Surface proteins that promote adherence of Staphylococcus aureusto human desquamated nasal epithelial cells
© Corrigan et al; licensee BioMed Central Ltd. 2009
Received: 22 September 2008
Accepted: 30 January 2009
Published: 30 January 2009
The natural habitat of Staphylococcus aureus is the moist squamous epithelium in the anterior nares. About 20% of the human population carry S. aureus permanently in their noses and another 60% of individuals are intermittent carriers. The ability of S. aureus to colonize the nasal epithelium is in part due to expression of surface proteins clumping factor B (ClfB) and the iron-regulated surface determinant A (IsdA), which promote adhesion to desquamated epithelial cells present in the anterior part of the nasal vestibule. S. aureus strain Newman defective in IsdA and ClfB exhibited reduced but not completely defective adherence to squamous cells in indicating that other cell surface components might also contribute.
Surface proteins IsdA, ClfB, and the serine-aspartic acid repeat proteins SdrC, SdrD and SdrE were investigated to determine their contribution to the adherence of S. aureus to desquamated nasal epithelial cells. This was achieved by expression of ClfB, IsdA, SdrC, SdrD and SdrE on the surface of the surrogate Gram-positive host Lactococcus lactis and by isolating mutants of S. aureus Newman defective in one or more factor. The level of adherence of strains to squamous cells isolated from the nares of volunteers was measured. Results consistently showed that ClfB, IsdA, SdrC and SdrD each contributed to the ability of S. aureus to adhere to squamous cells. A mutant lacking all four proteins was completely defective in adherence.
The ability of S. aureus Newman to adhere to desquamated nasal epithelial cells is multifactorial and involves SdrD and SdrC as well as ClfB and IsdA.
Staphylococcus aureus causes community-acquired and nosocomial infections. Although multiple body sites such as the axilla and the perineum can be colonized, the most frequent site of carriage is the moist squamous epithelium of the anterior nares. About 20% of the human population carry S. aureus permanently in their noses and another 60% of individuals are intermittent carriers . The reasons for the variable tropism of S. aureus for the human nares are unclear. Higher carriage rates occur in white people , in men , in certain age groups  and in dialysis , diabetic  and AIDS patients . Infection rates are higher in carriers than in non-carriers and invasive disease is often caused by a patients' carried strain . However when infected, carriers suffer significantly fewer fatalities, suggesting that carriage stimulates a degree of protective immunity .
It has been suggested that the ability of S. aureus to adhere to human desquamated nasal epithelial cells is an important factor in determining nasal colonization . Both clumping factor B (ClfB) and iron regulated surface determinant protein A (IsdA) are expressed on the bacterial cell surface and promote adhesion to desquamated epithelial cells in vitro and colonization of the nares of rodents in in vivo models [10, 11], and in the case of ClfB , humans. Protection against colonization was elicited by active immunization of rodents with recombinant ClfB or IsdA, and in the case of ClfB, with a function-blocking monoclonal antibody.
The surface protein SasG can also promote adhesion to desquamated nasal epithelial cells in vitro [13, 14]. However SasG is not expressed by many strains including Newman . A mutant of S. aureus strain Newman defective in IsdA and ClfB had reduced adherence to squamous cells but still bound at about 40% of the level of the wild-type . Since SasG is not expressed by strain Newman , other cell surface components are likely to be involved. It had been noted that the serine-aspartic acid repeat proteins SdrC and SdrD can also promote adhesion to squamous cells , although this has never been examined in detail. In this paper the role of surface proteins IsdA, ClfB, SdrC and SdrD in adhesion to desquamated cell has been systematically analyzed in order to determine the contribution of each under the same conditions. This was achieved by expression of ClfB, IsdA, SdrC and SdrD on the surface of the Gram-positive surrogate host Lactococcus lactis and by testing single and combined mutants of S. aureus Newman.
