Limits...
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.

Show MeSH

Related in: MedlinePlus

Esp treatment of GST-Atl hybrids.A, diagram illustrating the primary structure of GST hybrids with Atl domains including GST-AM, GST-AMΔR1R2, GST-GL, and GST-GLΔR3. 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-AMΔR1R2, GST-GL, and GST-GLΔR3. 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3795244&req=5

Figure 3: Esp treatment of GST-Atl hybrids.A, diagram illustrating the primary structure of GST hybrids with Atl domains including GST-AM, GST-AMΔR1R2, GST-GL, and GST-GLΔR3. 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-AMΔR1R2, GST-GL, and GST-GLΔR3. 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.

Mentions: To determine which of the functional domains of Atl are cleaved by Esp, we purified AM (N-acetylmuramoyl-l-alanine amidase), AMΔR1R2 (lacking the C-terminal repeat domains R1 and R2 of AM), GL (N-acetylglucosamine-N-acetylmuramic acid glucosaminidase), and GLΔR3 (lacking the N-terminal R3 domain of GL) as hybrids fused to the C-terminal end of GST (Fig. 3. Esp treatment cut AM, AMΔR1R2, and GL, but not GLΔR3 (Fig. 3B). Esp treatment generated several cleavage fragments from AM, AMΔR1R2, or GL, suggesting that the protease can cut at multiple sites within the amidase and the R1-R3 domains (Fig. 3B). Edman degradation of cleaved peptides identified glutamic acid residues (for example Glu862 in GL) as Esp cleavage sites (Fig. 3B).


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)

Esp treatment of GST-Atl hybrids.A, diagram illustrating the primary structure of GST hybrids with Atl domains including GST-AM, GST-AMΔR1R2, GST-GL, and GST-GLΔR3. 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-AMΔR1R2, GST-GL, and GST-GLΔR3. 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3795244&req=5

Figure 3: Esp treatment of GST-Atl hybrids.A, diagram illustrating the primary structure of GST hybrids with Atl domains including GST-AM, GST-AMΔR1R2, GST-GL, and GST-GLΔR3. 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-AMΔR1R2, GST-GL, and GST-GLΔR3. 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.
Mentions: To determine which of the functional domains of Atl are cleaved by Esp, we purified AM (N-acetylmuramoyl-l-alanine amidase), AMΔR1R2 (lacking the C-terminal repeat domains R1 and R2 of AM), GL (N-acetylglucosamine-N-acetylmuramic acid glucosaminidase), and GLΔR3 (lacking the N-terminal R3 domain of GL) as hybrids fused to the C-terminal end of GST (Fig. 3. Esp treatment cut AM, AMΔR1R2, and GL, but not GLΔR3 (Fig. 3B). Esp treatment generated several cleavage fragments from AM, AMΔR1R2, or GL, suggesting that the protease can cut at multiple sites within the amidase and the R1-R3 domains (Fig. 3B). Edman degradation of cleaved peptides identified glutamic acid residues (for example Glu862 in GL) as Esp cleavage sites (Fig. 3B).

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