Immunoprophylaxis and Immunotherapy of S. Epidermidis
Immunoprophylaxis and Immunotherapy of S. Epidermidis
As mentioned, device-related S. epidermidis infections are related to its capacity to form biofilms. Because S. epidermidis biofilm formation and its regulation, including quorum-sensing control, were recently reviewed, we will only briefly describe the process of biofilm formation emphasizing factors with value for immunoprophylaxis or immunotherapy of S. epidermidis infections.
The first step of biofilm formation is bacterial adherence to the surface. Direct binding to an abiotic implant surface is mediated by electrostatic and hydrophobic interactions, van der Waals forces and affected by physicochemical variables. In this interaction, surface proteins such as Ssp-1, Ssp-2 and AtlE may play a role. The first two proteins, for which molecular properties have not been described, form fimbria-like appendages protruding from the bacterial cell surface. It has recently been suggested that those proteins may be identical to two isoforms of the accumulation-associated protein, Aap. AtlE mediates the adhesion of S. epidermidis to a polystyrene surface and acts as an autolysin. Therefore, AtlE may be indirectly involved in cell adhesion via liberation of DNA. Addition of DNase I revealed that cell-free DNA is contributing to the attachment of S. epidermidis to both plastic and glass surfaces in the initial phase of biofilm development, and thus dismissing the role of AtlE. Teichoic acids (TAs), because of their polyanionic properties, also affect initial adherence. The role of TAs in S. epidermidis biofilm formation was confirmed by deletion of tagO, encoding an enzyme involved in TA biosynthesis. This increased the cell surface hydrophobicity and resulted in an impaired biofilm formation.
Indirect binding to a surface already coated with host plasma and matrix proteins such as fibrinogen (Fg), fibronectin (Fn) or collagen (Cn) can be mediated via microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Proteins such as extracellular matrix-binding protein (Embp), GehD, SdrG (also known as Fbe) and SdrF, the AtlE and Aae autolysins and cell wall TAs have already been described to possess matrix-binding properties. Consequently, all these surface-located components are good candidates for vaccine development aiming at the inhibition of the initial step of biofilm formation.
After adherence to the implant surface, biofilms develop through intercellular aggregation, which is realized by several components and finally results in a multilayered S. epidermidis biofilm. The major factor involved in intercellular adhesion is polysaccharide intercellular adhesin (PIA), which is identical to poly-N-acetylglucosamine. It is a homoglycan of β-1,6-linked N-acetylglucosamine positively charged due to 15–20% non-N-acetylated residues and with negative charges due to O-succinoyl ester groups. The de-acetylation of PIA is not only essential for biofilm formation, but also crucial for S. epidermidis virulence (see further). PIA biosynthesis depends on the expression of the icaADBC operon, which is controlled by a complex regulatory network (for the biosynthesis and composition of PIA, see reviews ). The presence of a functional icaADBC operon is clearly linked to enhanced virulence of S. epidermidis.
Nevertheless, S. epidermidis strains lacking icaADBC but still producing biofilm were isolated, indicating the existence of an ica-independent mechanism of cell accumulation. Later, a proteinaceous intercellular adhesin involved in cell accumulation during biofilm formation was discovered. This cell wall-anchored (CWA) accumulation-associated protein (Aap) is composed of several domains, of which domain A shows binding activity to corneocytes, rendering it important for skin colonization. In contrast, domain B consists of the so-called G5 domain repeats. Domain B has been proposed to be involved in N-acetylglucosamine binding, thus forming a protein–polysaccharide biofilm network. However, more recently, Zn-dependent dimerization of two G5 domains was described. Through a 'zinc zipper' mechanism, self-association of G5 domains in opposing Aap molecules occurs. To gain this intercellular aggregative function, the fibrillar Aap proteins have to be proteolytically processed by means of staphylococcal or host proteases, removing most of the A domain. Aap may be functionally involved in PIA-independent biofilm formation and has clinical relevance as shown for prosthetic joint infections. The use of Aap as a vaccine component to combat biofilm-related S. epidermidis infections will be discussed below.
Alternative proteins possibly mediating proteinaceous biofilm accumulation without the necessity of icaABCD are the biofilm-associated protein (Bap) and the biofilm-associated homolog protein (Bhp). Although Bap has been linked to mastitis-derived S. epidermidis isolates, Bhp has been found in human isolates. Both CWA proteins may play a role in cellular adhesion. Recently, Embp – already known as an Fn-binding MSCRAMM – was identified as a proteinaceous intercellular adhesin sufficient for mediating biofilm formation.
In addition to polysaccharides and protein networks, TAs and extracellular DNA are adhesive elements constituting the extracellular polymeric substance matrix of mature S. epidermidis biofilms. Because of their polyanionic character, they most likely act as structure-stabilizing factors by linking molecules together. Recently, it has been shown that the addition of DNase I to the growth medium during biofilm formation leads to an altered community, although the enzyme had no effect on preformed biofilms.
