Secreted proteases control autolysin-mediated biofilm growth of Staphylococcus aureus

Abstract

Staphylococcus epidermidis, a commensal of humans, secretes Esp protease to prevent Staphylococcus aureus biofilm formation and colonization. Blocking S. aureus colonization may reduce the incidence of invasive infectious diseases; however, the mechanism whereby Esp disrupts biofilms is unknown. We show here that Esp cleaves autolysin (Atl)-derived murein hydrolases and prevents staphylococcal release of DNA, which serves as extracellular matrix in biofilms. The three-dimensional structure of Esp was revealed by x-ray crystallography and shown to be highly similar to that of S. aureus V8 (SspA). Both atl and sspA are necessary for biofilm formation, and purified SspA cleaves Atl-derived murein hydrolases. Thus, S. aureus biofilms are formed via the controlled secretion and proteolysis of autolysin, and this developmental program appears to be perturbed by the Esp protease of S. epidermidis.

Keywords: Bacterial Pathogenesis; Biofilm; Extracellular Matrix; Serine Protease; Staphylococcus aureus.

FIGURE 1.

Purified, recombinant Esp displays protease activity, inhibits S. aureus biofilm formation and cleaves Atl. A, diagram illustrating the primary structure of pro-Esp, Esp, and the variant Esp^S235A that were purified from E. coli. The arrow indicates the thermolysin cleavage site. B, purified pro-Esp and Esp were separated by SDS-PAGE and stained with Coomassie Blue. C, Esp activity assay using azocasein substrate and measuring product absorbance at 440 nm. Enzyme activity measurements were averaged from three independent determinations, and the standard error of the means was determined (brackets). Statistical significance was determined with the two-tailed Student's t test. ***, p < 0.0001. D, purified pro-Esp, Esp, or Esp^S235A was incubated with S. aureus Newman during assembly of biofilms on fibronectin-coated microtiter plates at 37 °C with 5% CO₂ over 24 h. Following incubation, the plates were washed and stained with crystal violet to measure biofilm formation as absorbance at 595 nm. Biofilm data were averaged from three independent determinations, and the standard error of the means was calculated (brackets). Statistical significance was assessed with the two-tailed Student's t test in pairwise comparison with mock treated samples. ***, p < 0.0001; **, p < 0.001. E, mock, Esp, or Esp^S235A treated S. aureus Newman biofilms were dispersed, and proteins were analyzed by Coomassie-stained SDS-PAGE. Protein species that were absent in Esp treated samples were identified by LC-MS/MS. Arrows identify the migratory position of Atl (AM).

FIGURE 2.

Esp treatment of S. aureus Newman wild-type and atl mutant biofilms. A, purified Esp or mock treatment were added during S. aureus wild-type (wt) or atl mutant biofilm assembly on fibronectin-coated microtiter plates at 37 °C with 5% CO₂ for 24 h. Following incubation, plates were washed and stained with crystal violet to measure biofilm formation as absorbance at 595 nm (A₅₉₅). Biofilm data were averaged from three independent determinations. The standard error of the means is indicated as brackets. Statistical significance was assessed with the two-tailed Student's t test. **, p < 0.001; *, p < 0.05. B, mock or 25 μg/ml Esp were added to tryptic soy broth inoculated with S. aureus Newman wt or atl mutant strains, incubated with rotation at 37 °C and growth measured via absorbance at 600 nm (A₆₀₀). C, purified Esp or mock treatment was added during S. aureus Newman wt or atl mutant biofilm assembly on fibronectin-coated microtiter plates at 37 °C with 5% CO₂ for 24 h. Plates were washed, viable staphylococci were stained with SYTO 9, and fluorescence was captured via fluorescence microscopy. D, differential interference contrast (DIC) and fluorescence microscopy images acquired in C were quantified with ImageJ. The data were averaged from three independent determinations. The standard error of the means is indicated as brackets. Statistical significance was assessed with the two-tailed Student's t test. ***, p < 0.0001.

FIGURE 3.

