A novel Staphylococcus aureus biofilm phenotype mediated by the fibronectin-binding proteins, FnBPA and FnBPB

Abstract

Device-associated infections involving biofilm remain a persistent clinical problem. We recently reported that four methicillin-resistant Staphylococcus aureus (MRSA) strains formed biofilm independently of the icaADBC-encoded exopolysaccharide. Here, we report that MRSA biofilm development was promoted under mildly acidic growth conditions triggered by the addition of glucose to the growth medium. Loss of sortase, which anchors LPXTG-containing proteins to peptidoglycan, reduced the MRSA biofilm phenotype. Furthermore introduction of mutations in fnbA and fnbB, which encode the LPXTG-anchored multifunctional fibrinogen and fibronectin-binding proteins, FnBPA and FnBPB, reduced biofilm formation by several MRSA strains. However, these mutations had no effect on biofilm formation by methicillin-sensitive S. aureus strains. FnBP-promoted biofilm occurred at the level of intercellular accumulation and not primary attachment. Mutation of fnbA or fnbB alone did not substantially affect biofilm, and expression of either gene alone from a complementing plasmid in fnbA fnbB mutants restored biofilm formation. FnBP-promoted biofilm was dependent on the integrity of SarA but not through effects on fnbA or fnbB transcription. Using plasmid constructs lacking regions of FnBPA to complement an fnbAB mutant revealed that the A domain alone and not the domain required for fibronectin binding could promote biofilm. Additionally, an A-domain N304A substitution that abolished fibrinogen binding did not affect biofilm. These data identify a novel S. aureus biofilm phenotype promoted by FnBPA and FnBPB which is apparently independent of the known ligand-binding activities of these multifunctional surface proteins.

FIG. 1.

Structural organization of FnBPA from S. aureus 8325-4 and diagrammatic illustration of plasmid constructs lacking regions of FnBPA. Regions B, C, and D (tandem repeats 1 to 11) are required for fibronectin binding. Region A (comprising the subdomains N1, N2, and N3) binds fibrinogen and elastin. The LPXTG motif, required for sortase-mediated anchoring of the protein to the cell wall peptidoglycan, is indicated. The secretory signal sequence (S), wall-spanning (W), and membrane-spanning (M) domains are indicated.

FIG. 2.

Low pH-induced MRSA biofilm formation is triggered by supplementing the growth medium with 1% glucose. (A) Measurement of culture pH (over 24 h) of BH1CC grown in BHI and BHI-glucose media, BHI medium supplemented with 200 mM disodium phosphate (buffer), and BHI-glucose medium supplemented with 200 mM disodium phosphate (buffer). (B) Activation of BH1CC biofilm development under mildly acidic growth conditions. The starting pH of BHI medium was altered using acetic acid. (C) Biofilm development by BH1CC grown in BHI and BHI-glucose media, BHI medium supplemented with 200 mM disodium phosphate (buffer), and BHI-glucose medium supplemented with 200 mM disodium phosphate (buffer). Biofilm formation after growth for 24 h in tissue culture-treated 96-well plates was measured three times, and standard deviations are indicated.

FIG. 3.

Contribution of the srtA gene to biofilm regulation in S. aureus. (A) Impact of an srtA::Tc^r mutation and carriage of multicopy psrtA (srtA) and pLI50 (control) plasmids on biofilm development in the MRSA isolate BH1CC. (B and C) Impact of an srtA::Tc^r mutation on biofilm regulation in the MRSA isolates BH18, BH10, BH4, and BH38 (B) and the MSSA isolates BH48, BH49, and 8325-4 (C). The strains were grown in BHI medium or in BHI medium supplemented with 4% NaCl or 1% glucose at 37°C for 24 h. Biofilm formation in tissue culture-treated 96-well plates was measured three times, and standard deviations, which were less than 15%, are indicated.

FIG. 4.

Contribution of the fnbA and fnbB genes to biofilm regulation in S. aureus. (A and B) Impact of a fnbAB::Tc^r mutation on biofilm regulation in the MRSA isolates BH10, BH1CC, and BH4 (A) and the MSSA isolates BH48, BH49, and BH51 (B). The strains were grown in BHI, BHI-4% NaCl, and BHI-1% glucose media at 37°C for 24 h. (C) Impact of fnbA and fnbB single and double mutations and carriage of multicopy fnbA (pFnBPA4) and fnbB (pFnBPB4) plasmids on biofilm development in the MRSA isolates BH1CC, BH10, BH3, BH4, and B30. The strains were grown in BHI medium supplemented with 1% glucose at 37°C for 24 h. Biofilm formation in tissue culture-treated 96-well plates was measured three times, and standard deviations, which were less than 20%, are indicated where appropriate. (D) Biofilm development under flow conditions by the MRSA strains BH1CC (vector control), BH1CC fnbAB::Tc^r, and BH1CC fnbAB::Tc^r (pFnBPA4) using a Biosurface Technologies flow system. Strains were grown for 24 h at 37°C in BHI broth supplemented with 1% glucose, and the flow cells were photographed after 24 h.

