Determination of the membrane topology of PORCN, an O-acyl transferase that modifies Wnt signalling proteins.

Wnt gradients elicit distinct cellular responses, such as proliferation, specification, differentiation and survival in a dose-dependent manner. Porcupine (PORCN), a membrane-bound O-acyl transferase (MBOAT) that resides in the endoplasmic reticulum, catalyses the addition of monounsaturated palmitate to Wnt proteins and is required for Wnt gradient formation and signalling. In humans, PORCN mutations are causal for focal dermal hypoplasia (FDH), an X-linked dominant syndrome characterized by defects in mesodermal and endodermal tissues. PORCN is also an emerging target for cancer therapeutics. Despite the importance of this enzyme, its structure remains poorly understood. Recently, the crystal structure of DltB, an MBOAT family member from bacteria, was solved. In this report, we use experimental data along with homology modelling to DltB to determine the membrane topology of PORCN. Our studies reveal that PORCN has 11 membrane domains, comprising nine transmembrane spanning domains and two reentrant domains. The N-terminus is oriented towards the lumen while the C-terminus is oriented towards the cytosol. Like DltB, PORCN has a funnel-like structure that is encapsulated by multiple membrane-spanning helices. This new model for PORCN topology allows us to map residues that are important for biological activity (and implicated in FDH) onto its three-dimensional structure.

Diversity of Functionally Distinct Clonal Sets of Human Conventional Memory B Cells That Bind Staphylococcal Protein A.

Staphylococcus aureus, a common cause of serious and often fatal infections, is well-armed with secreted factors that disarm host immune defenses. Highly expressed in vivo during infection, Staphylococcal protein A (SpA) is reported to also contribute to nasal colonization that can be a prelude to invasive infection. Co-evolution with the host immune system has provided SpA with an Fc-antibody binding site, and a Fab-binding site responsible for non-immune superantigen interactions via germline-encoded surfaces expressed on many human BCRs. We wondered whether the recurrent exposures to S. aureus commonly experienced by adults, result in the accumulation of memory B-cell responses to other determinants on SpA. We therefore isolated SpA-specific class-switched memory B cells, and characterized their encoding VH : VL antibody genes. In SpA-reactive memory B cells, we confirmed a striking bias in usage for VH genes, which retain the surface that mediates the SpA-superantigen interaction. We postulate these interactions reflect co-evolution of the host immune system and SpA, which during infection results in immune recruitment of an extraordinarily high prevalence of B cells in the repertoire that subverts the augmentation of protective defenses. Herein, we provide the first evidence that human memory responses are supplemented by B-cell clones, and circulating-antibodies, that bind to SpA determinants independent of the non-immune Fc- and Fab-binding sites. In parallel, we demonstrate that healthy individuals, and patients recovering from S. aureus infection, both have circulating antibodies with these conventional binding specificities. These findings rationalize the potential utility of incorporating specially engineered SpA proteins into a protective vaccine.

Convergent Evolution of Neutralizing Antibodies to Staphylococcus aureus γ-Hemolysin C That Recognize an Immunodominant Primary Sequence-Dependent B-Cell Epitope.

Staphylococcus aureus infection is a major public health threat in part due to the spread of antibiotic resistance and repeated failures to develop a protective vaccine. Infection is associated with production of virulence factors that include exotoxins that attack host barriers and cellular defenses, such as the leukocidin (Luk) family of bicomponent pore-forming toxins. To investigate the structural basis of antibody-mediated functional inactivation of Luk toxins, we generated a panel of murine monoclonal antibodies (MAbs) that neutralize host cell killing by the γ-hemolysin HlgCB. By biopanning these MAbs against a phage-display library of random Luk peptide fragments, we identified a small subregion within the rim domain of HlgC as the epitope for all the MAbs. Within the native holotoxin, this subregion folds into a conserved ß-hairpin structure, with exposed key residues, His252 and Tyr253, required for antibody binding. On the basis of the phage-display results and molecular modeling, a 15-amino-acid synthetic peptide representing the minimal epitope on HlgC (HlgC241-255) was designed, and preincubation with this peptide blocked antibody-mediated HIgCB neutralization. Immunization of mice with HlgC241-255 or the homologous LukS246-260 subregion peptide elicited serum antibodies that specifically recognized the native holotoxin subunits. Furthermore, serum IgG from patients who were convalescent for invasive S. aureus infection showed neutralization of HlgCB toxin activity ex vivo, which recognized the immunodominant HlgC241-255 peptide and was dependent on His252 and Tyr253 residues. We have thus validated an efficient, rapid, and scalable experimental workflow for identification of immunodominant and immunogenic leukotoxin-neutralizing B-cell epitopes that can be exploited for new S. aureus-protective vaccines and immunotherapies.

Unbiased Identification of Immunogenic Staphylococcus aureus Leukotoxin B-Cell Epitopes.

