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Secreted proteases control autolysin-mediated biofilm growth of Staphylococcus aureus.

Chen C, Krishnan V, Macon K, Manne K, Narayana SV, Schneewind O - J. Biol. Chem. (2013)

Bottom Line: Blocking S. aureus colonization may reduce the incidence of invasive infectious diseases; however, the mechanism whereby Esp disrupts biofilms is unknown.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.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Microbiology, University of Chicago, Chicago, Illinois 60637.

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.

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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 EspS235A 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 EspS235A 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 EspS235A 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).
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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 EspS235A 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 EspS235A 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 EspS235A 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).

Mentions: Following signal peptide cleavage, the pro-form of Esp (pro-Esp) is cleaved in the extracellular medium of S. epidermidis cultures to generate mature Esp protease, which mediates the disassembly of S. aureus biofilms (7). We expressed six-histidyl-tagged pro-Esp in E. coli and purified recombinant protein by affinity chromatography (Fig. 1A). Thermolysin cleavage and gel filtration chromatography were used to obtain purified Esp (Fig. 1B). The variant EspS235A harbors an alanyl substitution at the active site serine residue of Esp (Fig. 1A). When examined for protease activity with azocasein substrate (40), Esp cleaved significantly more substrate than pro-Esp, whereas EspS235A did not display protease activity (Fig. 1C). Wild-type S. aureus strain Newman was used to form staphylococcal biofilms using human fibronectin as a matrix, which were quantified by crystal violet staining (41). Treatment with Esp, but not pro-Esp or EspS235A, triggered disassembly of staphylococcal biofilms (Fig. 1D). Proteins in biofilms with or without Esp treatment were separated by 10–20% gradient SDS-PAGE, stained with Coomassie Brilliant Blue, and identified via LC-MS/MS (Fig. 1E). Treatment with Esp, but not EspS235A, caused Atl degradation (Fig. 1E). Our experiments revealed that Esp cleaved 18 additional polypeptides, including FnbA, FnbB, Eap, and SpA, that had previously been identified as Esp substrates (26) (Table 2). Although the genes for some of these secreted proteins contribute to S. aureus biofilm formation, they are not essential for this developmental process. Of note, in S. aureus Newman biofilms, Atl is a highly abundant component and effectively degraded during Esp treatment (Fig. 1E). Considering the importance of Atl in biofilm development, we focused our experimental approach on the interactions between Esp and Atl.


Secreted proteases control autolysin-mediated biofilm growth of Staphylococcus aureus.

Chen C, Krishnan V, Macon K, Manne K, Narayana SV, Schneewind O - J. Biol. Chem. (2013)

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 EspS235A 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 EspS235A 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 EspS235A 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).
© Copyright Policy - open-access
Related In: Results  -  Collection

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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 EspS235A 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 EspS235A 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 EspS235A 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).
Mentions: Following signal peptide cleavage, the pro-form of Esp (pro-Esp) is cleaved in the extracellular medium of S. epidermidis cultures to generate mature Esp protease, which mediates the disassembly of S. aureus biofilms (7). We expressed six-histidyl-tagged pro-Esp in E. coli and purified recombinant protein by affinity chromatography (Fig. 1A). Thermolysin cleavage and gel filtration chromatography were used to obtain purified Esp (Fig. 1B). The variant EspS235A harbors an alanyl substitution at the active site serine residue of Esp (Fig. 1A). When examined for protease activity with azocasein substrate (40), Esp cleaved significantly more substrate than pro-Esp, whereas EspS235A did not display protease activity (Fig. 1C). Wild-type S. aureus strain Newman was used to form staphylococcal biofilms using human fibronectin as a matrix, which were quantified by crystal violet staining (41). Treatment with Esp, but not pro-Esp or EspS235A, triggered disassembly of staphylococcal biofilms (Fig. 1D). Proteins in biofilms with or without Esp treatment were separated by 10–20% gradient SDS-PAGE, stained with Coomassie Brilliant Blue, and identified via LC-MS/MS (Fig. 1E). Treatment with Esp, but not EspS235A, caused Atl degradation (Fig. 1E). Our experiments revealed that Esp cleaved 18 additional polypeptides, including FnbA, FnbB, Eap, and SpA, that had previously been identified as Esp substrates (26) (Table 2). Although the genes for some of these secreted proteins contribute to S. aureus biofilm formation, they are not essential for this developmental process. Of note, in S. aureus Newman biofilms, Atl is a highly abundant component and effectively degraded during Esp treatment (Fig. 1E). Considering the importance of Atl in biofilm development, we focused our experimental approach on the interactions between Esp and Atl.

Bottom Line: Blocking S. aureus colonization may reduce the incidence of invasive infectious diseases; however, the mechanism whereby Esp disrupts biofilms is unknown.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.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Microbiology, University of Chicago, Chicago, Illinois 60637.

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.

Show MeSH
Related in: MedlinePlus