Final Report Summary - PROTEIN-BIOFILM (Characterization of protein-dependent biofilms by Staphylococcus aureus)
In natural environments bacteria grow as multicellular communities, called biofilms, embedded in extracellular matrix and adhered to inert surfaces or living tissues. Exopolysaccharides are continually described as being essential components of the matrix, however increasing number of evidences indicate the existence of clinical S. aureus isolates able to produce proteinaceous-dependent biofilm matrix. The research project was focused on the investigation of protein-mediated biofilm development in S. aureus.
The ultimate goal was to understand the influence that biofilm matrix nature (polysaccharidic or proteinaceuos) has on embedded bacteria. By using different approaches we have identified and characterised three protein-mediated biofilm in S. aureus:
Identification of Protein A as a main component of the biofilm matrix in S. aureus. Using two-dimensional nano-liquid chromatography and mass spectrometry we analysed the composition of the proteinaceus biofilm matrix of the S. aureus agr arlRS double mutant and we identified protein A as a main component of the biofilm matrix. We demonstrated that secreted protein A promotes bacterial aggregation and biofilm formation under static and continuous flow culture conditions independently of the exopolysaccharide PIA-PNAG. Biofilm formation mediated by protein A was inhibited in a dose dependent manner by the addition of serum, purified immunoglobulin G or anti-protein A antibodies. Moreover, protein A showed an important role in the colonisation of subcutaneously implanted catheters.
Role of Fibronectin-binding proteins in S. aureus biofilm formation.
From a collection of 63 S. aureus clinical strains we selected a meticillin resistant S. aureus strain (MRSA 132) that form a PIA/PNAG dependent biofilm in osmotic conditions or a proteinaceus dependent biofilm when it was grown in presence of glucose. With the aim to identify the proteinaceus compound that mediates biofilm formation we systematically disrupted each of the 20 proteins of the LPXTG family anchored to the cell wall. Biofilm formation of the resulting mutants showed that only fibronecting binding protein mutant lost the capacity to form biofilm suggesting that fibronecting binding proteins among all LPXTG proteins are essential components of the protein-dependent matrix. Moreover, using a murine model of subcutaneous catheter infection we showed that a fibronecting binding protein mutant displayed a lower capacity to form a biofilm on the implanted catheters than the PIA/PNAG mutant suggesting that fibronecting binding proteins play a more relevant role than PIA/PNAG polysaccharide in the colonisation of implanted devices.
The role of the Bap protein of S. aureus in host cell interactions. Bap (biofilm-associated protein) is a 2,276-aminoacid surface protein able to induce a protein-dependent biofilm matrix in some strains of S. aureus. We have performed adhesion and invasion assays using bovine mammary epithelial cell line (MAC-T) and human hepatocyte cell line (Hep3B). Bap promotes bacterial adhesion to both epithelial cell lines. However, invasion was more efficient in the absence than in the presence of Bap. Using a ligand overlay approach and mass spectrometry we identify Gp96, a member of the Hsp90 family of molecular chaperones, as a cell surface receptor of Bap. The interaction of Bap with Gp96 was demonstrated in vitro using pull-down assays with S. aureus strains expressing Bap or purified Bap protein incubated with recombinant Gp96. Furthermore, we showed using two cell lines that do not express Gp96 (Vero and Gpc16) together with their corresponding cell lines transfected with GP96, that Bap-mediated invasion inhibition correlated with the presence of Gp96 at the cell surface.
The ultimate goal was to understand the influence that biofilm matrix nature (polysaccharidic or proteinaceuos) has on embedded bacteria. By using different approaches we have identified and characterised three protein-mediated biofilm in S. aureus:
Identification of Protein A as a main component of the biofilm matrix in S. aureus. Using two-dimensional nano-liquid chromatography and mass spectrometry we analysed the composition of the proteinaceus biofilm matrix of the S. aureus agr arlRS double mutant and we identified protein A as a main component of the biofilm matrix. We demonstrated that secreted protein A promotes bacterial aggregation and biofilm formation under static and continuous flow culture conditions independently of the exopolysaccharide PIA-PNAG. Biofilm formation mediated by protein A was inhibited in a dose dependent manner by the addition of serum, purified immunoglobulin G or anti-protein A antibodies. Moreover, protein A showed an important role in the colonisation of subcutaneously implanted catheters.
Role of Fibronectin-binding proteins in S. aureus biofilm formation.
From a collection of 63 S. aureus clinical strains we selected a meticillin resistant S. aureus strain (MRSA 132) that form a PIA/PNAG dependent biofilm in osmotic conditions or a proteinaceus dependent biofilm when it was grown in presence of glucose. With the aim to identify the proteinaceus compound that mediates biofilm formation we systematically disrupted each of the 20 proteins of the LPXTG family anchored to the cell wall. Biofilm formation of the resulting mutants showed that only fibronecting binding protein mutant lost the capacity to form biofilm suggesting that fibronecting binding proteins among all LPXTG proteins are essential components of the protein-dependent matrix. Moreover, using a murine model of subcutaneous catheter infection we showed that a fibronecting binding protein mutant displayed a lower capacity to form a biofilm on the implanted catheters than the PIA/PNAG mutant suggesting that fibronecting binding proteins play a more relevant role than PIA/PNAG polysaccharide in the colonisation of implanted devices.
The role of the Bap protein of S. aureus in host cell interactions. Bap (biofilm-associated protein) is a 2,276-aminoacid surface protein able to induce a protein-dependent biofilm matrix in some strains of S. aureus. We have performed adhesion and invasion assays using bovine mammary epithelial cell line (MAC-T) and human hepatocyte cell line (Hep3B). Bap promotes bacterial adhesion to both epithelial cell lines. However, invasion was more efficient in the absence than in the presence of Bap. Using a ligand overlay approach and mass spectrometry we identify Gp96, a member of the Hsp90 family of molecular chaperones, as a cell surface receptor of Bap. The interaction of Bap with Gp96 was demonstrated in vitro using pull-down assays with S. aureus strains expressing Bap or purified Bap protein incubated with recombinant Gp96. Furthermore, we showed using two cell lines that do not express Gp96 (Vero and Gpc16) together with their corresponding cell lines transfected with GP96, that Bap-mediated invasion inhibition correlated with the presence of Gp96 at the cell surface.