Esp treatment of GST-Atl hybrids. A, diagram illustrating the primary structure of GST hybrids with Atl domains including GST-AM, GST-AM_ΔR1R2, GST-GL, and GST-GL_ΔR3. B, purified GST hybrids (5 μg) were incubated with 400 nm Esp (+) or mock treated (−) for 20 min at 37 °C. Proteins were separated on SDS-PAGE followed by Coomassie Blue staining. Black arrowheads identify the migratory positions of GST-AM, GST-AM_ΔR1R2, GST-GL, and GST-GL_ΔR3. The white arrowhead identifies an Esp cleavage species of GST-GL, which had been cut after glutamyl 862 (E/VKTTQK), as identified by Edman degradation.

FIGURE 4.

Peptidoglycan hydrolase and biofilm promoting activity of GST-Atl hybrids in the presence or absence of Esp treatment. A, S. aureus Newman murein sacculi were obtained with a bead beater instrument and extracted with detergent as well as hydrofluoric acid to remove membranes and wall teichoic acids, respectively. Cleavage of peptidoglycan by 5 μg of purified lysostaphin (lyso), GST-AM (AM), GST-AM_ΔR1R2 (AM_ΔR1R2), GST-GL (GL), or GST-GL_ΔR3 (GL_ΔR3) in the presence (+) or absence (−) of 400 nm Esp was monitored by measuring absorbance at 600 nm (A₆₀₀). The data represent averages of three independent experimental determinations, and the standard error of the means is indicated by brackets. Statistical significance was assessed in pairwise comparison with the two-tailed Student's t test. ***, p < 0.0001; **, p < 0.001; *, p < 0.05). B, biofilm formation of the atl mutant on fibronectin-coated microtiter plates at 37 °C with 5% CO₂ for 24 h was analyzed in the presence or absence (mock) of 5 μg of purified GST-AM (AM), GST-AM_ΔR1R2 (AM_ΔR1R2), GST-GL (GL), GST-GL_ΔR3 (GL_ΔR3), or 400 nm Esp (+ or −). Following incubation, the plates were washed and stained with crystal violet to measure biofilm formation as absorbance at 595 nm (A₅₉₅). Biofilm data were averaged from three independent determinations. The standard error of the means is indicated as brackets. Statistical significance was assessed with the two-tailed Student's t test. **, p < 0.001; *, p < 0.05.

FIGURE 5.

Effect of Esp treatment on the staphylococcal cluster dispersing activity of GST-Atl hybrids. A, overnight cultures of S. aureus Newman wild-type (wt) or atl variant were diluted to A₆₀₀ 0.05 in 1 ml of TSB and incubated at 37 °C for 2 h in the presence or absence of 25 μg of purified GST-AM (AM), GST-AM_ΔR1R2 (AM_ΔR1R2), GST-GL (GL), GST-GL_ΔR3 (GL_ΔR3), or 400 nm Esp (+ or −). Staphylococci were fixed with 4% paraformaldehyde, washed, suspended with 1 ml of PBS, and analyzed by flow cytometry. B, percentage of bacteria in large cell clusters were quantified for wild-type and atl mutant staphylococci with or without Atl hybrids and Esp treatment.

FIGURE 6.

Esp treatment and the release of extracellular DNA in S. aureus biofilms. A, purified 400 nm Esp or DNase I were incubated with S. aureus Newman wild-type (wt) or its atl variant during biofilm assembly on fibronectin-coated microtiter plates at 37 °C with 5% CO₂ for 24 h. Following incubation, plates were washed and stained with PI to reveal extracellular DNA or SYTO 9 to reveal viable staphylococci and analyzed via DIC and fluorescence microscopy. DIC, differential interference contrast. B, fluorescence intensity staining of PI, SYTO 9, or PI/SYTO 9 staining in samples from A was quantified with ImageJ. The data were averaged from three independent determinations, and the standard error of the means is indicated as brackets. Statistical significance was assessed in pairwise comparison using the two-tailed Student's t test. ***, p < 0.0001; **, p < 0.001; *, p < 0.05.