FIG. 5.

Susceptibility of fnbA- and fnbB-induced MRSA (A) and MSSA (B) biofilms to treatment with sodium metaperiodate and proteinase K. All biofilms of strains carrying multicopy fnbA (pFnBPA4) and fnbB (pFnBPA4) plasmids were grown in BHI medium supplemented with 1% glucose at 37°C for 24 h prior to treatment.

FIG. 6.

Contribution of sarA to MRSA biofilm development, fnbA and fnbB transcription, and binding to immobilized human fibronectin. (A) Biofilm development by the MRSA isolates BH1CC, BH3, and BH4 and derivatives lacking the sarA gene and the sarA mutants with the multicopy fnbA (pFnBPA4) or fnbB (pFnBPB4) plasmid grown at 37°C for 24 h in BHI medium supplemented with 1% glucose. Biofilm formation was measured at least three times, and standard deviations, which were less than 20%, are indicated. (B) Comparative measurement of fnbA, fnbB, and gyrB (control) transcription in the MRSA isolates BH1CC, BH3, and BH4 and their isogenic ΔsarA mutants. RT-PCR analysis was performed on total RNA extracted from cultures grown at 37°C or 30°C to an A₆₀₀ of 2.0 in BHI medium supplemented with 1% glucose. These experiments were performed separately for each strain and its isogenic mutant, and representative results have been assembled. (C) Binding of the MRSA isolate BH1CC and BH1CC derivatives harboring mutations in the sarA and fnbAB loci to immobilized human fibronectin. Values represent the mean of triplicate wells.

FIG. 7.

Susceptibility of S. aureus biofilm development to treatment with exogenous V8 protease. (A) Biofilm development by the MRSA isolate BH1CC grown in BHI and BHI-glucose media with or without exogenous V8 protease (5 units/ml). (B) Biofilm development by the laboratory MSSA strain RN4220 grown in BHI, BHI-NaCl, and BHI-glucose media with or without exogenous V8 protease (5 units/ml). (C) Biofilm development by MRSA isolates DAR217 (CC5), DAR22 (CC5), DAR35 (CC8), BH4 (CC8), BH10 (CC22), DAR113 (CC22), DAR141 (CC30), DAR199 (CC30), and DAR70 (CC45) grown in BHI-glucose medium with or without exogenous V8 protease (5 units/ml). Biofilm formation was measured at least three times, and standard deviations, which were less than 20%, are indicated.

FIG. 8.

Role of FnBPA structural domains in MRSA biofilm development and binding to immobilized human fibronectin. (A) Complementation of biofilm development in MRSA fnbAB::Tc^r mutants by the A (pRM8) or BCD (pRM1) domains of the FnBPA protein. MRSA isolates BH1CC, BH10, BH3, BH4, and BH30 were grown in BHI medium supplemented with 1% glucose at 37°C for 24 h. (B) Complementation of fibronectin binding activity in MRSA fnbAB::Tc^r mutants by the A (pRM8) or BCD (pRM1) domains of the FnBPA protein. Cells of BH1CC, BH10, BH3, BH4, and BH30 were tested for their ability to bind to immobilized human fibronectin (5 μg/ml). (C) Inhibition of MRSA biofilm development by anti-FnBPA_37-544 A-domain antibodies. MRSA isolates BH1CC, BH10, BH4, and BH3 were grown in BHI medium supplemented with 1% glucose and various concentrations of polyclonal anti-FnBPA A-domain antibodies at 37°C for 24 h. Values represent the mean of triplicate wells. (D) Binding of the MRSA isolate BH1CC to immobilized human fibronectin in the presence and absence of polyclonal anti-FnBPA A-domain antibodies. BH1CC fnbAB::Tc^r was used as a negative control. Values represent the mean of triplicate wells.

FIG. 9.

Binding to human immobilized fibrinogen (A) and complementation of biofilm development (B) in MRSA fnbAB::Tc^r mutants by pRM8 (FnBPA A domain) carrying the N304A amino acid substitution. MRSA isolates BH1CC, BH10, BH3, and BH4 were grown in BHI medium supplemented with 1% glucose at 37°C for 24 h. Standard deviations are indicated.