During biofilm formation, fluid-filled channels are formed and the biofilm gets a specific 3D structure. Although the processes involved in S. epidermidis biofilm structuring have still not been completely unraveled, it is believed that quorum-sensing-controlled modulins play a key role. These phenol-soluble modulins (PSMs) are a class of surfactant-like peptides characterized by an amphipathic α-helical structure and divided into two types: the shorter α type of approximately 20 amino acids (PSMα, γ, δ and ɛ) and the longer β type of approximately 40 amino acids (PSMβs). During biofilm development, a downregulation of PSM expression was observed, indicating that the absence of PSMs has an important function in S. epidermidis biofilms. Recently, Wang et al. demonstrated that the β-type PSMs, all encoded in one operon, were nearly the only PSM type present in biofilms. At lower concentration, the PSMβs may form the molecular basis of cell–cell disruption, resulting in the formation of void spaces and channels.
In addition to the S. epidermidis autolysin proteins AtlE and Aae, the two-component regulatory system LytSR and the CidA/LrgA (holin/antiholin) system are regulating cell death and lysis inside S. epidermidis biofilms. Surrounding cells may benefit from these two processes because the released compounds may serve as nutrients.
Biofilm structuring finally leads to a chemically heterogeneous layer. Accordingly, mature biofilms consist of cells in at least four physiologically different states: cells growing aerobically; others growing fermentatively; approximately 10% dead cells; and, finally, phenotypic variants or dormant cells. The presence of diverse subpopulations might increase the range of conditions in which the community as a whole can flourish.
Once a mature staphylococcal biofilm has been established, individual cells or cell aggregates may be released, and these cells disperse, leading not only to embolisms, sepsis and hospital-acquired pneumonia, but also to biofilm formation at other sites. Several factors have been proposed to trigger biofilm disassembly, including mechanical forces, nutrient depletion, waste products, pH change, cessation of production of biofilm-building material and production of specific detachment factors. In a mature biofilm, increasing levels of PSMβ lead to biofilm cluster detachment, which in turn results in systemic spread of biofilm fragments. The key role of PSMβ in biofilm dissemination from S. epidermidis-colonized catheters was recently proven in a mouse model. In addition, PSMγ (identical to δ-toxin) has been proposed to act as a cell–cell interruption factor.
Biofilm Formation: The Major Virulence Factor
As mentioned, device-related S. epidermidis infections are related to its capacity to form biofilms. Because S. epidermidis biofilm formation and its regulation, including quorum-sensing control, were recently reviewed, we will only briefly describe the process of biofilm formation emphasizing factors with value for immunoprophylaxis or immunotherapy of S. epidermidis infections.
Primary Attachment
The first step of biofilm formation is bacterial adherence to the surface. Direct binding to an abiotic implant surface is mediated by electrostatic and hydrophobic interactions, van der Waals forces and affected by physicochemical variables. In this interaction, surface proteins such as Ssp-1, Ssp-2 and AtlE may play a role. The first two proteins, for which molecular properties have not been described, form fimbria-like appendages protruding from the bacterial cell surface. It has recently been suggested that those proteins may be identical to two isoforms of the accumulation-associated protein, Aap. AtlE mediates the adhesion of S. epidermidis to a polystyrene surface and acts as an autolysin. Therefore, AtlE may be indirectly involved in cell adhesion via liberation of DNA. Addition of DNase I revealed that cell-free DNA is contributing to the attachment of S. epidermidis to both plastic and glass surfaces in the initial phase of biofilm development, and thus dismissing the role of AtlE. Teichoic acids (TAs), because of their polyanionic properties, also affect initial adherence. The role of TAs in S. epidermidis biofilm formation was confirmed by deletion of tagO, encoding an enzyme involved in TA biosynthesis. This increased the cell surface hydrophobicity and resulted in an impaired biofilm formation.
Indirect binding to a surface already coated with host plasma and matrix proteins such as fibrinogen (Fg), fibronectin (Fn) or collagen (Cn) can be mediated via microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Proteins such as extracellular matrix-binding protein (Embp), GehD, SdrG (also known as Fbe) and SdrF, the AtlE and Aae autolysins and cell wall TAs have already been described to possess matrix-binding properties. Consequently, all these surface-located components are good candidates for vaccine development aiming at the inhibition of the initial step of biofilm formation.
