Staphylococcal Protein A Contributes to Persistent Colonization of Mice with Staphylococcus aureus.

Staphylococcus aureus persistently colonizes the nasopharynx in humans, which increases the risk for invasive diseases, such as skin infection and bacteremia. Nasal colonization triggers IgG responses against staphylococcal surface antigens; however, these antibodies cannot prevent subsequent colonization or disease. Here, we describe S. aureus WU1, a multilocus sequence type 88 (ST88) isolate that persistently colonizes the nasopharynx in mice. We report that staphylococcal protein A (SpA) is required for persistence of S. aureus WU1 in the nasopharynx. Compared to animals colonized by wild-type S. aureus, mice colonized with the Δspa variant mount increased IgG responses against staphylococcal colonization determinants. Immunization of mice with a nontoxigenic SpA variant, which cannot cross-link B cell receptors and divert antibody responses, elicits protein A-neutralizing antibodies that promote IgG responses against colonizing S. aureus and diminish pathogen persistence.IMPORTANCEStaphylococcus aureus persistently colonizes the nasopharynx in about one-third of the human population, thereby promoting community- and hospital-acquired infections. Antibiotics are currently used for decolonization of individuals at increased risk of infection. However, the efficacy of antibiotics is limited by recolonization and selection for drug-resistant strains. Here, we propose a model of how staphylococcal protein A (SpA), a B cell superantigen, modifies host immune responses during colonization to support continued persistence of S. aureus in the nasopharynx. We show that this mechanism can be thwarted by vaccine-induced anti-SpA antibodies that promote IgG responses against staphylococcal antigens and diminish colonization.

N-acetylglucosaminylation of serine-aspartate repeat proteins promotes Staphylococcus aureus bloodstream infection.

Staphylococcus aureus secretes products that convert host fibrinogen to fibrin and promote its agglutination with fibrin fibrils, thereby shielding bacteria from immune defenses. The agglutination reaction involves ClfA (clumping factor A), a surface protein with serine-aspartate (SD) repeats that captures fibrin fibrils and fibrinogen. Pathogenic staphylococci express several different SD proteins that are modified by two glycosyltransferases, SdgA and SdgB. Here, we characterized three genes of S. aureus, aggA, aggB (sdgA), and aggC (sdgB), and show that aggA and aggC contribute to staphylococcal agglutination with fibrin fibrils in human plasma. We demonstrate that aggB (sdgA) and aggC (sdgB) are involved in GlcNAc modification of the ClfA SD repeats. However, only sdgB is essential for GlcNAc modification, and an sdgB mutant is defective in the pathogenesis of sepsis in mice. Thus, GlcNAc modification of proteins promotes S. aureus replication in the bloodstream of mammalian hosts.

Structural and functional characterization of the Streptococcus pneumoniae RrgB pilus backbone D1 domain.

Streptococcus pneumoniae expresses on its surface adhesive pili, involved in bacterial attachment to epithelial cells and virulence. The pneumococcal pilus is composed of three proteins, RrgA, RrgB, and RrgC, each stabilized by intramolecular isopeptide bonds and covalently polymerized by means of intermolecular isopeptide bonds to form an extended fiber. RrgB is the pilus scaffold subunit and is protective in vivo in mouse models of sepsis and pneumonia, thus representing a potential vaccine candidate. The crystal structure of a major RrgB C-terminal portion featured an organization into three independently folded protein domains (D2-D4), whereas the N-terminal D1 domain (D1) remained unsolved. We have tested the four single recombinant RrgB domains in active and passive immunization studies and show that D1 is the most effective, providing a level of protection comparable with that of the full-length protein. To elucidate the structural features of D1, we solved the solution structure of the recombinant domain by NMR spectroscopy. The spectra analysis revealed that D1 has many flexible regions, does not contain any intramolecular isopeptide bond, and shares with the other domains an Ig-like fold. In addition, we demonstrated, by site-directed mutagenesis and complementation in S. pneumoniae, that the D1 domain contains the Lys residue (Lys-183) involved in the formation of the intermolecular isopeptide bonds and pilus polymerization. Finally, we present a model of the RrgB protein architecture along with the mapping of two surface-exposed linear epitopes recognized by protective antisera.

