σB Inhibits Poly- N-Acetylglucosamine Exopolysaccharide Synthesis and Biofilm Formation in Staphylococcus aureus

Abstract

Staphylococcus aureus clinical strains are able to produce at least two distinct types of biofilm matrixes: biofilm matrixes made of the polysaccharide intercellular adhesin (PIA) or poly-N-acetylglucosamine (PNAG), whose synthesis is mediated by the icaADBC locus, and biofilm matrixes built of proteins (polysaccharide independent). σ^B is a conserved alternative sigma factor that regulates the expression of more than 100 genes in response to changes in environmental conditions. While numerous studies agree that σ^B is required for polysaccharide-independent biofilms, controversy persists over the role of σ^B in the regulation of PIA/PNAG-dependent biofilm development. Here, we show that genetically unrelated S. aureus σ^B-deficient strains produced stronger biofilms under both static and flow conditions and accumulated higher levels of PIA/PNAG exopolysaccharide than their corresponding wild-type strains. The increased accumulation of PIA/PNAG in the σ^B mutants correlated with a greater accumulation of the IcaC protein showed that it was not due to adjustments in icaADBC operon transcription and/or icaADBC mRNA stability. Overall, our results reveal that in the presence of active σ^B, the turnover of Ica proteins is accelerated, reducing the synthesis of PIA/PNAG exopolysaccharide and consequently the PIA/PNAG-dependent biofilm formation capacity.IMPORTANCE Due to its multifaceted lifestyle, Staphylococcus aureus needs a complex regulatory network to connect environmental signals with cellular physiology. One particular transcription factor, named σ^B (SigB), is involved in the general stress response and the expression of virulence factors. For many years, great confusion has existed about the role of σ^B in the regulation of the biofilm lifestyle in S. aureus Our study demonstrated that σ^B is not necessary for exopolysaccharide-dependent biofilms and, even more, that S. aureus produces stronger biofilms in the absence of σ^B The increased accumulation of exopolysaccharide correlates with higher stability of the proteins responsible for its synthesis. The present findings reveal an additional regulatory layer to control biofilm exopolysaccharide synthesis under stress conditions.

Keywords: Biofilm; PNAG; SigB; Staphylococcus aureus; ica operon.

FIG 1

Mutation of σ^B in unrelated biofilm-positive S. aureus strains enhances biofilm formation. (a) Dot blot analysis of PNAG accumulation in different S. aureus strains. Cell surface extracts of biofilm cultures were spotted onto nitrocellulose filters. PNAG production was detected with anti-S. aureus PNAG antiserum. (b) Biofilm formation of the unrelated S. aureus strains and their respective mutants in σ^B (Δσ^B), icaADBC genes (Δica), and Δσ^B Δica. Bacteria were grown on polystyrene microtiter plates for 24 h. wt, wild type. (c) Biofilm formation of the wild-type S. aureus strains and their respective σ^B mutants on polystyrene microtiter plates after 6 h and 24 h. The bacterial cells were stained with crystal violet, and biofilms were quantified by solubilizing the crystal violet with alcohol-acetone and determining the absorbance at 595 nm. The error bars represent the standard deviations of the results of three independent experiments. *, P < 0.05; **, P < 0.01. (d) Colony morphologies of biofilm-positive strains and their isogenic σ^B mutants on Congo red agar after 24 h of incubation.

FIG 2

Influence of σ^B deletion on biofilm formation in continuous-flow microfermentors. (a) Biofilm development of the wild-type strain (wt) and its isogenic Δσ^B mutants on glass slides of microfermentors after 24 h. (b) Quantification of biofilms adhering to glass slides. The glass slides were placed into 10 ml of PBS. Cells were removed from the slides by vigorous vortexing. The optical density of the solution was measured at 650 nm (OD₆₅₀). The biofilm formation of S. aureus 10833 wt and Δσ^B was represented as 10 times the OD₆₅₀ of the glass slides. The box and whisker plot indicates high and low values, medians, and interquartile ranges. Each group contained 4 or 5 microfermentors. Statistical differences were determined with Mann-Whitney tests. The asterisks indicate differences in competition indexes greater than 1 (P < 0.05).

