Genetic evidence for an alternative citrate-dependent biofilm formation pathway in Staphylococcus aureus that is dependent on fibronectin binding proteins and the GraRS two-component regulatory system - PubMed (original) (raw)

Genetic evidence for an alternative citrate-dependent biofilm formation pathway in Staphylococcus aureus that is dependent on fibronectin binding proteins and the GraRS two-component regulatory system

Robert M Q Shanks et al. Infect Immun. 2008 Jun.

Abstract

We reported previously that low concentrations of sodium citrate strongly promote biofilm formation by Staphylococcus aureus laboratory strains and clinical isolates. Here, we show that citrate promotes biofilm formation via stimulating both cell-to-surface and cell-to-cell interactions. Citrate-stimulated biofilm formation is independent of the ica locus, and in fact, citrate represses polysaccharide adhesin production. We show that fibronectin binding proteins FnbA and FnbB and the global regulator SarA, which positively regulates fnbA and fnbB gene expression, are required for citrate's positive effects on biofilm formation, and citrate also stimulates fnbA and fnbB gene expression. Biofilm formation is also stimulated by several other tricarboxylic acid (TCA) cycle intermediates in an FnbA-dependent fashion. While aconitase contributes to biofilm formation in the absence of TCA cycle intermediates, it is not required for biofilm stimulation by these compounds. Furthermore, the GraRS two-component regulator and the GraRS-regulated efflux pump VraFG, identified for their roles in intermediate vancomycin resistance, are required for citrate-stimulated cell-to-cell interactions, but the GraRS regulatory system does not impact the expression of the fnbA and fnbB genes. Our data suggest that distinct genetic factors are required for the early steps in citrate-stimulated biofilm formation. Given the role of FnbA/FnbB and SarA in virulence in vivo and the lack of a role for ica-mediated biofilm formation in S. aureus catheter models of infection, we propose that the citrate-stimulated biofilm formation pathway may represent a clinically relevant pathway for the formation of these bacterial communities on medical implants.

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Figures

FIG. 1.

Citrate stimulates cell-to-surface and cell-to-cell interactions. (A) The number of cells per microscopic field was determined at 45 min postinoculation by phase-contrast microscopy in the absence (no add'n) and presence of 0.2% citrate. *, P < 0.01 compared to no addition. (B) Visual aggregation assay with the WT and the fnbB mutant grown overnight with 0% or 0.2% citrate. The white arrow indicates the typical WT aggregate, and the black arrow indicates the altered aggregate formed by the fnbB mutant. (C) PIA/PNAG levels measured by dot blot assay. Extracts of the WT strain grown in the absence or presence of citrate were prepared as described in Materials and Methods and spotted in a series of fivefold dilutions. (D) Biofilm formation of the WT and fnb mutants was assessed in a 96-well plate assay in the absence or in the presence of 0.2% citrate (+Citrate). (E and F) Biofilm formation (E) and aggregation (F) phenotypes were assessed for the WT strain grown in the presence and absence of citrate and/or heparin. Biofilm formation and aggregation assays were performed as described in Materials and Methods.

FIG. 2.

Citrate-mediated gene expression. (A) qRT-PCR analysis of fnb gene expression in the WT strain in the absence (black bar) and presence (gray bar) of citrate. *, P < 0.05. (B) qRT-PCR analysis of fnb gene expression in the Δ_graR_ mutant in the absence (black bar) and presence (gray bar) of citrate. *, P < 0.05. The expression of the fnb genes is normalized to gyrB expression.

FIG. 3.

The GraRS two-component regulatory system is required for citrate-stimulated biofilm formation and aggregation. (A) Biofilm formation in the absence and presence of 0.2% citrate for the indicated strain. *, P < 0.001 compared to WT with no citrate; **, P = 0.026 compared to WT with no citrate; #, P = 0.98 compared to the graR mutant without citrate. (B) Aggregation assay after overnight growth for the indicated strains without and with 0.2% citrate. The arrow indicates the aggregate formed by the WT grown with citrate. (C) Quantification of an aggregation assay for citrate-stimulated aggregation by the WT and the Δ_graS_ mutant. *, P < 0.001 compared to the WT with no citrate. (D) VISA isolate Mu50 requires a functional graR gene for citrate-stimulated biofilm formation in a 96-well plate assay. Biofilm and aggregation assays were performed as described in Materials and Methods.

FIG. 4.

TCA cycle intermediates stimulate biofilm formation. (A) Diagram of the TCA cycle. Boxed compounds stimulate biofilm formation in an FnbA-dependent manner. (B) Biofilm formation by the WT (black bars) and the fnbA mutant (white bars) in the presence of TCA cycle intermediates. Saline served as a control in these studies. The biofilm assay was performed as described in Materials and Methods. (C) Biofilm formation by the WT (black bars) and the acn mutant (white bars) in the presence of TCA cycle intermediates. These assays were performed as described above (B). (D) Expression of the _fnbA_-gfp fusion was assessed in a solution containing TSB plus 0.2% glucose supplemented with saline or the indicated TCA cycle intermediates. Expression is presented as relative fluorescence units (RFU).

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Genetic evidence for an alternative citrate-dependent biofilm formation pathway in Staphylococcus aureus that is dependent on fibronectin binding proteins and the GraRS two-component regulatory system - PubMed (original) (raw)