FIGURE 7.

S. aureus V8 protease and biofilm formation. A, protein sequence alignment of mature Esp and V8. B, GST-AM, GST-AM_ΔR1R2, GST-GL, and GST-GL_ΔR3 (5 μg) were incubated with 400 nm Esp (Esp), V8 protease (V8), or mock treatment (−) for 20 min at 37 °C. Proteins were separated on SDS-PAGE followed by Coomassie Blue staining. Arrowheads identify the migratory positions of GST-AM, GST-AM_ΔR1R2, GST-GL, and GST-GL_ΔR3. C, purified Esp, V8, or mock treatment were added during biofilm formation of S. epidermidis RP62a and S. aureus Newman wild-type and atl and sspA mutant strains on fibronectin-coated microtiter plates at 37 °C with 5% CO₂ for 24 h. Following incubation, plates were washed and stained with crystal violet, and biofilm formation was measured as absorbance at 595 nm. D, S. aureus Newman wild-type (wt) or its sspA mutant without plasmid (−) or with psspA or vector control (pWW412) was incubated on fibronectin-coated microtiter plates at 37 °C with 5% CO₂ for 24 h. Following incubation, the plates were washed and stained with crystal violet to measure biofilm formation as absorbance at 595 nm (A₅₉₅). Biofilm data were averaged from three independent determinations. The standard error of the means is indicated as brackets. Statistical significance was assessed with the two-tailed Student's t test. **, p < 0.001; *, p < 0.05; NS, no significant difference. E, the activity of extracellular proteases secreted by S. aureus wild-type (wt), atl and sspA mutant cultures were quantified with the azocasein assay, and product cleavage was measured as absorbance at 440 nm. The protease activity data were averaged from three independent determinations. The standard error of the means is indicated as brackets. Statistical significance was assessed with the two-tailed Student's t test. NS, no significant difference.

FIGURE 8.

Comparison between Esp and V8 crystal structures. a, ribbon representation of the refined crystal structure of active Esp. The α-helices are represented in cyan, β-strands are in magenta, and loop regions are in light brown. The putative catalytic His¹¹⁷, Asp¹⁵⁹, and Ser²³⁵ residue side chains are represented as green sticks. b, superposition of Esp (PDB code 4JCN, represented in magenta) and pancreatic trypsin (PDB code 1TRM, in cyan) crystal structures. Surface loops (A, C, D, 1, 2, and 3) that dictate substrate specificity for trypsin are labeled, and the disulfide bonds (in yellow) that hold its structure together are also shown. c, superposition of Esp (magenta) and V8 (ivory, PDB code 1QY6) crystal structures. d, significant residue differences observed between Esp (magenta) and V8 (ivory) crystal structures are shown in their respective positions. Side chains are shown as sticks: green for V8 and magenta for Esp residues.

FIGURE 9.

Model illustrating S. aureus atl-dependent biofilm formation and the impact of serine proteases, i.e., S. epidermidis Esp or S. aureus V8 (SspA), on controlling Atl activity and biofilm disassembly. The model distinguishes five steps in the biofilm developmental process: attachment, eDNA release, maturation, detachment, and dissemination. Three surface proteins (Eap, FnbA, and FnbB) are thought to promote S. aureus attachment to fibronectin (attachment). The secretion of Atl promotes the release of eDNA as an extracellular matrix for biofilm formation (eDNA release). Activation of secreted SspA (V8 protease) inactivates Atl, thereby promoting staphylococcal replication in the newly formed matrix (biofilm maturation). The continued activation of SspA promotes the detachment of staphylococcal cells from the biofilm (detachment). Detached staphylococci disseminate and adhere elsewhere by binding to fibronectin and establishing another biofilm. S. aureus biofilm formation is perturbed by the S. epidermidis secreted protease Esp. We propose that exuberant expression of S. epidermidis Esp (unlike S. aureus SspA) perturbs biofilm formation of S. aureus.