Expression of IsdA, ClfB, SdrC and SdrD by the surrogate host L. lactis
Adherence of S. aureusmutants to desquamated nasal epithelial cells
In order to determine the role of IsdA in adherence, mutants were grown to stationary phase in the iron limited medium RPMI and tested for adhesion to squamous cells. Newman wild-type and Newman clfA adhered at similar levels of ca 1300 bacteria per 100 squamous cells (Figure 4B). This confirms that ClfA does not promote adhesion to squamous cells. Disruption of ClfB, IsdA or SdrCDE in the clfA mutant host each caused a drop in adherence to ca 800 bacteria per 100 squamous cells (Figure 4B). The decrease was statistically significant for IsdA (P = 0.0389, compared to Newman clfA) but not for ClfB or SdrCDE (P = 0.0662 and 0.1852, respectively compared to Newman clfA). Combining the isdA and sdrCDE mutations, the clfB and sdrCDE mutations or the isdA and clfB mutations decreased adherence further (Figure 4B, P = 0.0352, 0.0135 and 0.0183, respectively compared to Newman clfA). Finally when a mutant lacking ClfA, ClfB, IsdA and SdrCDE was tested, only 200 bacteria adhered per 100 squamous cells. Of the Sdr proteins only SdrD and SdrE were expressed by Newman growing in RPMI (Figure 3) (IsdA and ClfB are also expressed under these conditions [12, 15]) and as SdrE does not promote adhesion it can be concluded that the decrease associated with the deletion of sdrCDE was due to the loss of the SdrD protein. In conclusion, these results are consistent with the data obtained with L. lactis and demonstrate a similar role for ClfB, IsdA, SdrC and SdrD in adhesion to squamous cells.
When the strains were grown under iron restricted conditions in RPMI, complementation with ClfB, IsdA, SdrC or SdrD each promoted adhesion (Figure 5B, P = 0.029, ClfB; P = 0.0536, SdrC; P = 0.0908, SdrD; P = 0.0384, IsdA). The conclusion about IsdA was drawn by comparing the level of adhesion promoted by the plasmid expressing both IsdA and IsdB with that expressing IsdB alone. Attempts to express IsdA alone in pCU1 were unsuccessful. These results were statistically significant except for those involving SdrC and SdrD. Expression of SdrE did not promote adhesion (Figure 5B). These results confirm that ClfB, IsdA, SdrC and SdrD are all important in adherence of S. aureus to desquamated nasal epithelial cells under growth conditions that pertain in vivo.
S. aureus is a commensal of the moist squamous epithelium of the anterior nares of a significant proportion of the population. Colonization is a known risk factor for the development of staphylococcal infections in the community and hospital. The causes of intermittent and persistent carriage are believed to be different. Persistent carriers are often colonised by a single strain of S. aureus over a long period of time, while intermittent carriers tend to carry different strains for briefer time periods [16, 17]. Persistent carriers also carry a higher load of bacteria in the nares than intermittent carriers [18, 19]. When volunteers were decolonized and were then inoculated with a mixture of S. aureus strains non-carriers eliminated the bacteria, whereas persistent carriers selected their original S. aureus colonizing strain from the mixture . These results indicate that certain host characteristics determine carriage status in particular individuals. This view is supported by the fact that certain age or ethnic groups seem to be predisposed to carriage [2, 3]. One determinant of varying patterns of nasal carriage may be differing expression levels of ligands for S. aureus on the surface of desquamated nasal epithelial cells.
In this study we used three donors to provide the desquamated nasal epithelial cells for adhesion experiments. They were selected because their cells supported a consistent level of adhesion. It has been noted that cells from different donors can provide widely variable levels of adhesion . The reason for this is not known. One possibility is different levels of expression of the ligands responsible for adherence promoted by one or more of the S. aureus surface proteins. It is imperative to perform a detailed comparative study of the ability of the surface proteins described here to support adhesion of bacteria to squamous cells from donors who are persistent carriers and those who are non-carriers. This could contribute to the knowledge of the contribution of host factors to carriage.
