Biofilm Matrix Exoproteins Induce a Protective Immune Response against Staphylococcus aureus Biofilm Infection

Abstract

The Staphylococcus aureus biofilm mode of growth is associated with several chronic infections that are very difficult to treat due to the recalcitrant nature of biofilms to clearance by antimicrobials. Accordingly, there is an increasing interest in preventing the formation of S. aureus biofilms and developing efficient antibiofilm vaccines. Given the fact that during a biofilm-associated infection, the first primary interface between the host and the bacteria is the self-produced extracellular matrix, in this study we analyzed the potential of extracellular proteins found in the biofilm matrix to induce a protective immune response against S. aureus infections. By using proteomic approaches, we characterized the exoproteomes of exopolysaccharide-based and proteinbased biofilm matrices produced by two clinical S. aureus strains. Remarkably, results showed that independently of the nature of the biofilm matrix, a common core of secreted proteins is contained in both types of exoproteomes. Intradermal administration of an exoproteome extract of an exopolysaccharide-dependent biofilm induced a humoral immune response and elicited the production of interleukin 10 (IL-10) and IL-17 in mice. Antibodies against such an extract promoted opsonophagocytosis and killing of S. aureus. Immunization with the biofilm matrix exoproteome significantly reduced the number of bacterial cells inside a biofilm and on the surrounding tissue, using an in vivo model of mesh-associated biofilm infection. Furthermore, immunized mice also showed limited organ colonization by bacteria released from the matrix at the dispersive stage of the biofilm cycle. Altogether, these data illustrate the potential of biofilm matrix exoproteins as a promising candidate multivalent vaccine against S. aureus biofilm-associated infections.

The Extracellular Matrix of Staphylococcus aureus Biofilms Comprises Cytoplasmic Proteins That Associate with the Cell Surface in Response to Decreasing pH

“…These 11 proteins are characterized by roles in central metabolism, such as glycolysis and fermentation ( 14 ). Consistent with our findings, several of these proteins (for example, enolase, transketolase, acetolactate synthase, GAPDH [glyceraldehyde-3-phosphate dehydrogenase], and lactate dehydrogenase) were previously found to be abundant under biofilm-inducing conditions and in the biofilm exoproteome ( 15 – 17 ) in studies that did not investigate their role in biofilm formation. In addition to identifying proteins that were enriched under biofilm-inducing conditions, we also detected extracellular proteins that were either more abundant under the non-biofilm-inducing conditions or showed equal levels of abundance under the two sets of conditions (having a 14 N/ 15 N ratio of between 0.5 and 2) (see Table S1 in the supplemental material).…”

The Extracellular Matrix of Staphylococcus aureus Biofilms Comprises Cytoplasmic Proteins That Associate with the Cell Surface in Response to Decreasing pH

“…These 11 proteins are characterized by roles in central metabolism, such as glycolysis and fermentation ( 14 ). Consistent with our findings, several of these proteins (for example, enolase, transketolase, acetolactate synthase, GAPDH [glyceraldehyde-3-phosphate dehydrogenase], and lactate dehydrogenase) were previously found to be abundant under biofilm-inducing conditions and in the biofilm exoproteome ( 15 – 17 ) in studies that did not investigate their role in biofilm formation. In addition to identifying proteins that were enriched under biofilm-inducing conditions, we also detected extracellular proteins that were either more abundant under the non-biofilm-inducing conditions or showed equal levels of abundance under the two sets of conditions (having a 14 N/ 15 N ratio of between 0.5 and 2) (see Table S1 in the supplemental material).…”

Evaluation of mucosal barrier disruption due to Staphylococcus lugdunensis and Staphylococcus epidermidis exoproteins in patients with chronic rhinosinusitis

“…Previous research demonstrated that immunization with the biofilm matrix produced by S. aureus substantially decreased the bacterial cell count within a biofilm and surrounding tissue, as evidenced by an in vivo model (mice) of mesh-associated biofilm infection. 23 In addition, our results showed that the exoproteins secreted by S. epidermidis planktonic isolates had more profound effects on the mucosal barrier compared with exoproteins secreted by matched biofilm forms. Many studies have shown that S. epidermidis biofilms are highly resistant to antibiotics or induce higher production of proinflammatory cytokines compared to their planktonic counterparts; however, these studies assessed the effect of the actual biofilm or cells released from biofilm.…”

Characterization of Staphylococcus lugdunensis biofilm reveals key differences according to clonal lineage and iron availability

“…These proteins have been described in S. aureus: one (SLGD_02212) is a protease (Clp) (46), this type of enzyme being involved in biofilm dispersal phase (12), and the other (SLGD_00206) is an IsaB analog protein, an extracellular nucleic acid binding protein whose role in virulence is not established in S. aureus (46). KEGG analyzes revealed that the biofilm matrix of the two S. lugdunensis strains mostly contained proteins involved in metabolic pathways, particularly in carbohydrate metabolism, as previously described in S. lugdunensis and other staphylococci (22,41,47,48). In particular, we have identified a large number of proteins involved in glycolysis and gluconeogenesis for the formation of pyruvate, in line with the results obtained for S. epidermidis (41) or S. aureus biofilms (48).…”

