Figure 1.
ClfB promotes adherence of S. aureus Newman and L. lactis to loricrin.
S. aureus Newman, Newman ΔclfB, L. lactis MG1363 (pKS80) and L. lactis MG1363 (pKS80:clfB) were tested for binding to ligands immobilized on 96-well plates. Bacteria were added to wells coated with immobilized GST-tagged recombinant human loricrin (Hlor), murine loricrin (MLor)) human K10 peptide (HK10) and murine K10 peptide (MK10)) (1 µM). Bacterial adherence was measured by staining with crystal violet and measurement of the absorbance at 570 nm. Values represent the mean ± SD of triplicate wells. The data shown is representative of two individual experiments. Statistical analysis was performed using an unpaired t test. * p<0.05, ** p<0.005, ***p<0.0005 versus binding of ClfB-expressing bacteria.
Figure 2.
Surface Plasmon Resonance analysis of the interaction of ClfB with loricrin and keratin.
Representative sensorgrams display binding of rClfB201–542 to and dissocation from (A) GST-HLor, (B) GST-HK10, (C) GST-MLor and (D) GST-MK10 in a single cycle kinetics assay. GST-tagged ligands were captured onto a CM5 chip coated with anti-GST IgG and were exposed to increasing concentrations of rClfB201–542. Binding is measured as response units (RU) against time. The affinities were calculated from curve fitting to a plot of the RU values against concentrations of rClfB201–542. Arrows indicate the time at which rClfB201–542 is injected. The data shown is representative of 3 individual experiments.
Table 1.
Affinities of ClfB N2N3201–542 for loricrin, keratin and fibrinogen using surface plasmon resonance.
Figure 3.
Inhibition of bacterial adherence and rClfB binding to immobilized ligands.
(A) L. lactis NZ9800 (pNZ8037), L. lactis NZ9800 (pNZ8037:clfB) and L. lactis NZ9800 (pNZ8037:clfBQ235A) were added to wells containing immobilized GST-tagged Hlor, MLor, HK10, MK10 (0.5 µM). Bacterial adherence was measured by staining with crystal violet and measurement of the absorbance at 570 nm. The data shown is representative of two individual experiments (B). S. aureus Newman pre-incubated with GST or HK10 (2 µM) was added to loricrin-coated microtitre wells (0.5 µM). Bacterial adherence was measured by staining with crystal violet and measurement of the absorbance at 570 nm and was expressed as a percentage of total binding. Values represent the mean ± SD of triplicate wells. The data shown is representative of two individual experiments. (C) Recombinant ClfB N23201–542 was pre-incubated with GST, HK10 or L2v (14 µM) before being added to loricrin-coated microtitre wells (0.5 µM). Bound protein was detected using HRP-conjugated anti-his antibodies and was expressed as a percentage of total bound protein. Values represent the mean ± SD of triplicate wells. The values shown are representative of 3 individual experiments. Statistical analysis was performed using an unpaired t-test. *** p<0.0005 versus binding of ClfB-expressing bacteria (A) or pre-incubation with GST (B, C).
Figure 4.
HLor region L2v blocks ClfB-mediated adherence of S. aureus to human desquamated epithelial cells.
S. aureus strains were grown to exponential phase in TSB (A) or in RPMI (B). Washed cells were incubated with recombinant GST or recombinant L2v-GST, or just resuspended in PBS, before being incubated with human nasal epithelial cells. Adherent bacteria were enumerated by microscopy and were expressed as a percentage of the positive control. Results are expressed as the mean ± SD of 3 independent experiments. Statistical analysis was performed using an unpaired t test.
Figure 5.
Nasal colonisation of S. aureus in the FVB wild-type and Lor−/− mouse.
Mice were inoculated intra-nasally with S. aureus Newman (2×108 CFU). Mice were euthanized and bacterial burden in the noses established on days 1, 3, 10 and 21. Results expressed as mean Log CFU per nose (n = 15, per group). Statistical analysis was performed using the Mann-Whitney test.
Table 2.
Systemic infection in Lor−/− mice.
Figure 6.
Expression of loricrin and cytokeratin during S. aureus nasal colonisation.
Nasal tissue from WT and Lor−/− mice was excised and homogenised in PBS. Soluble proteins were extracted and analysed by Western immunoblotting using rabbit anti-murine loricrin IgG followed by HRP-conjugated goat anti-rabbit IgG. Bound antibody was removed and the filter was re-probed with rabbit anti-murine K10 IgG followed by HRP-conjugated protein A. Band intensity was measured using ImageQuant software and was expressed as a percentage of the highest intensity band. Data represents mean ± SD, n = 4 mice, per group.
Figure 7.
Nasal colonisation of L. lactis expressing ClfB and Newman ΔclfB− in the FVB wild-type and Lor−/− mice.
(A) Mice were inoculated intra-nasally with L. lactis MG1363 (pKS80) or L. lactis MG1363 (pKS80:clfB) (2×1011 CFU). Mice were euthanized after 24 hours and the bacterial burden in noses was established. Inoculation with the empty vector (pKS80) did not result in significant colonisation (>5 CFU per nose) in either WT or Lor−/− mice. Statistical analysis was performed using the Mann-Whitney test. (B) Mice were inoculated intra-nasally with Newman or Newman ΔclfB (2×108 CFU). After 10 days, mice were euthanized and bacterial burden in the noses was established. Each dot indicates the number of CFU/nose for a single mouse. Results expressed as Log CFU per nose, median indicated by bar (n = 15–20 per group). Statistical analysis was performed using the Krustal-wallis test and Dunns Multiple Comparisons test. *p<0.05, **p<0.005.