FIG 3

Analysis of PNAG accumulation in σ^B mutants. (a) Dot-blot analysis of PNAG accumulation in S. aureus wild-type and Δσ^B mutant strains. Cell surface extracts of biofilm cultures were treated with proteinase K and spotted onto nitrocellulose filters at different dilutions (1:10 to 1:5,000). PNAG production was detected with anti-S. aureus PNAG antiserum. (b) PNAG levels of the S. aureus 15981 Δica mutant and the Δσ^B Δica double mutant complemented with pSC18 plasmid, which carries the ica locus. For dot blot analysis, samples diluted 1:100, 1:1,000, and 1:5,000 were spotted onto nitrocellulose membranes, and PNAG production was detected with an anti-PNAG antiserum.

FIG 4

Analysis of ica operon expression in σ^B mutants. (a) PNAG accumulation at different growth stages: exponential (Exp) and stationary (ON). Samples diluted 1:100 or 1:5,000 were spotted onto nitrocellulose membranes. PNAG production was detected with anti-PNAG antiserum. (b) Effects of σ^B deletion on ica promoter activity in S. aureus strains 15981 and 132 (wt) and their respective Δσ^B mutants carrying pCN52-Pica_gfp at exponential and stationary phases. Expression of GFP under the control of the ica promoter (Pica_gfp) was determined by Western blotting using monoclonal antibodies. (c) Biofilm formation by S. aureus 15981 Phyp_ica and 132 Phyp_ica and their respective σ^B mutants on polystyrene microtiter plates after 4 h of incubation. The bacterial cells were stained with crystal violet. (d) PNAG accumulation in cell extracts of S. aureus 15981 Phyp_ica and 132 Phyp_ica and their respective σ^B mutants. PNAG production was detected by dot blot analysis using anti-S. aureus PNAG antiserum.

FIG 5

Analysis of the posttranscriptional regulation of the ica operon by σ^B. (a) Fusion of the 5ʹ UTR of the ica operon to gfpmut2 under the control of the Phyper promoter. Levels of GFP were determined in the S. aureus 132 and 15981 wild-type strains and their respective σ^B mutants by Western blotting using monoclonal antibodies against GFP. (b) Northern blot analysis of RNA harvested from S. aureus 15981 and 132 wild-type strains and their corresponding σ^B mutants. The strains were grown in TSB-gluc at 37°C until exponential phase. The lower gels show 16S ribosome bands stained with ethidium bromide as a loading control. The blots were probed with a riboprobe specific for icaA and icaC transcripts. The positions of RNA standards in kilobases are indicated.

FIG 6

σ^B influence on IcaC protein levels. (a) Immunodetection of IcaC-tagged protein in the wt and the σ^B mutants of S. aureus 15981 and 132 at exponential phase. Whole-cell bacterial lysates were subjected to electrophoretic separation in an SDS-12% polyacrylamide gel. The proteins were transferred onto a nitrocellulose membrane and probed with anti-Flag M2 MAb conjugated with peroxidase. Relative quantification of the Flag protein was obtained using the ImageJ program. A Coomassie-stained gel portion is shown as a loading control. (b) Immunodetection of IcaC-tagged protein upon blocking protein biosynthesis in exponentially growing cells. (c) Relative IcaC levels upon blocking protein biosynthesis as an average of the results of two independent experiments; the error bars indicate standard deviations.

FIG 7

Proposed model of σ^B regulatory effect on S. aureus biofilm formation. The transcriptional regulator σ^B induces PNAG-independent biofilm formation by repressing extracellular proteases, by inhibiting nuclease secretion, and/or by the activation of Atl. On the other hand, σ^B regulates PNAG-dependent multicellular behavior by modulating PNAG levels by at least repressing IS256 and translationally regulating Ica enzymes.