Staphylococcus aureus Fibronectin Binding Protein A Mediates Biofilm Development and Infection

Abstract

Implanted medical device-associated infections pose significant health risks, as they are often the result of bacterial biofilm formation. Staphylococcus aureus is a leading cause of biofilm-associated infections which persist due to mechanisms of device surface adhesion, biofilm accumulation, and reprogramming of host innate immune responses. We found that the S. aureus fibronectin binding protein A (FnBPA) is required for normal biofilm development in mammalian serum and that the SaeRS two-component system is required for functional FnBPA activity in serum. Furthermore, serum-developed biofilms deficient in FnBPA were more susceptible to macrophage invasion, and in a model of biofilm-associated implant infection, we found that FnBPA is crucial for the establishment of infection. Together, these findings show that S. aureus FnBPA plays an important role in physical biofilm development and represents a potential therapeutic target for the prevention and treatment of device-associated infections.

Keywords: S. aureus; biofilm; fibronectin binding protein; infection.

FIG 1

Sae-mediated biofilm development in fetal bovine serum. GFP⁺ S. aureus strains LAC, UAMS-1, and Newman, including their respective isogenic mutants, were cultured statically in chambered cover glass at 37°C for 24 h in RPMI with/without 10% or 50% fetal bovine serum (FBS) or in 100% FBS. (A) Representative three-dimensional (3D) Z-stack confocal microscopy images of WT LAC biofilms developed in RPMI alone or with FBS as indicated. COMSTAT2 quantification of average biofilm thickness (in microns) and dimensionless roughness coefficients in LAC (B and C), UAMS-1 (D and E), and Newman (F and G) strains. Data are from three independent experiments and presented as means ± standard deviations (SDs). Statistical analyses were performed using a two-way analysis of variance (ANOVA) with Tukey’s post hoc multiple-comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, no statistically significant differences across the groups.

FIG 2

FnBPA-dependent biofilm development in FBS and human serum (HS). GFP⁺ S. aureus strain LAC and its isogenic ΔfnbA and ΔfnbB mutants were cultured statically in chambered cover glass at 37°C for 24 h in RPMI with/without 10% or 50% FBS or in 100% FBS (A and B) or HS (C and D) followed by COMSTAT2 quantification of average biofilm thickness (in microns) and dimensionless roughness coefficients. Data are from three independent experiments and presented as means ±SDs. Statistical analyses were performed using a two-way ANOVA with Tukey’s post hoc multiple-comparison test. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 3

Fibronectin binding protein gene expression in serum. Analysis of fnbA (A) and fnbB (B) expression in S. aureus LAC WT and isogenic ΔsaeS and SaeS^L18P mutant biofilms developed in RPMI with/without 10% or 50% FBS or 50% HS after 5 h. Data are from at least two independent experiments and presented as mean fold changes from LAC in RPMI ± SDs. Statistical analysis was performed using a two-way ANOVA with Tukey’s post hoc multiple-comparison test. *, P < 0.05; **, P < 0.01; ns, no statistically significant difference across the groups.

FIG 4

Immobilized human Fn binding. Biofilm-associated cells from S. aureus strains LAC (A), UAMS-1 (B), and Newman (C), including their respective isogenic mutants, cultured in RPMI with/without 10% or 50% FBS or in 100% FBS were collected and assessed for their binding capacity to immobilized human Fn as measured by crystal violet staining. Data are from at least two independent experiments and presented as mean absorbances at 595 nm (OD₅₉₅) ± SDs. Statistical analyses were performed using a two-way ANOVA with Tukey’s post hoc multiple-comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, no statistically significant difference across the groups.

FIG 5

FnBPA-mediated inhibition of macrophage biofilm invasion. CellTracker violet-labeled bone marrow-derived macrophage (MΦ) were added to WT LAC and ΔfnbA biofilms cultured in RPMI plus 50% FBS and assessed for MΦ invasion after 4 h. (A) Representative 3D Z-stack confocal images of BMDM (blue) invasion into LAC (top) and ΔfnbA (bottom) biofilms developed in RPMI plus 50% FBS. White arrows indicate BMDMs not clearly visible. (B) Quantified BMDM invasion as percentage of visible BMDMs in a sterile sample within the same x/y/z dimensions. Data are from three independent experiments and presented as means ± SDs. Statistical analyses were performed using a two-way ANOVA with Tukey’s post hoc multiple-comparison test. ****, P < 0.0001.

FIG 6

FnBPA is required for S. aureus catheter-associated infection. Implanted subcutaneous catheters were infected with 10³ WT LAC, ΔfnbA, ΔsaeS, or SaeS^L18P mutant strains. Mice were sacrificed 7 days postinfection, whereupon bacterial burdens associated with the catheter (A) and surrounding soft tissue (B) were determined. Data are from two independent experiments, and the horizonal lines represent the mean CFUs. Each symbol denotes an individual mouse, and gray symbols indicate bacterial titers falling below the limit of detection (dashed lines). Statistical analyses were performed using a Kruskal-Wallis one-way ANOVA with an uncorrected Dunn’s test for multiple comparisons. *, P < 0.05; ***, P < 0.001.