Interconnections between Sigma B, agr, and proteolytic activity in Staphylococcus aureus biofilm maturation

Abstract

Staphylococcus aureus is a proficient biofilm former on host tissues and medical implants. We mutagenized S. aureus strain SH1000 to identify loci essential for ica-independent mechanisms of biofilm maturation and identified multiple insertions in the rsbUVW-sigB operon. Following construction and characterization of a sigB deletion, we determined that the biofilm phenotype was due to a lack of sigma factor B (SigB) activity. The phenotype was conserved in a sigB mutant of USA300 strain LAC, a well-studied community-associated methicillin-resistant S. aureus isolate. We determined that agr RNAIII levels were elevated in the sigB mutants, and high levels of RNAIII expression are known to have antibiofilm effects. By introducing an agr mutation into the SH1000 or LAC sigB deletion strain, S. aureus regained biofilm capacity, indicating that the biofilm phenotype was agr dependent. Protease activity is linked to agr activity and ica-independent biofilm formation, and we observed that the protease inhibitors phenylmethylsulfonyl fluoride and alpha-macroglobulin could reverse the sigB biofilm defect. Similarly, inactivating genes encoding both the aureolysin and Spl extracellular proteases in the sigB mutant restored biofilm capacity. Due to the growing link between murein hydrolase activity and biofilm maturation, autolysin zymography was performed, which revealed an altered profile in the sigB mutant; again, the phenotype could be repaired through protease inactivation. These findings indicate that the lack of SigB activity results in increased RNAIII expression, thus elevating extracellular protease levels and altering the murein hydrolase activity profile. Altogether, our observations demonstrate that SigB is an essential regulator of S. aureus biofilm maturation.

FIG. 1.

Biofilm phenotypes of transposon insertions in the rsbUVW-sigB locus. (A) Microtiter biofilm assays of the wild-type (SH1000), three transposon mutants in rsbUV genes, and constructed ΔsigB and ΔsigB Δagr mutants. The sarA::Kan mutant was included as a negative control. The crystal violet in each well was solubilized and the OD₅₉₅ was determined. (B) Graphic map of approximate locations of transposon insertions in the rsbUVW-sigB locus.

FIG. 2.

Confirmation of the constructed ΔsigB mutant. Plasmid pAH12 (P_asp23-mCherry) was used to measure SigB activity from the asp23 promoter in strains SH1000 (sigB⁺) and AH1012 (ΔsigB). Fluorescence readings for the mCherry reporter were taken after 20 h of growth of cells. For complementation, strain AH1012 (with pAH12) was transformed with plasmid pALC2109 (P_tet-sigB), and sigB expression from the tet promoter was induced with 50 ng/ml anhydrotetracycline.

FIG. 3.

Biofilm phenotypes of the ΔsigB mutant. For flow cells, strains SH1000 (sigB⁺) (A) and AH1012 (ΔsigB) (B) were transformed with plasmid pALC2084 (induced with 20 ng/ml anhydrotetracycline) to image biofilms with GFP. Biofilms were grown for 4 days in a once-through flow cell setup, and a z-series of images was taken by CLSM and reconstructed with Volocity software. Each side of a grid square is 20 μm in the image reconstruction. For complementation of the ΔsigB mutant, strain AH1012 was transformed with plasmid pALC2109. Complementation was assessed with microtiter (C) and flow cell (D) biofilm assays. In the microtiter biofilm, levels of uninduced and induced (50 ng/ml anhydrotetracycline) sigB expression were compared. For the flow cell, a biofilm of strain AH1012 with the sigB expression plasmid pALC2109 (induced with 50 ng/ml anhydrotetracycline) was grown and poststained with Syto9.

FIG. 4.

PIA production in ΔsigB mutants. Quantitative dot blot s analysis was performed with anti-PIA antiserum. S. epidermidis R97-03 was used as a positive control producer. For S. aureus, MN8m was used as a positive control, and a Δica mutation served as a negative control for the MN8, SH1000, and LAC genetic backgrounds. For testing, dot blot S assays were performed on strains SH1000 (wild-type [WT]), AH1012 (SH1000 ΔsigB), LAC, and AH1096 (LAC ΔsigB), along with the indicated controls. The dot blot s results were quantitated with ImageGauge software (Fuji) and plotted relative to S. epidermidis R97-03 (S. epi). P values of <0.05 as calculated by a Student's t test were considered statistically significant.

