Surface proteins that promote adherence of Staphylococcus aureus to human desquamated nasal epithelial cells

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Figure 1

Adherence of L. lactis expressing different surface proteins to desquamated nasal epithelial cells. L. lactis (pKS80), L. lactis (pKS80clfB⁺), L. lactis (pKS80sdrC⁺), L. lactis (pKS80sdrD⁺), L. lactis (pKS80sdrE⁺) and L. lactis (pKS80isdA⁺) grown to stationary phase were tested for their ability to bind to human desquamated epithelial cells. Counts represent the number of bacterial cells adhering to 100 squamous cells. Results are expressed as the mean of triplicate experiments +/- standard deviations.

Figure 2

Western immunoblot to detect expression of ClfB, SdrC, SdrD and SdrE. A-D. Cell wall associated proteins from strains Newman, Newman clfA, Newman clfA clfB, Newman clfA isdA clfB, Newman clfA clfB sdrCDE, Newman clfA isdA clfB sdrCDE, Newman clfA sdrCDE, Newman clfA isdA sdrCDE and Newman clfA isdA clfB sdrCDE (pCU1) and (A) Newman clfA isdA clfB sdrCDE (pCU1clfB⁺), (B) Newman clfA isdA clfB sdrCDE (pCU1sdrC⁺), (C) Newman clfA isdA clfB sdrCDE (pCU1sdrD⁺), (D) Newman clfA isdA clfB sdrCDE (pCU1sdrE⁺) were grown to the exponential phase in TSB, cell wall associated proteins were solubilised by lysostaphin and run on 7.5% SDS-PAGE gels. Western immunoblotting was performed with (A) rabbit anti-ClfB antibodies, (B) rabbit anti-SdrC antibodies, (C) rabbit anti-SdrD antibodies and (D) rabbit anti-SdrE antibodies and subsequently with HRP-conjugated protein A-peroxidase.

Figure 3

Western immunoblot to detect expression of surface protein under iron-limiting conditions. Bacteria were grown to stationary phase in RPMI. Cell wall associated proteins were solubilized with lysostaphin and separated on a 7.5% SDS-PAGE gel and detected with rabbit antibodies followed by HRP-conjugated protein A-peroxidase. (A). Newman wild-type, Newman clfA, Newman clfA clfB, Newman clfA isdA clfB, Newman clfA clfB sdrCDE, Newman clfA isdA clfB sdrCDE, Newman clfA isdA clfB sdrCDE (pCU1) and Newman clfA isdA clfB sdrCDE (pCU1clfB⁺). (B). Newman wild type, Newman clfA, Newman clfA isdA, Newman clfA isdA clfB, Newman clfA isdA sdrCDE, Newman clfA isdA clfB sdrCDE, Newman clfA isdA clfB sdrCDE (pCU1) and Newman clfA isdA clfB sdrCDE (pCU1isdAB⁺). (C). Newman clfA, Newman clfA sdrCDE, Newman clfA isdA sdrCDE, Newman clfA clfB sdrCDE, Newman clfA isdA clfB sdrCDE, Newman clfA isdA clfB sdrCDE (pCU1), Newman clfA isdA clfB sdrCDE (pCU1sdrC⁺), Newman clfA isdA clfB sdrCDE (pCU1sdrD⁺) and Newman clfA isdA clfB sdrCDE (pCU1sdrE⁺). The primary antibodies used were (A) rabbit anti-ClfB (B) rabbit anti-IsdA and (C) rabbit anti-SdrD B repeats.

Figure 4

Adherence of Newman mutants to desquamated nasal epithelial cells. The ability of (A) Newman clfA, Newman clfA clfB, Newman clfA sdrCDE and Newman clfA clfB sdrCDE grown to exponential phase in TSB and (B) Newman, Newman clfA, Newman clfA clfB, Newman clfA sdrCDE, Newman clfA isdA, Newman clfA isdA sdrCDE, Newman clfA clfB sdrCDE, Newman clfA isdA clfB, and Newman clfA isdA clfB sdrCDE grown to stationary phase in RPMI to adhere to desquamated human nasal epithelial cells was measured. The tenth track is a control without S. aureus showing background due to adherent bacteria from the donor. Counts represent the number of bacterial cells adhering to 100 squamous cells. Results are expressed as the mean of triplicate experiments +/- standard deviations.

Figure 5

Adherence of S. aureus Newman complemented mutants grown in TSB and iron restricted conditions to desquamated nasal epithelial cells. The ability of mutants of strain Newman carrying complementing pCU1 plasmids carrying surface protein genes to adhere to desquamated nasal epithelial cells was tested. Strains Newman clfA, Newman clfA isdA clfB sdrCDE, Newman clfA isdA clfB sdrCDE (pCU1), Newman clfA isdA clfB sdrCDE (pCU1clfB⁺), Newman clfA isdA clfB sdrCDE (pCU1sdrC⁺), Newman clfA isdA clfB sdrCDE (pCU1sdrD⁺), Newman clfA isdA clfB sdrCDE (pCU1sdrE⁺),) Newman clfA isdA clfB sdrCDE (pCU1isdAB⁺) and Newman clfA isdA clfB sdrCDE (pCU1isdB⁺) grown to the exponential phase in (A) TSB and to the stationary phase in (B) RPMI were tested for adherence. Counts represent the number of bacterial cells adhering to 100 squamous cells. Results are expressed as the mean of triplicate experiments +/- standard deviations.