Surface proteins ClfB and IsdA have previously been shown to promote adhesion to squamous epithelial cells [9, 15] and are required for colonization of the nares of rodents [11, 15]. Both ClfB and IsdA have been shown to bind to proteins present in the envelope of cornified squamous epithelial cells. IsdA and ClfB both bind to cytokeratin 10 and loricrin  (Clarke, S. Walsh, E. J. Andre, G. Dufrene, Y. Foster, T. J. Foster, S. J. manuscript submitted). Loricrin accounts for 70 – 85% of the cornified envelope [23–25]. It is possible that differences in the level of expression of these proteins could contribute to the variation in carriage of S. aureus in the nares.
To investigate the contribution of each of five surface proteins (IsdA, ClfB, SdrC, SdrD and SdrE) to squamous cell adhesion, the proteins were expressed from the surrogate host L. lactis. Expression of IsdA, ClfB, SdrC and SdrD each resulted in increased adherence. Gene disruption and complementation experiments in S. aureus also showed a role for IsdA, ClfB, SdrC and SdrD in adhesion. SdrE did not promote adhesion by either L. lactis or S. aureus. Schaffer et al 2006 investigated whether SdrC or SdrD had a role in colonization of the nares in a mouse model. Mutants defective in SdrC or SdrD colonized mice to the same extent as the wild-type indicating that these proteins do not play a role colonization of the nares of mice . However, this does not necessarily mean that SdrC and SdrD have no role to play in colonization of the human nares.
Adherence to desquamated epithelial cells from the anterior nares is clearly multifactorial. When expression of IsdA, ClfB, SdrC and SdrD was disrupted in strain Newman the level of adherence was reduced to background. This does not support a role for wall teichoic acid (WTA) in promoting adhesion to squamous cells [21, 26]. SasG did not play a role in adherence of Newman to squamous cells because this protein was not expressed detectably by this strain despite the intact sasG gene being present . SasG might play a role in clinical isolates where expression occurs at higher levels.
It has been reported that WTA plays a prominent part in nasal colonization of the cotton rat model . The authors also demonstrated that teichoic acid promoted bacterial adhesion to normal epithelial cells. However the WTA apparently does not contribute to bacterial adhesion to desquamated nasal cells epithelial cells . This is consistent with the data reported here which indicates that only surface proteins are required for adhesion to squames. A multiple mutant defective in ClfB, SdrC, SdrD and IsdA did not adhere. If WTA contributed to adherence the multiple mutant would still have bound above background levels. Thus colonization of the cotton rat requires both WTA  and surface proteins  albeit at different stages in the process  and in different parts of the nares.
Eradication of carriage of S. aureus has been shown to reduce infection rates in dialysis, diabetic and AIDS patients [4–6]. Vaccination with IsdA and ClfB was effective in reducing S. aureus carriage in animal models [11, 15]. It has been suggested that immune responses in part determine the ability of humans to carry S. aureus in the nares. This study has confirmed the role of ClfB and IsdA in adhesion to desquamated nasal epithelial cells and has revealed important roles for SdrC and SdrD. Vaccination against two or more of these surface proteins could provide significant reduction in carriage and could potentially reduce the rate of infection and dissemination.
Escherichia coli strains were grown on Luria (L) agar or in L-broth (Difco). S. aureus strains were grown on tryptic soy agar (TSA; Oxoid), tryptic soy broth (TSB) or RPMI 1640 (Sigma). Cultures were grown in an orbital shaker at 200 rpm at 37°C. RPMI cultures were subcultured into fresh broth and grown for a further 15 h before harvesting. L. lactis strains were cultured in M17 medium (Difco) containing 0.5% (v/v) glucose without shaking at 30°C. Antibiotics (Sigma) were added when needed as follows: ampicillin (100 μg ml-1), erythromycin (10 μg ml-1), chloramphenicol (10 μg ml-1) or tetracycline (2 μg ml-1).
