Dermatol. 1986; 122: 166-169Crossref PubMed Scopus (92) Google Scholar). This technique, however, unfortunately lacks the possibility to test a defined layer of cells concerning the bilateral expression of adhesins. The assay described here provides a rapid and reproducible method for the quantitation of staphylococcal adherence factors to cultured human keratinocytes using the biotin/streptavidine system. In addition, the investigation of genetically engineered mutants of the pathogenetic S. aureus strain Newman allows the identification of bacterial structures playing a role in the colonization process by disabling the bacteria in their expression of defined surface proteins. Already previous studies suggested specific receptor/ligand binding as a major adhesion factor for S. aureus to keratinocytes and a variety of structures have been supposed as a specific counterpart for the staphylococcal protein A (Bibel et al., 1983Bibel D.J. Aly R. Shinefield H.R. Maibach H.I. The Staphylococcus aureus receptor for fibronectin.J Invest Dermatol. 1983; 80: 494-496Abstract Full Text PDF PubMed Scopus (29) Google Scholar;Cole and Silverberg, 1986Cole G.W. Silverberg N.L. The adherence of Staphylococcus aureus to human corneocytes.Arch Dermatol. 1986; 122: 166-169Crossref PubMed Scopus (92) Google Scholar). To approach this question we used various genetically defined mutants of strain Newman that have been generated in a battery of experiments dealing with the colonization of S. aureus to various human tissues (Patel et al., 1987Patel A.H. Nowlan P. Weavers E.D. Foster T. Virulence of Protein A-deficient and alpha-toxin-deficient mutants of Staphylococcus aureus isolated by allele replacement.Infect Immun. 1987; 55: 3103-3110Crossref PubMed Google Scholar;Phonimdaeng et al., 1990Phonimdaeng P. O’reilly M. Nowlan P. Bramkley A.J. Foster T.J. The coagulase of Staphylococcus aureus 8325–4: sequence analysis and virulence of site-specific coagulase-deficient mutants.Mol Microbiol. 1990; 4: 393-404Crossref PubMed Scopus (112) Google Scholar;McDevitt et al., 1994McDevitt D. Francois P. Vaudaux P. Foster T.J. Molecular characterization of the clumping factor (fibrinogen receptor) of Staphylococcus aureus.Mol Microbiol. 1994; 11: 237-248Crossref PubMed Scopus (428) Google Scholar;Greene et al., 1995Greene C. McDevitt D. Francois P. Vaudaux P.E. Lew D.P. Foster T.J. Adhesion properties of mutants of Staphylococcus aureus in fibronectin-binding proteins and studies on the expression of the fnb genes.Mol Microbiol. 1995; 17: 1143-1152Crossref PubMed Scopus (232) Google Scholar). To some extent we could find parallels to keratinocytes. The important role of Protein A for the staphylococcal adhesion to human epidermal cells has been previously described (Cole and Silverberg, 1986Cole G.W. Silverberg N.L. The adherence of Staphylococcus aureus to human corneocytes.Arch Dermatol. 1986; 122: 166-169Crossref PubMed Scopus (92) Google Scholar). This result was confirmed by the low adhesion capacities of Protein A-negative mutant DU5873 to the cultured monolayers. Although its counterpart on human keratinocytes and corneocytes has still not been identified so far, the strong affinity of Protein A for the Fc-terminus of human IgG-antibodies in inflamed tissue might enable S. aureus to gain access to the human epidermis. As with our model the presence of such antibodies could be ruled out, a second, maybe not receptor-specific adhesion mechanism has to be considered. The diminished adherence of strain DU5886 impaired in the elaboration of the fibronectin-binding proteins A and B is consistent with the findings ofBibel et al., 1983Bibel D.J. Aly R. Shinefield H.R. Maibach H.I. The Staphylococcus aureus receptor for fibronectin.J Invest Dermatol. 1983; 80: 494-496Abstract Full Text PDF PubMed Scopus (29) Google Scholar, who previously described an essential role for this matrix protein in the adhesion process by using purified fibronectin. Their finding that Protein A is not the ligand for human fibronectin has been supported by the identification of the genes responsible for the staphylococcal fibronectin-binding protein A and B (Jönsson et al., 1991Jönsson K. Signäs C. Müller H-P Lindberg M. Two different genes encode fibronectin binding-proteins in Staphylococcus aureus..Eur J Biochem. 1991; 202: 1041-1048Crossref PubMed Scopus (291) Google Scholar). This group could show that two different genes separated by a stretch of 682 bp on the chromosome of S. aureus are responsible for fibronectin binding. Important work was added byGreene et al., 1995Greene C. McDevitt D. Francois P. Vaudaux P.E. Lew D.P. Foster T.J. Adhesion properties of mutants of Staphylococcus aureus in fibronectin-binding proteins and studies on the expression of the fnb genes.Mol Microbiol. 1995; 17: 1143-1152Crossref PubMed Scopus (232) Google Scholar, who demonstrated that deletion in only one of these genes did not lead to a significant decrease in the binding of S. aureus to fibronectin-coated coverslips, whereas the double mutant almost completely lost its binding capacity. Restoring either one of the fibronectin-binding proteins on shuttle plasmids resulted in full adhesion to the coverslips. As a consequence of these findings we used the double mutant DU5886 in our assay. The decreased adherence of this mutant to the keratinocytes could be partially restored when either a fnbA- or a fnbB-carrying plasmid was introduced into DU5886, although neither restoration resulted in complete regain of adherence; however, these results failed statistical significance. These findings confirm an important role for human fibronectin as ligand for S. aureus on human keratinocytes, as further confirmed by the immunohistochemical colocalization in the photomicrographs. Investigating the coagulase-negative and clumping factor-negative variants DU5852 and DU5854 revealed some interesting findings. Although the main ligand for these virulence factors is represented by the plasma protein fibrinogen (McDevitt et al., 1992McDevitt D. Vaudaux P. Foster T.J. Genetic evidence that bound coagulase of Staphylococcus aureus is not clumping factor.Infect Immun. 