Accumulation
After adherence to the implant surface, biofilms develop through intercellular aggregation, which is realized by several components and finally results in a multilayered S. epidermidis biofilm. The major factor involved in intercellular adhesion is polysaccharide intercellular adhesin (PIA), which is identical to poly-N-acetylglucosamine. It is a homoglycan of β-1,6-linked N-acetylglucosamine positively charged due to 15–20% non-N-acetylated residues and with negative charges due to O-succinoyl ester groups. The de-acetylation of PIA is not only essential for biofilm formation, but also crucial for S. epidermidis virulence (see further). PIA biosynthesis depends on the expression of the icaADBC operon, which is controlled by a complex regulatory network (for the biosynthesis and composition of PIA, see reviews ). The presence of a functional icaADBC operon is clearly linked to enhanced virulence of S. epidermidis.
Nevertheless, S. epidermidis strains lacking icaADBC but still producing biofilm were isolated, indicating the existence of an ica-independent mechanism of cell accumulation. Later, a proteinaceous intercellular adhesin involved in cell accumulation during biofilm formation was discovered. This cell wall-anchored (CWA) accumulation-associated protein (Aap) is composed of several domains, of which domain A shows binding activity to corneocytes, rendering it important for skin colonization. In contrast, domain B consists of the so-called G5 domain repeats. Domain B has been proposed to be involved in N-acetylglucosamine binding, thus forming a protein–polysaccharide biofilm network. However, more recently, Zn-dependent dimerization of two G5 domains was described. Through a 'zinc zipper' mechanism, self-association of G5 domains in opposing Aap molecules occurs. To gain this intercellular aggregative function, the fibrillar Aap proteins have to be proteolytically processed by means of staphylococcal or host proteases, removing most of the A domain. Aap may be functionally involved in PIA-independent biofilm formation and has clinical relevance as shown for prosthetic joint infections. The use of Aap as a vaccine component to combat biofilm-related S. epidermidis infections will be discussed below.
Alternative proteins possibly mediating proteinaceous biofilm accumulation without the necessity of icaABCD are the biofilm-associated protein (Bap) and the biofilm-associated homolog protein (Bhp). Although Bap has been linked to mastitis-derived S. epidermidis isolates, Bhp has been found in human isolates. Both CWA proteins may play a role in cellular adhesion. Recently, Embp – already known as an Fn-binding MSCRAMM – was identified as a proteinaceous intercellular adhesin sufficient for mediating biofilm formation.
Biofilm Structuring & Maturation
In addition to polysaccharides and protein networks, TAs and extracellular DNA are adhesive elements constituting the extracellular polymeric substance matrix of mature S. epidermidis biofilms. Because of their polyanionic character, they most likely act as structure-stabilizing factors by linking molecules together. Recently, it has been shown that the addition of DNase I to the growth medium during biofilm formation leads to an altered community, although the enzyme had no effect on preformed biofilms.
During biofilm formation, fluid-filled channels are formed and the biofilm gets a specific 3D structure. Although the processes involved in S. epidermidis biofilm structuring have still not been completely unraveled, it is believed that quorum-sensing-controlled modulins play a key role. These phenol-soluble modulins (PSMs) are a class of surfactant-like peptides characterized by an amphipathic α-helical structure and divided into two types: the shorter α type of approximately 20 amino acids (PSMα, γ, δ and ɛ) and the longer β type of approximately 40 amino acids (PSMβs). During biofilm development, a downregulation of PSM expression was observed, indicating that the absence of PSMs has an important function in S. epidermidis biofilms. Recently, Wang et al. demonstrated that the β-type PSMs, all encoded in one operon, were nearly the only PSM type present in biofilms. At lower concentration, the PSMβs may form the molecular basis of cell–cell disruption, resulting in the formation of void spaces and channels.
In addition to the S. epidermidis autolysin proteins AtlE and Aae, the two-component regulatory system LytSR and the CidA/LrgA (holin/antiholin) system are regulating cell death and lysis inside S. epidermidis biofilms. Surrounding cells may benefit from these two processes because the released compounds may serve as nutrients.
Biofilm structuring finally leads to a chemically heterogeneous layer. Accordingly, mature biofilms consist of cells in at least four physiologically different states: cells growing aerobically; others growing fermentatively; approximately 10% dead cells; and, finally, phenotypic variants or dormant cells. The presence of diverse subpopulations might increase the range of conditions in which the community as a whole can flourish.
Biofilm Detachment & Dispersal
Once a mature staphylococcal biofilm has been established, individual cells or cell aggregates may be released, and these cells disperse, leading not only to embolisms, sepsis and hospital-acquired pneumonia, but also to biofilm formation at other sites. Several factors have been proposed to trigger biofilm disassembly, including mechanical forces, nutrient depletion, waste products, pH change, cessation of production of biofilm-building material and production of specific detachment factors. In a mature biofilm, increasing levels of PSMβ lead to biofilm cluster detachment, which in turn results in systemic spread of biofilm fragments. The key role of PSMβ in biofilm dissemination from S. epidermidis-colonized catheters was recently proven in a mouse model. In addition, PSMγ (identical to δ-toxin) has been proposed to act as a cell–cell interruption factor.