The wild-type strain S. aureus strain Newman (10) and Newman isdA (RC107 ΔisdA ) were subjected to allele replacement mutagenesis with the temperature sensitive plasmid pJH1  forming strains DU5999 clfA5  and DU6020 clfA5 isdA. The clfB::Emr mutation  and the ΔsdrCDE::Tcr mutation  were introduced by transduction using phage 85  in order to construct the following mutants of Newman: DU6000 clfA5 clfB::Emr , DU6021 clfA5 ΔsdrCDE::Tcr, DU6001 clfA5 clfB::Emr ΔsdrCDE::Tcr , DU6022 clfA5 isdA ΔsdrCDE::Tcr, DU6023 clfA5 isdA clfB::Emr ΔsdrCDE::Tcr. For each transductant the mutations were verified by PCR and Southern blotting.
Complementation analysis was performed by transferring into DU6023 clfA5 isdA clfB::Emr ΔsdrCDE::Tcr plasmid pCU1 containing the full length structural genes for S. aureus surface proteins as follows: pCU1sdrC+, pCU1sdrD+, pCU1sdrE+, pCU1clfB+ , pCU1isdAisdB+ and pCU1isdB+. The plasmids were maintained by selecting resistance to chloramphenicol (10 μg ml-1). In each case the gene was amplified from genomic DNA of strain Newman to include the promoter region and the downstream transcription terminator. In the case of isd proteins both the closely linked isdA and isdB genes and their cognate promoters were cloned together. The primers are described below.
Expression of surface proteins in L. lactis MG1363  was achieved by cloning open reading frames from Newman genomic DNA in-frame into the expression vector pKS80  forming pKS80sdrC+ (25), pKS80sdrD+ (25), pKS80sdrE+ (25), pKS80clfB+ (25) and pKS80isdA+ (this study). Plasmid transformants were selected and maintained in M17 medium containing erythromycin.
Standard procedures were used . Restriction enzymes and ligase (New England Biolabs or Roche) were used according to manufacturer's protocol, as was Pfu DNA polymerase (Roche). Oligonucleotides were purchased from Sigma-Genosys.
Plasmid and strain construction
Primers pCU1sdrCF (5'-CGGGGATCCAAGCTTAGATTAAAAGTGAG-'3) and pCU1sdrCR (5'-GCTCTAGACTGGGAATTTCTAAACAG-'3), pCU1sdrDF (5'-CGGGGATCCTTCTGTTTAGAAATTCCCAG-'3) and pCU1sdrDR (5'-GCTCTAGACCAGGCCTCACGGAC-'3) and pCU1sdrEF (5'-CCGGATCCGTAGAAACGAATAAGAAAAAGC-'3) and pCU1sdrER (5'-GCTCTAGAGTAATTCATATTATCGCCTC-'3) which all incorporate a 5' BamHI and '3 XbaI site, respectively, were used to amplify the sdrC, sdrD and sdrE genes, respectively, from strain Newman genomic DNA. The DNA containing the sdrC, sdrD and sdrE genes were digested with BamHI and XbaI and cloned between the BamHI and XbaI sites of plasmid pCU1.
Primers pCU1isdBF (5'-CAGCTGCAGCCTATGTCATAGATATTTCATAATC-'3) and pCU1isdBR (5'-CAGGAGCTCAGAGATTCTAAACGTATTCGTAAG-'3) which incorporate a 5' PstI and 3' XbaI site, respectively, were used to amplify the isdB coding sequence including the upstream promoter and Fur consensus sequence from strain Newman genomic DNA. The isdB coding sequence is located 203 bp downstream of the isdA coding sequence on the S. aureus chromosome. Primers pCU1isdAF (5'-CAGCTGCAGACATAATCCTCCTTTTTATGATTG-'3) and pCU1isdBR (5'-CAGGAGCTCAGAGATTCTAAACGTATTCGTAAG-'3) were used to amplify the isdA and isdB coding sequence including the upstream promoter and Fur consensus sequence of both genes. The 2.3 kb isdB and 3.6 kb isdAB coding sequences were digested with PstI and XbaI and cloned between the PstI and XbaI sites of plasmid pCU1. Plasmids pCU1isdB+ and pCU1isdAB+ were sequenced and screened by restriction mapping.