Unbiased identification of individual immunogenic B-cell epitopes in major antigens of a pathogen remains a technology challenge for vaccine discovery. We therefore developed a platform for rapid phage display screening of deep recombinant libraries consisting of as few as one major pathogen antigen. Using the bicomponent pore-forming leukocidin (Luk) exotoxins of the major pathogen Staphylococcus aureus as a prototype, we randomly fragmented and separately ligated the hemolysin gamma A (HlgA) and LukS genes into a custom-built phage display system, termed pComb-Opti8. Deep sequence analysis of barcoded amplimers of the HlgA and LukS gene fragment libraries demonstrated that biopannng against a cross-reactive anti-Luk monoclonal antibody (MAb) recovered convergent molecular clones with short overlapping homologous sequences. We thereby identified an 11-amino-acid sequence that is highly conserved in four Luk toxin subunits and is ubiquitous in representation within S. aureus clinical isolates. The isolated 11-amino-acid peptide probe was predicted to retain the native three-dimensional (3D) conformation seen within the Luk holotoxin. Indeed, this peptide was recognized by the selecting anti-Luk MAb, and, using mutated peptides, we showed that a particular amino acid side chain was essential for these interactions. Furthermore, murine immunization with this peptide elicited IgG responses that were highly reactive with both the autologous synthetic peptide and the full-length Luk toxin homologues. Thus, using a gene fragment- and phage display-based pipeline, we have identified and validated immunogenic B-cell epitopes that are cross-reactive between members of the pore-forming leukocidin family. This approach could be harnessed to identify novel epitopes for a much-needed S. aureus-protective subunit vaccine.

Hierarchy of human IgG recognition within the Staphylococcus aureus immunome.

Staphylococcus aureus is an opportunistic pathogen that causes a range of serious infections associated with significant morbidity, by strains increasingly resistant to antibiotics. However, to date all candidate vaccines have failed to induce protective immune responses in humans. We need a more comprehensive understanding of the antigenic targets important in the context of human infection. To investigate infection-associated immune responses, patients were sampled at initial presentation and during convalescence from three types of clinical infection; skin and soft tissue infection (SSTI), prosthetic joint infection (PJI) and pediatric hematogenous osteomyelitis (PHO). Reactivity of serum IgG was tested with an array of recombinant proteins, representing over 2,652 in-vitro-translated open reading frames (ORFs) from a community-acquired methicillin-resistant S. aureus USA300 strain. High-level reactivity was demonstrated for 104 proteins with serum IgG in all patient samples. Overall, high-level IgG-reactivity was most commonly directed against a subset of secreted proteins. Although based on limited surveys, we found subsets of S. aureus proteins with differential reactivity with serum samples from patients with different clinical syndromes. Together, our studies have revealed a hierarchy within the diverse proteins of the S. aureus "immunome", which will help to advance efforts to develop protective immunotherapeutic agents.

Human Memory B Cells Targeting Staphylococcus aureus Exotoxins Are Prevalent with Skin and Soft Tissue Infection.

Staphylococcus aureus is a Gram-positive opportunistic pathogen that causes superficial and invasive infections in the hospital and community. High mortality from infection emphasizes the need for improved methods for prevention and treatment. Although S. aureus possesses an arsenal of virulence factors that contribute to evasion of host defenses, few studies have examined long-term humoral and B-cell responses. Adults with acute-phase skin and soft tissue infections were recruited; blood samples were obtained; and S. aureus isolates, including methicillin-resistant strains, were subjected to genomic sequence analysis. In comparisons of acute-phase sera with convalescent-phase sera, a minority (37.5%) of patients displayed 2-fold or greater increases in antibody titers against three or more S. aureus antigens, whereas nearly half exhibited no changes, despite the presence of toxin genes in most infecting strains. Moreover, enhanced antibody responses waned over time, which could reflect a defect in B-cell memory or long-lived plasma cells. However, memory B cells reactive with a range of S. aureus antigens were prevalent at both acute-phase and convalescent-phase time points. While some memory B cells exhibited toxin-specific binding, those cross-reactive with structurally related leucocidin subunits were dominant across patients, suggesting the targeting of conserved epitopes. Memory B-cell reactivity correlated with serum antibody levels for selected S. aureus exotoxins, suggesting a relationship between the cellular and humoral compartments. Overall, although there was no global defect in the representation of anti-S. aureus memory B cells, there was evidence of restrictions in the range of epitopes recognized, which may suggest potential therapeutic approaches for augmenting host defenses.IMPORTANCE The contribution of B-cell memory and long-term antibody responses to host defenses against S. aureus exotoxins remains poorly understood. Our studies confirmed that infection did not commonly lead to enhanced long-term humoral responses. Whereas circulating memory B cells against S. aureus secreted exotoxins were prevalent, they were dominated by cross-reactivity with structurally related leucocidin subunits, consistent with recognition of conserved epitopes. These findings also provide the first evidence of a relationship between the reactivity of antistaphylococcal circulating memory B cells and serum antibody levels. In general, infection was not associated with a global defect in B-cell memory for S. aureus secreted factors, and responses were highly dominated by cross-reactivity to structurally related exotoxins, which arguably may alone be suboptimal in providing host defenses. Our studies illuminate aspects of the S. aureus-host relationship that may better inform strategies for the development of an effective protective vaccine.