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.

ABSTRACTStaphylococcus aureusis a leading cause of human soft tissue infections and bacterial sepsis. The emergence of antibiotic-resistant strains (methicillin-resistantS. aureus[MRSA]) has prompted research into staphylococcal vaccines and preventive measures. The envelope ofS. aureusis decorated with staphylococcal protein A (SpA), which captures the Fcγ portion of immunoglobulins to prevent opsonophagocytosis and associates with the Fab portion of VH3-type B cell receptors to trigger B cell superantigen activity. Nontoxigenic protein A (SpAKKAA), when used as an immunogen in mice, stimulates humoral immune responses that neutralize the Fcγ and the VH3+Fab binding activities of SpA and provide protection from staphylococcal abscess formation in mice. Here, we isolated monoclonal antibodies (MAbs) against SpAKKAAthat, by binding to the triple-helical bundle fold of its immunoglobulin binding domains (IgBDs), neutralize the Fcγ and Fab binding activities of SpA. SpAKKAAMAbs 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, SpAKKAAMAbs may be useful for the prevention and therapy of staphylococcal disease in humans.

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 Å 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.

ABSTRACTDuring infection,Staphylococcus aureussecretes 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 differentS. aureusisolates, 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 ofS. aureusclotting 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-typeS. aureusstrains in mice was derived.

Vaccines and antibody therapeutics targeting staphylococcal surface molecules have failed to achieve clinical efficacy against MRSA infection. Here, Thomer et al. show that the R domain of prothrombin directs fibrinogen to the surface of S. aureus, which generates a protective coat for the pathogen, inhibiting phagocytosis by immune cells. The use of R-specific antibodies allows for immune cell recognition and protects mice against lethal bloodstream infections by broad spectrum MRSA isolates.

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.

Background: Staphylococcus aureus agglutinates in plasma in a manner that requires host fibrinogen and clumping factor A, a bacterial surface protein with serine-aspartate (SD) repeats. Results: SdgB modifies serine residues in SD repeats with GlcNAc, and this glycosylation contributes to the pathogenesis of sepsis. Conclusion: Glycosylation of SD repeats aids bacterial escape from host defenses. Significance: Interference with glycosylation may alter staphylococcal infections.

persistently colonizes the nasopharynx of 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 WU1, a multi-locus sequence type ST88 isolate, that persistently colonizes the nasopharynx of mice. We report that staphylococcal protein A (SpA) is required for persistence of WU1 in the nasopharynx. Compared to animals colonized by wild-type , mice colonized with the Δ variant mount increased IgG responses against staphylococcal colonization determinants. Immunization of mice with a non-toxigenic SpA variant, which cannot crosslink B cell receptors and divert antibody responses, elicits protein A-neutralizing antibodies that promote IgG responses against colonizing and diminish pathogen persistence. persistently colonizes the nasopharynx of about a 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 whereby staphylococcal protein A (SpA), a B cell superantigen, modifies host immune responses during colonization to support continued persistence of 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.