Plasmids pCU1, pCU1clfB+, pCU1sdrC+, pCU1sdrD+, pCU1sdrE+, pCU1isdB+ and pCU1isdAB+ were transformed into S. aureus RN4220 and transduced into strain Newman clfA clfB isdA sdrCDE selecting for chloramphenicol resistance.
Primers FpKisdA (5'-CGCTGATCAAACATTATTTAAACAGTAAGTATC-'3) and RpKisdA (5'-CGCTGATCATTATTTAGATTCTTTTCTTTTGA-'3) which incorporate a 5' and a 3' BclI site, respectively, were used to amplify the isdA coding sequence from genomic DNA. The PCR product was digested with BclI and cloned into BclI digested pKS80. This resulted in the open reading frame of isdA being fused to the ATG codon of the expression cassette to optimize translation and created the plasmid pKS80isdA+. The plasmid was sequenced, screened by restriction mapping and electroporated into competent L. lactis strain MG1363.
Western immunoblot analysis
Cell wall-associated proteins of S. aureus and L. lactis were prepared as previously described [35, 22]. For S. aureus exponential phase cultures were grown to an OD600 of 0.6. Stationary phase cultures were grown for 16 – 24 h. Cells were harvested, washed in PBS and resuspended to an OD600 of 1 in lysis buffer (50 mM Tris/HCl, 20 mM MgCl2, pH 7.5) supplemented with 30% (w/v) raffinose and 40 μl ml-1 protease inhibitors (Roche). Cell wall proteins were solubilized by incubation with lysostaphin (200 μgml-1) for 10 minutes at 37°C. Cell wall fractions were separated on 7.5% (w/v) polyacrylamide gels, electrophoretically transferred onto PVDF membranes (Roche), blocked in 10% (w/v) skimmed milk (Marvel) and probed with anti-ClfB antibodies (1:5,000; , anti-IsdA antibodies (1:2,000; a gift from Prof. P. Speziale, Department of Biochemistry, University of Pavia, Pavia, Italy) and anti-SdrC, anti-SdrD, anti-SdrE or anti-Sdr region B antibodies (1:2,000) . The specificity of each antibody is indicated by the fact that no immnocrossreactive bands appeared in mutant strains lacking the relevant antigen. Membranes were washed three times with gentle agitation for 15 min in TS-Tween (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.05% (v/v) Tween 20 (Sigma)). Bound antibodies were detected using horseradish peroxidase-conjugated protein A-peroxidase (1:500; Sigma). Proteins were visualised using the LumiGLO™ Reagent and peroxide detection system (Cell Signalling Technology®). Membranes were detected using Kodak X-ray film. The exposed films were fixed and developed using a Kodak X-OMAT 1000 Processor developing machine.
Bacterial adherence to desquamated epithelial cells
Bacterial adherence assays were performed as previously described . Briefly desquamated nasal epithelial cells were harvested from three healthy donors by vigorous swabbing of the anterior nares. One donor was a carrier of S. aureus while the other two were not. After washing in PBS, cells were adjusted to 1 × 105cell ml-1. Bacterial cells were washed with PBS and adjusted to 1 × 109cells ml-1. 100 μl volumes of bacterial and epithelial cells were mixed and incubated at 37°C for 1 h with occasional shaking before being captured on 12 μm isopore polycarbonate filters, washed with PBS, fixed and stained with 5% (w/v) crystal violet. The filters were mounted onto glass slides. The number of bacteria per 100 squames was counted using light microscopy.
Statistical analyses were determined by the Student t-test, using the online GraphPad software. Differences were considered significant if p values were less than 0.05.
Grants from Science Foundation Ireland and the Health Research Board are acknowledged. We thank Professor Simon Foster (University of Sheffield) for sending the isdA mutant of S. aureus Newman
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