1992; 60: 1514-1523Crossref PubMed Google Scholar;Dickinson et al., 1995Dickinson R.B. Nagel J.A. McDevitt D. Foster T.J. Proctor R.A. Cooper S.L. Quantitative comparison of clumping factor- and coagulase-mediated Staphylococcus aureus adhesion to surface-bound fibrinogen under flow.Infect Immun. 1995; 63: 3143-3150PubMed Google Scholar;Moreillon et al., 1995Moreillon P. Entenza J.M. Francioli P. McDevitt D. Foster T.J. Francois P. Vaudaux P. Role of Staphylococcus aureus coagulase and clumping factor in pathogenesis of experimental endocarditis.Infect Immun. 1995; 63: 4738-4743Crossref PubMed Google Scholar;Vaudaux et al., 1995Vaudaux P.E. Francois P. Proctor R.A. et al.Use of adhesion-defective mutants of Staphylococcus aureus to define the role of specific plasma proteins in promoting bacterial adhesion to canine arterovenous shunts.Infect Immun. 1995; 63: 585-590Crossref PubMed Google Scholar), the defective mutants showed decreased adherence capacities to the cultured cells. Additionally to the serum fibrinogen binding, a second cellular-based adhesion mechanism has to be considered. In the clinical setting of chronically inflamed skin the action of these two staphylococcal virulence factors can be explained by the increased presence of fibrinogen on epidermal structures. Introducing the wild-type plasmid into the clumping-factor defective variant interestingly resulted in a slightly increased adherence of 109%. This phenomenon, however, might be the consequence of a multicopy effect that undergo bacterial plasmids when duplicated independently from the chromosomal duplication process. These results demonstrate that more than one adhesin of S. aureus plays a role in the adhesion to the human epidermis. Recent findings in the molecular organization of the bacterial genome revealed important insights of the regulation of a variety of staphylococcal proteins. Regulating operons as agr and sar are able to control the expression of surface structures and enable this microorganism to increase the production of adhesins (Kornblum et al., 1990Kornblum J. Kreiswirth B. Projan S.J. Ross H. Novick R.P. Agr: a polycistronic locus regulating exoprotein synthesis in Staphylococcus aureus, p.in: Novick RP Molecular Biology of the Staphylococci. VCH Publishers, New York1990: 373-402Google Scholar;Cheung and Projan, 1994Cheung A.L. Projan S.J. Cloning and sequencing of sarA of S. aureus, a gene required for the expression of agr.J Bacteriol. 1994; 173: 1827-1830Google Scholar, Cheung et al., 1997Cheung A.L. Eberhardt K. Heinrichs J.H. Regulation of Protein A synthesis by the sar and agr loci of Staphylococcus aureus.Infect Immun. 1997; 65: 2243-2249Crossref PubMed Google Scholar). In addition, environmental factors may also have an impact on bacterial colonization. One of these is the skin surface pH for which we could show an increased adherence of strain Newman at alkaline pH values, which are characteristic for diseases with epidermal barrier dysfunction such as atopic dermatitis. Changes in the pH on the other hand are known to have an impact on the expression of the above mentioned regulatory systems in S. aureus (Regassa et al., 1992Regassa L.B. Betley M.J. Novick R.P. Glucose and nonmaintained pH decrease expression of the accessory gene regulator (agr) in Staphylococcus aureus.Infect Immun. 1992; 60: 3381-3388PubMed Google Scholar). As in recent years a variety of additional human matrix proteins have been identified to act as receptors for staphylococcal proteins such as fibronectin, vitronectin, and laminin (Lopes et al., 1985Lopes J.D. dos Reis M. Brentani R.R. Presence of laminin receptors in Staphylococcus aureus..Science. 1985; 229: 275-276Crossref PubMed Scopus (174) Google Scholar;Jönsson et al., 1991Jönsson K. Signäs C. Müller H-P Lindberg M. Two different genes encode fibronectin binding-proteins in Staphylococcus aureus..Eur J Biochem. 1991; 202: 1041-1048Crossref PubMed Scopus (291) Google Scholar;Liang et al., 1993Liang O.D. Maccarena M. Flock J-I Paulsson M. Preissner K.T. Waldström T. Multiple interactions between human vitronectin and Staphylococcus aureus.Biochim Biophys Acta. 1993; 1225: 57-63Crossref PubMed Scopus (34) Google Scholar); further genetic studies should focus on S. aureus mutants defective in the respective binding proteins alone and in combination. A possible therapeutic approach to prevent staphylococci to pathogenetically colonize the human skin could result in blocking the responsible structures. Parts of this work have been presented at the ESDR meeting in Rome on October 4–7 1997. The authors wish to thank T. Schäfer and W.-I. Worret for their help in preparing the manuscript. The technical assistance of S. Bogner is gratefully acknowledged. This work was supported by grant KKF/F-16–97 (M.M.) from the Technical University, Munich.
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