Proteomics-Based Identification of Anchorless Cell Wall Proteins as Vaccine Candidates against Staphylococcus aureus

Staphylococcus aureus is an important human pathogen with increasing clinical impact due to the extensive spread of antibiotic-resistant strains. Therefore, development of a protective polyvalent vaccine is of great clinical interest. We employed an intravenous immunoglobulin (IVIG) preparation as a source of antibodies directed against anchorless S. aureus surface proteins for identification of novel vaccine candidates. In order to identify such proteins, subtractive proteome analysis (SUPRA) of S. aureus anchorless cell wall proteins was performed. Proteins reacting with IVIG but not with IVIG depleted of S. aureus-specific opsonizing antibodies were considered vaccine candidates. Nearly 40 proteins were identified by this preselection method using matrix-assisted laser desorption ionization—time of flight analysis. Three of these candidate proteins, enolase (Eno), oxoacyl reductase (Oxo), and hypothetical protein hp2160, were expressed as glutathione S-transferase fusion proteins, purified, and used for enrichment of corresponding immunoglobulin Gs from IVIG by affinity chromatography. Use of affinity-purified anti-Eno, anti-Oxo, and anti-hp2160 antibodies resulted in opsonization, phagocytosis, and killing of S. aureus by human neutrophils. High specific antibody titers were detected in mice immunized with recombinant antigens. In mice challenged with bioluminescent S. aureus, reduced staphylococcal spread was measured by in vivo imaging. The recovery of S. aureus CFU from organs of immunized mice was diminished 10- to 100-fold. Finally, mice immunized with hp2160 displayed statistically significant higher survival rates after lethal challenge with clinically relevant S. aureus strains. Taken together, our data suggest that anchorless cell wall proteins might be promising vaccine candidates and that SUPRA is a valuable tool for their identification.Staphylococcus aureus is an opportunistic, nosocomial, community-acquired pathogen which causes several diseases ranging from minor skin infections to serious life-threatening infections like sepsis, endocarditis, pneumonia, and toxic shock syndrome (30). The rapid emergence of both hospital-associated methicillin (meticillin)-resistant S. aureus (MRSA) and community-acquired MRSA (CA-MRSA) is a major epidemiological problem worldwide (5, 25). A further threatening trend concerning S. aureus infections is the emergence of isolates with resistance to vancomycin, currently the antibiotic of choice against MRSA strains, and also to newly introduced drugs, such as daptomycin and linezolid (47). Hence, it is not surprising that interest in developing alternative approaches to prevent and treat staphylococcal infections has increased in recent years (34, 48).The major effector mechanism of the human immune system against S. aureus infection is comprised of professional phagocytes, such as neutrophils, that ingest and eliminate bacteria (16). However, phagocytosis of S. aureus relies on the opsonization of bacteria by antibodies and complement (7). Recognition of opsonizing antibodies bound to the surface of S. aureus via Fcγ receptors of neutrophils is a prerequisite for induction of the oxidative burst and therefore for killing of the phagocytosed bacteria (23) and induction of a long-term immune response (38). On the other hand, the presence of antistaphylococcal antibodies does not guarantee protection against reinfection. The reason for this apparent discrepancy is still not well understood. However, it was reported recently that antibodies against certain staphylococcal antigens present in healthy donors were missing or underrepresented in patient sera, indicating that antibodies reacting to these antigens are more efficient for induction of phagocytosis and for subsequent elimination of S. aureus than other antibodies (10, 12). Because of this, identification of protective antigens is a crucial step for vaccine development.Until now, most strategies for vaccination against S. aureus, active or passive, concentrated on single-component vaccines based on capsular polysaccharides or well-known virulence factors possessing the LPXTG motif, such as fibronectin binding protein (FnBP), collagen binding protein (CnBP), or clumping factor A (ClfA), as vaccination targets (14, 44). However, despite promising vaccination results obtained with animal models, so far most of the potential vaccines tested in clinical trials have failed to provide significant protection against S. aureus infection (48).It turns out that the efficacy of a monovalent vaccine may be hampered by the functional redundancy of adhesion proteins (17) or the appearance of escape mutants (56). Recently, it has been shown that a multivalent vaccine consisting of four antigenic determinants provides protection against lethal challenge with S. aureus in mice, whereas single-component immunization was much less effective (55). Therefore, identification of novel targets for an effective S. aureus vaccine has repeatedly been recognized as a high priority by experts in this field (20, 28, 34, 40, 43, 48). Indeed, numerous staphylococcal surface proteins predicted to be promising antigenic targets have been identified so far using recently adopted technologies, like proteomics (19, 36, 57) or protein selection methods based on expression libraries (10, 13, 58, 59). Unfortunately, most studies have not provided functional proof that identified proteins are vaccine candidates.Due to the fact that most of the previous experimental vaccine studies concentrated on candidates exhibiting the LPXTG sorting signal, we focused primarily on identification of noncovalently linked, cell wall-associated proteins, so-called anchorless cell wall (ACW) proteins. Proteins belonging to this class possess neither a conserved signal peptide nor an LPXTG motif and were recently recognized as novel virulence factors in gram-positive bacteria (9). Most of these ACW proteins are multifunctional; e.g., they are involved in different metabolic pathways and also in adhesion to extracellular matrix and invasion of host cells. Such proteins cannot be targeted by genome sequence screening due to the lack of conserved epitopes like LPXTG.For identification of new potential vaccine targets among ACW proteins we used intravenous immunoglobulin (IVIG) preparations to avoid limitation of the antibody source (i.e., individual sera). IVIG is a pool of immunoglobulins (Igs) from healthy persons that contains a broad spectrum of opsonizing antibodies against various pathogens, including S. aureus. By employing two-dimensional gel electrophoresis (2-DE), subtractive immunoblotting, and mass spectrometry, we identified several ACW proteins as novel vaccine targets. This method for identification of potential vaccines was called subtractive proteome analysis (SUPRA). The protective activity of antibodies raised against some of the identified proteins was evaluated in vitro and in a murine sepsis model.