Figure 1.
Growth media influences biofilm disassembly.
Confocal micrographs of S. aureus SH1000 biofilms grown in TSBg media (A) for 30 hours readily disassemble upon exposure to biofilm matrix degrading enzymes proteinase K, dispersin B, and DNaseI at 0.2 µg/mL each. S. aureus biofilms grown in PNG media (B) for 30 hours fail to disassemble upon exposure to matrix-degrading enzymes. Images are representative of three separate experiments and each side of a grid square represents 20 µm. (C) Biofilms at the air-liquid interface of test tube cultures withstand 1% SDS exposure when grown in PNG media but disassemble when grown in TSBg. Top images show stained test tube biofilms; graph below is quantification of biofilm biomass. * P<0.002 compared to no SDS treatment.
Figure 2.
S. aureus produces extracellular fibers during biofilm growth in PNG media.
TEM micrographs of cells from S. aureus SH1000 biofilms grown in TSBg medium (A) versus cells from biofilms grown in PNG media (B). High magnification reveals fibers are associated with the cell wall and approximately 12 nm in width (C). An agr mutant does not produce extracellular fibers (D). Bar length indicates 1 µm in A, B, and D, and 250 nm in C.
Figure 3.
Fibers are composed of phenol soluble modulins.
(A) S. aureus biofilm cells were lysed and run into a 12% SDS-PAGE gel (TSBg first lane or PNG second lane); protein that did not migrate through the gel (indicated by arrow) was extracted from the staking gel, treated with formic acid to break up aggregated proteins, and finally run on a new 12% SDS-PAGE gel (B). Bands that appeared after formic acid treatment (1–4) were excised and analyzed via LC-MS/MS. (C) TEM micrograph of purified fiber sample that was then exposed to extensive pepsin digestion and analyzed via LC-MS/MS. Bar indicates 250 nm. (D) Peptides identified by mass spectrometry analysis and their relative abundance factors in the sample (NSAF).
Figure 4.
Phenol soluble modulins are small peptides expressed from three discrete regions of the S. aureus genome.
(A) Phenol soluble modulins (PSMs) are encoded in two operons, the alpha (αPSM1–4) and beta (βPSM1–2) operons, and δ-toxin is encoded within the Agr regulatory RNA, RNAIII (hld). (B) PSMs are small hydrophobic peptides with highly similar amino acid content.
Figure 5.
Mutants unable to produce α and βPSMs fail to form fibers during biofilm growth.
TEM micrographs of S. aureus biofilm cells grown for five days in PNG media. (A) wildtype (strain SH1000), (B) Δαβpsm (strain BB2388), (C) Δαβpsm complemented (strain BB2408). (D–F) TEM micrographs of fiber preparations from wildtype (D), Δαβpsm (E), and Δαβpsm complemented (F). Bars indicate 500 nm.
Figure 6.
Synthetic phenol soluble modulin peptides bind ThT and polymerize into amyloid-like fibers.
(A) Normalized fluorescence intensity of [white circle] 0.1 mg/mL of each PSM peptide or [black circle] 0.05 mg/mL of each PSM peptide in 2 mM ThT. Fluorescence emission was measured at 495 nm after excitation at 438 nm. Assays were repeated in triplicate and all demonstrated a similar trend. (B) 48 hours after mixing 100 µg/mL each of the seven PSM peptides (α1–4, β1–2, and δ-toxin), fibril structures are readily observed by TEM. (C) PSM fibers [black circle] display a ThT fluorescence peak around 482 nm compared to a ThT-only blank [grey circle]. (D) PSM fibers [black circle] produce a characteristic Congo red (CR) absorbance “red-shift” associated with amyloid binding compared to a CR-only blank [grey circle]. (E) Pelleted PSM fibers [grey circle] display a greater β-sheet content than the remaining supernatant [black circle]. Assays were repeated in triplicate and displayed similar trends. Bar indicates 500 nm.
Figure 7.
An αβPSM mutant forms biofilms susceptible to disassembly by matrix degrading enzymes and mechanical stress.
Confocal micrographs of Δαβpsm mutant (A) (strain BB2388) versus complemented mutant expressing α and βpsm operons in trans (B) (strain BB2408) flow cell biofilms grown for 30 hours prior to proteinase K, dispersin B, and DNaseI exposure (at 0.2 µg/mL each). Images are representative of three separate experiments and each side of a grid square represents 20 µm. (C) Analysis of biofilm development at the air-liquid interface of test tube cultures in PNG media after vortexing. Graph shows quantification of biofilm biomass (OD A595) and images below show stained test tube biofilms. * P<0.005 compared to wildtype.
Figure 8.
Amyloid fiber formation modulates PSM activity.
(A) S. aureus wildtype biofilms were grown in microtiter plates for 24 hours then washed and exposed to increasing concentrations of soluble αPSM1 or αPSM1 fibers at concentrations of 10, 50 or 100 µg/mL for six hours. Biofilms were then washed, stained and remaining biofilm biomass was visualized (images of wells below graph) and quantitated (OD at A595). (B & C) TEM micrographs of αPSM1 samples used in the experiment demonstrate the absence (B) and presence (C) of fibers. * P<0.002 compared to control no αPSM1 treatment. We verified that αPSM1 fibers bind CR (D) and ThT (E) similar to amyloid fibers.
Table 1.
Strains and plasmids used in the present study.