Protein A-specific monoclonal antibodies and prevention of Staphylococcus aureus disease in mice.

Staphylococcus aureus is a leading cause of human soft tissue infections and bacterial sepsis. The emergence of antibiotic-resistant strains (methicillin-resistant S. aureus [MRSA]) has prompted research into staphylococcal vaccines and preventive measures. The envelope of S. aureus is decorated with staphylococcal protein A (SpA), which captures the Fcγ portion of immunoglobulins to prevent opsonophagocytosis and associates with the Fab portion of V(H)3-type B cell receptors to trigger B cell superantigen activity. Nontoxigenic protein A (SpA(KKAA)), when used as an immunogen in mice, stimulates humoral immune responses that neutralize the Fcγ and the V(H)3(+) Fab binding activities of SpA and provide protection from staphylococcal abscess formation in mice. Here, we isolated monoclonal antibodies (MAbs) against SpA(KKAA) that, by binding to the triple-helical bundle fold of its immunoglobulin binding domains (IgBDs), neutralize the Fcγ and Fab binding activities of SpA. SpA(KKAA) MAbs promoted opsonophagocytic killing of MRSA in mouse and human blood, provided protection from abscess formation, and stimulated pathogen-specific immune responses in a mouse model of staphylococcal disease. Thus, SpA(KKAA) MAbs may be useful for the prevention and therapy of staphylococcal disease in humans.

Coagulases as determinants of protective immune responses against Staphylococcus aureus.

During infection, Staphylococcus aureus secretes two coagulases (Coa and von Willebrand factor binding protein [vWbp]), which, following an association with host prothrombin and fibrinogen, form fibrin clots and enable the establishment of staphylococcal disease. Within the genomes of different S. aureus isolates, coagulase gene sequences are variable, and this has been exploited for a classification of types. We show here that antibodies directed against the variable prothrombin binding portion of coagulases confer type-specific immunity through the neutralization of S. aureus clotting activity and protection from staphylococcal disease in mice. By combining variable portions of coagulases from North American isolates into hybrid Coa and vWbp proteins, a subunit vaccine that provided protection against challenge with different coagulase-type S. aureus strains in mice was derived.

The two variants of the Streptococcus pneumoniae pilus 1 RrgA adhesin retain the same function and elicit cross-protection in vivo.

Thirty percent of Streptococcus pneumoniae isolates contain pilus islet 1, coding for a pilus composed of the backbone subunit RrgB and two ancillary proteins, RrgA and RrgC. RrgA is the major determinant of in vitro adhesion associated with pilus 1, is protective in vivo in mouse models, and exists in two variants (clades I and II). Mapping of the sequence variability onto the RrgA structure predicted from X-ray data showed that the diversity was restricted to the "head" of the protein, which contains the putative binding domains, whereas the elongated "stalk" was mostly conserved. To investigate whether this variability could influence the adhesive capacity of RrgA and to map the regions important for binding, two full-length protein variants and three recombinant RrgA portions were tested for adhesion to lung epithelial cells and to purified extracellular matrix (ECM) components. The two RrgA variants displayed similar binding abilities, whereas none of the recombinant fragments adhered at levels comparable to those of the full-length protein, suggesting that proper folding and structural arrangement are crucial to retain protein functionality. Furthermore, the two RrgA variants were shown to be cross-reactive in vitro and cross-protective in vivo in a murine model of passive immunization. Taken together, these data indicate that the region implicated in adhesion and the functional epitopes responsible for the protective ability of RrgA may be conserved and that the considerable level of variation found within the "head" domain of RrgA may have been generated by immunologic pressure without impairing the functional integrity of the pilus.

A second pilus type in Streptococcus pneumoniae is prevalent in emerging serotypes and mediates adhesion to host cells.