FIG. 5.

Interconnection between the SigB and agr regulatory systems. (A) Effect of the ΔsigB mutation on agr expression. Plasmid pAH8 (P3_agr-mCherry) was used to monitor agr activity in strains SH1000 (sigB⁺) and AH1012 (ΔsigB). Fluorescence readings for the mCherry reporter were taken after 8 h of growth in TSB. For complementation, strain AH1012 (with pAH8) was transformed with plasmid pALC2109 (P_tet-sigB), and sigB expression was induced with 50 ng/ml anhydrotetracycline. (B) Analysis of RNAIII levels with quantitative RT-PCR using strains SH1000, AH1012, and AH1012 with complementation plasmid pALC2109. RNAIII levels are relative to 16S RNA control transcript. (C) Flow cell biofilm of strain AH1106 (ΔsigB Δagr). Flow cell biofilms were grown for 4 days in a once-through setup, and cells were visualized with CLSM using GFP labeling with plasmid pALC2084 (induced with 20 ng/ml anhydrotetracycline). Each side of a grid square is 20 μm in the CLSM image reconstruction.

FIG. 6.

Effect of PMSF on biofilm formation in a sarA mutant and SigB-defective strains. Microtiter biofilm assays were performed with SH1000 (sarA⁺ sigB⁺), SH1002 (sarA::Kan), AH528 (rsbU::Tn5), and AH1012 (ΔsigB). Biofilms were grown with TSB alone, 2% DMSO, or 400 μM PMSF dissolved in DMSO. Biofilm quantitation is plotted relative to the OD₅₉₅ of SH1000 biofilms.

FIG. 7.

Effect of protease mutations on biofilm formation in a ΔsigB mutant. (A) Microtiter biofilm assays of SH1000, AH1012 (ΔsigB), AH750 (Δaur Δspl), and AH1136 (ΔsigB Δaur Δspl) strains. (B) Flow cell biofilm of strain AH1136. Flow cell biofilms were grown for 4 days in a once-through setup, and cells were visualized with CLSM using GFP labeling with plasmid pALC2084 (induced with 20 ng/ml anhydrotetracycline). Each side of a grid square is 20 μm in the CLSM image reconstruction.

FIG. 8.

LAC wild-type and ΔsigB mutant phenotypes. (A) Plasmid pAH6 (P_asp23-mCherry) was used to measure SigB activity from the asp23 promoter in strains LAC (sigB⁺) and AH1096 (ΔsigB). Fluorescence readings for the mCherry reporter were taken after 20 h of growth of cells. (B) Plasmid pAH1 (P3_agr-mCherry) was used to monitor agr activity in LAC and AH1096 after 8 h of growth in TSB. (C to G) CLSM reconstructions of flow cell biofilms grown for 4 days. Strains LAC, AH1096, AH1203, and AH1204 were visualized by CLSM using GFP labeling with plasmid pALC2084 (induced with 20 ng/ml anhydrotetracycline). For complementation assessment (E), a biofilm of strain AH1096 with the sigB expression plasmid pALC2109 (induced with 50 ng/ml anhydrotetracycline) was grown in the flow cell and poststained with Syto9. Each side of a grid square is 20 μm in the CLSM image reconstruction.

FIG. 9.

Protease and autolysin zymography in ΔsigB mutants. Gel images are black-white inverted to aid visualization. (A) Extracellular protease zymography using 8% SDS-PAGE supplemented with 0.2% gelatin as a substrate. (B) Autolysin zymography using 8% SDS-PAGE supplemented with 0.2% M. luteus cells as a substrate. Lanes are identical in both protease and autolysin gels. Labels at the top of panel A indicate the genetic background. The presence or absence of SigB is indicated as follows: +, chromosomal sigB; +_C, plasmid-encoded sigB. Lane 1, AH1136 (ΔsigB Δaur Δspl::Erm); lane 2, SH1000 with pALC2073; lane 3, AH1012 with pALC2073; lane 4, AH1012 with pALC2109; lane 5, LAC with pALC2073; lane 6, AH1096 with pALC2073; lane 7, AH1096 with pALC2109. Expression of sigB from plasmid pALC2109 was induced with 50 ng/ml anhydrotetracycline.