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Staphylococcus aureus is one of the most important human pathogens worldwide. Its high antibiotic resistance profile reinforces the need for new interventions like vaccines in addition to new antibiotics. Vaccine development efforts against S. aureus have failed so far however, the findings from these human clinical and non-clinical studies provide potential insight for such failures. Currently, research is focusing on identifying novel vaccine formulations able to elicit potent humoral and cellular immune responses. Translational science studies are attempting to discover correlates of protection using animal models as well as in vitro and ex vivo models assessing efficacy of vaccine candidates. Several new vaccine candidates are being tested in human clinical trials in a variety of target populations. In addition to vaccines, bacteriophages, monoclonal antibodies, centyrins and new classes of antibiotics are being developed. Some of these have been tested in humans with encouraging results. The complexity of the diseases and the range of the target populations affected by this pathogen will require a multipronged approach using different interventions, which will be discussed in this review., Competing Interests: JC is a PhD fellow who is enrolled in the School of Biochemistry and Immunology at Trinity College Dublin and participates in a postgraduate studentship program at GSK. ES, SR, FB, and SP are employees of the GSK group of companies. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Clegg, Soldaini, McLoughlin, Rittenhouse, Bagnoli and Phogat.)

The skin is an immunocompetent tissue that harbors several kinds of immune cells and a plethora of commensal microbes constituting the skin microbiome. Staphylococcus aureus is a prominent skin pathogen that colonizes a large proportion of the human population. We currently have an incomplete understanding of the correlates of protection against S. aureus infection, however genetic and experimental evidence has shown that CD4 + T cells play a key role in orchestrating a protective anti- S. aureus immune response. A high S. aureus -specific memory CD4 + T cell response has been reported in the blood of healthy subjects. Since T cells are more abundant in the skin than in blood, we hypothesized that S. aureus -specific CD4 + T cells could be present in the skin of healthy individuals. Indeed, we observed proliferation of tissue-resident memory CD4 + T cells and production of IL-17A, IL-22, IFN-γ and TNF-β by cells isolated from abdominal skin explants in response to heat-killed S. aureus . Remarkably, these cytokines were produced also during an ex vivo epicutaneous S. aureus infection of human skin explants. These findings highlight the importance of tissue-resident memory CD4 + T cells present at barrier sites such as the skin, a primary entry site for S. aureus . Further phenotypical and functional characterization of these cells will ultimately aid in the development of novel vaccine strategies against this elusive pathogen., Competing Interests: AH, MEM, and ARC are Ph.D. fellows and are enrolled in the Infection and Immunity Ph.D. program, part of the graduate school of Life Sciences at Utrecht University and participated in a post graduate studentship program at GSK. BC was a PhD student from the University of Siena funded by GSK. ST, FB, and ES are employees of the GSK group of companies. FB hold shares in the GSK group of companies and holds pending and issued patents (WO/2019/158537, WO/2015/144691, WO/2015/144653, WO/2015/144655, WO/2014/033190, WO/2014/033191, WO/2014/033192, WO/2014/033193, WO/2013/030378, and WO/2010/119343) on S. aureus vaccine formulations., (Copyright © 2021 Hendriks, Mnich, Clemente, Cruz, Tavarini, Bagnoli and Soldaini.)

Tissue-resident memory T cells are important in adaptive immunity against many infections, rendering these cells attractive potential targets in vaccine development. Genetic and experimental evidence highlights the importance of cellular immunity in protection from Staphylococcus aureus skin infections, yet skin-resident memory T cells are, thus far, an untested component of immunity during such infections. Novel methods of generating and sampling vaccine-induced skin memory T cells are paralleled by discoveries of global, skin-wide immunosurveillance. We propose skin-resident memory CD4 + T cells as a potential missing link in the search for correlates of protection during S. aureus infections. A better appreciation of their phenotypes and functions could accelerate the development of preventive vaccines against this highly virulent and antibiotic-resistant pathogen., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Understanding Staphylococcus aureus ( S. aureus )-host immune system interaction is crucial to meet the tremendous medical need associated with this life-threatening bacterial infection. Given the crucial role of dendritic cells (DC) in dictating immune responses upon microbial challenge, we investigated how the bacterial viability and the conservation of structural integrity influence the response of human DC to S. aureus . To this end, human primary DC were stimulated with the methicillin-resistant S. aureus USA300 live strain, USA300 inactivated by heat (HI), ultraviolet irradiation (UVI), or paraformaldehyde treatment (PFAI) and subsequently analyzed for cell phenotype and immune-modulatory properties. Although no differences in terms of DC viability and maturation were observed when DC were stimulated with live or inactivated bacteria, the production of IL-12, IL-23, and other cytokines differed significantly. The Th1 and Th17 expansion was also more pronounced in response to live vs. inactivated S. aureus . Interestingly, cytokine production in DC treated with live and inactivated USA300 required phagocytosis, whereas blocking endosomal Toll-like receptor signaling mainly reduced the cytokine release by live and HI USA300. A further analysis of IFN-β signaling revealed the induction of a cyclic GMP-AMP synthase stimulator of interferon genes (cGAS-STING)-independent and IRF3-dependent signaling pathway(s) in UVI-stimulated DC. This study underscores the capacity of human DC to discriminate between live and inactivated S. aureus and, further, indicates that DC may represent a valuable experimental setting to test different inactivation methods with regard to the retention of S. aureus immunoregulatory properties. These and further insights may be useful for the development of novel therapeutic and prophylactic anti- S. aureus vaccine strategies., (Copyright © 2019 Cruciani, Sandini, Etna, Giacomini, Camilli, Severa, Rizzo, Bagnoli, Hiscott and Coccia.)