Analysis of publicly available genomes of Streptococcus pneumoniae has led to the identification of a new genomic element containing genes typical of gram-positive pilus islets (PIs). Here, we demonstrate that this genomic region, herein referred to as PI-2 (consisting of pitA, sipA, pitB, srtG1, and srtG2) codes for a second functional pilus in pneumococcus. Polymerization of the PI-2 pilus requires the backbone protein PitB as well as the sortase SrtG1 and the signal peptidase-like protein SipA. Presence of PI-2 correlates with the genotype as defined by multilocus sequence typing and clonal complex (CC). The PI-2-positive CCs are associated with serotypes 1, 2, 7F, 19A, and 19F, considered to be emerging serotypes in both industrialized and developing countries. Interestingly, strains belonging to CC271 (where sequence type 271 is the predicted founder of the CC) contain both PI-1 and PI-2, as revealed by genome analyses. In these strains both pili are surface exposed and independently assembled. Furthermore, in vitro experiments provide evidence that the pilus encoded by PI-2 of S. pneumoniae is involved in adherence. Thus, pneumococci encode at least two types of pili that play a role in the initial host cell contact to the respiratory tract and are potential antigens for inclusion in a new generation of pneumococcal vaccines.

Antibodies against a secreted product of Staphylococcus aureus trigger phagocytic killing.

Host immunity against bacteria typically involves antibodies that recognize the microbial surface and promote phagocytic killing. Methicillin-resistant Staphylococcus aureus (MRSA) is a frequent cause of lethal bloodstream infection; however, vaccines and antibody therapeutics targeting staphylococcal surface molecules have thus far failed to achieve clinical efficacy. S. aureus secretes coagulase (Coa), which activates host prothrombin and generates fibrin fibrils that protect the pathogen against phagocytosis by immune cells. Because of negative selection, the coding sequence for the prothrombin-binding D1-D2 domain is highly variable and does not elicit cross-protective immune responses. The R domain, tandem repeats of a 27-residue peptide that bind fibrinogen, is conserved at the C terminus of all Coa molecules, but its functional significance is not known. We show here that the R domain enables bloodstream infections by directing fibrinogen to the staphylococcal surface, generating a protective fibrin shield that inhibits phagocytosis. The fibrin shield can be marked with R-specific antibodies, which trigger phagocytic killing of staphylococci and protect mice against lethal bloodstream infections caused by a broad spectrum of MRSA isolates. These findings emphasize the critical role of coagulase in staphylococcal escape from opsonophagocytic killing and as a protective antigen for S. aureus vaccines.

A monoclonal antibody that recognizes the E domain of staphylococcal protein A.

Staphylococcal protein A (SpA) binds Fcγ and VH3 clan Fab domains of human and animal immunoglobulin (Ig) with each of its five Ig binding domains (IgBDs), thereby supporting Staphylococcus aureus escape from opsonophagocytic killing and suppressing adaptive B cell responses. The variant SpAKKAA cannot bind Ig yet retains antigenic properties that elicit SpA-neutralizing antibodies and disease protection in mice, whereas S. aureus infection or SpA-immunization cannot elicit neutralizing antibodies. As a test for this model, we analyzed here mAb 358A76, which was isolated from SpA-immunized mice. Unlike SpAKKAA-derived mAbs, mAb 358A76 binds only the first IgBD (E) but not any of the other four IgBDs (D-A-B-C) and its binding can neutralize only the E domain of SpA, which does not provide disease protection in mice. These results are in agreement with a model whereby wild-type SpA-immunization generates a limited antibody response without neutralizing and/or disease protective attributes.

Supramolecular organization of the repetitive backbone unit of the Streptococcus pneumoniae pilus.

Streptococcus pneumoniae, like many other Gram-positive bacteria, assembles long filamentous pili on their surface through which they adhere to host cells. Pneumococcal pili are formed by a backbone, consisting of the repetition of the major component RrgB, and two accessory proteins (RrgA and RrgC). Here we reconstruct by transmission electron microscopy and single particle image reconstruction method the three dimensional arrangement of two neighbouring RrgB molecules, which represent the minimal repetitive structural domain of the native pilus. The crystal structure of the D2-D4 domains of RrgB was solved at 1.6 A resolution. Rigid-body fitting of the X-ray coordinates into the electron density map enabled us to define the arrangement of the backbone subunits into the S. pneumoniae native pilus. The quantitative fitting provide evidence that the pneumococcal pilus consists uniquely of RrgB monomers assembled in a head-to-tail organization. The presence of short intra-subunit linker regions connecting neighbouring domains provides the molecular basis for the intrinsic pilus flexibility.