The Extracellular Matrix of Staphylococcus aureus Biofilms Comprises Cytoplasmic Proteins That Associate with the Cell Surface in Response to Decreasing pH

“…These 11 proteins are characterized by roles in central metabolism, such as glycolysis and fermentation ( 14 ). Consistent with our findings, several of these proteins (for example, enolase, transketolase, acetolactate synthase, GAPDH [glyceraldehyde-3-phosphate dehydrogenase], and lactate dehydrogenase) were previously found to be abundant under biofilm-inducing conditions and in the biofilm exoproteome ( 15 – 17 ) in studies that did not investigate their role in biofilm formation. In addition to identifying proteins that were enriched under biofilm-inducing conditions, we also detected extracellular proteins that were either more abundant under the non-biofilm-inducing conditions or showed equal levels of abundance under the two sets of conditions (having a 14 N/ 15 N ratio of between 0.5 and 2) (see Table S1 in the supplemental material).…”

Evaluation of mucosal barrier disruption due to Staphylococcus lugdunensis and Staphylococcus epidermidis exoproteins in patients with chronic rhinosinusitis

“…Previous research demonstrated that immunization with the biofilm matrix produced by S. aureus substantially decreased the bacterial cell count within a biofilm and surrounding tissue, as evidenced by an in vivo model (mice) of mesh-associated biofilm infection. 23 In addition, our results showed that the exoproteins secreted by S. epidermidis planktonic isolates had more profound effects on the mucosal barrier compared with exoproteins secreted by matched biofilm forms. Many studies have shown that S. epidermidis biofilms are highly resistant to antibiotics or induce higher production of proinflammatory cytokines compared to their planktonic counterparts; however, these studies assessed the effect of the actual biofilm or cells released from biofilm.…”

Characterization of Staphylococcus lugdunensis biofilm reveals key differences according to clonal lineage and iron availability

“…These proteins have been described in S. aureus: one (SLGD_02212) is a protease (Clp) (46), this type of enzyme being involved in biofilm dispersal phase (12), and the other (SLGD_00206) is an IsaB analog protein, an extracellular nucleic acid binding protein whose role in virulence is not established in S. aureus (46). KEGG analyzes revealed that the biofilm matrix of the two S. lugdunensis strains mostly contained proteins involved in metabolic pathways, particularly in carbohydrate metabolism, as previously described in S. lugdunensis and other staphylococci (22,41,47,48). In particular, we have identified a large number of proteins involved in glycolysis and gluconeogenesis for the formation of pyruvate, in line with the results obtained for S. epidermidis (41) or S. aureus biofilms (48).…”

The Extracellular Matrix of Staphylococcus aureus Biofilms Comprises Cytoplasmic Proteins That Associate with the Cell Surface in Response to Decreasing pH

“…These 11 proteins are characterized by roles in central metabolism, such as glycolysis and fermentation ( 14 ). Consistent with our findings, several of these proteins (for example, enolase, transketolase, acetolactate synthase, GAPDH [glyceraldehyde-3-phosphate dehydrogenase], and lactate dehydrogenase) were previously found to be abundant under biofilm-inducing conditions and in the biofilm exoproteome ( 15 – 17 ) in studies that did not investigate their role in biofilm formation. In addition to identifying proteins that were enriched under biofilm-inducing conditions, we also detected extracellular proteins that were either more abundant under the non-biofilm-inducing conditions or showed equal levels of abundance under the two sets of conditions (having a 14 N/ 15 N ratio of between 0.5 and 2) (see Table S1 in the supplemental material).…”

Evaluation of mucosal barrier disruption due to Staphylococcus lugdunensis and Staphylococcus epidermidis exoproteins in patients with chronic rhinosinusitis

“…Previous research demonstrated that immunization with the biofilm matrix produced by S. aureus substantially decreased the bacterial cell count within a biofilm and surrounding tissue, as evidenced by an in vivo model (mice) of mesh-associated biofilm infection. 23 In addition, our results showed that the exoproteins secreted by S. epidermidis planktonic isolates had more profound effects on the mucosal barrier compared with exoproteins secreted by matched biofilm forms. Many studies have shown that S. epidermidis biofilms are highly resistant to antibiotics or induce higher production of proinflammatory cytokines compared to their planktonic counterparts; however, these studies assessed the effect of the actual biofilm or cells released from biofilm.…”

Characterization of Staphylococcus lugdunensis biofilm reveals key differences according to clonal lineage and iron availability

“…These proteins have been described in S. aureus: one (SLGD_02212) is a protease (Clp) (46), this type of enzyme being involved in biofilm dispersal phase (12), and the other (SLGD_00206) is an IsaB analog protein, an extracellular nucleic acid binding protein whose role in virulence is not established in S. aureus (46). KEGG analyzes revealed that the biofilm matrix of the two S. lugdunensis strains mostly contained proteins involved in metabolic pathways, particularly in carbohydrate metabolism, as previously described in S. lugdunensis and other staphylococci (22,41,47,48). In particular, we have identified a large number of proteins involved in glycolysis and gluconeogenesis for the formation of pyruvate, in line with the results obtained for S. epidermidis (41) or S. aureus biofilms (48).…”