Staphylococcus aureus is an opportunistic human pathogen and a major cause of invasive infections such as bacteremia, endocarditis, pneumonia, and wound infections. FhuD2 is a staphylococcal lipoprotein involved in the uptake of iron-hydroxymate and is under the control of the iron uptake regulator Fur. This protein is part of an investigational multicomponent vaccine formulation that has shown protective efficacy in several murine models of infection. Even though fhuD2 expression has been shown to be upregulated in murine kidneys infected with S. aureus , it is not known whether the bacterium undergoes increased iron deprivation during prolonged infection. Furthermore, different S. aureus infection niches might provide different environments and levels of iron availability, resulting in different fhuD2 expression patterns among organs of the same host. To address these questions, we characterized the in vitro expression of the fhuD2 gene and confirmed Fur-dependent regulation of its expression. We further investigated its expression in mice infected with a bioluminescent reporter strain of S. aureus expressing the luciferase operon under the control of the fhuD2 promoter. The emission of bioluminescence in different organs was followed over a 7-day time course, and quantitative real-time PCR analysis of the RNA transcribed from the endogenous fhuD2 gene was performed. Using this approach, we were able to show that fhuD2 expression was induced during infection in all organs analyzed and that differences in expression were observed at different time points and in different infected organs. Our data suggest that S. aureus undergoes increased iron deprivation during the progression of infection in diverse host organs and accordingly induces dedicated iron acquisition mechanisms. Since FhuD2 plays a central role in providing the pathogen with the required iron, further knowledge of the patterns of fhuD2 expression in vivo during infection will be instrumental in better defining the role of this antigen in S. aureus pathogenesis and as a vaccine antigen., (Copyright © 2017 American Society for Microbiology.)

The opportunistic pathogen Staphylococcus aureus ( S. aureus ) is a major cause of nosocomial- and community-acquired infections. In addition, many antibiotic-resistant strains are emerging worldwide, thus, there is an urgent unmet need to pinpoint novel therapeutic and prophylactic strategies. In the present study, we characterized the impact of infection with the pandemic methicillin-resistant USA300 S. aureus strain on human primary dendritic cells (DC), key initiators and regulators of immune responses. In particular, among staphylococcal virulence factors, the function of EsxA and EsxB, two small acidic dimeric proteins secreted by the type VII-like secretion system Ess (ESAT-6-like secretion system), was investigated in human DC setting. A comparative analysis of bacterial entry, replication rate as well as DC maturation, apoptosis, signaling pathway activation and cytokine production was performed by using wild type (wt) USA300 and three isogenic mutants carrying the deletion of esxA (Δ esxA ), esxB (Δ esxB ), or both genes (Δ esxAB ). The S. aureus mutant lacking only the EsxA protein (Δ esxA) stimulated a stronger pro-apoptotic phenotype in infected DC as compared to wt USA300, Δ esxAB , and Δ esxB strains. When the mutant carrying the esxB deletion (Δ esxB ) was analyzed, a higher production of both regulatory and pro-inflammatory mediators was found in the infected DC with respect to those challenged with the wt counterpart and the other esx mutants. In accordance with these data, supernatant derived from Δ esxB -infected DC promoted a stronger release of both IFN-γ and IL-17 from CD4 + T cells as compared with those conditioned with supernatants derived from wild type USA300-, Δ esxAB -, and Δ esxA -infected cultures. Although, the interaction of S. aureus with human DC is not yet fully understood, our data suggest that both cytokine production and apoptotic process are modulated by Esx factors, thus indicating a possible role of these proteins in the modulation of DC-mediated immunity to S. aureus .

Staphylococcus aureus is a versatile, opportunistic human pathogen that can asymptomatically colonize a human host but can also cause a variety of cutaneous and systemic infections. The ability of S. aureus to adapt to such diverse environments is reflected in the presence of complex regulatory networks fine-tuning metabolic and virulence gene expression. One of the most widely distributed mechanisms is the two-component signal transduction system (TCS) which allows a pathogen to alter its gene expression profile in response to environmental stimuli. The simpler TCSs consist of only a transmembrane histidine kinase (HK) and a cytosolic response regulator. S. aureus encodes a total of 16 conserved pairs of TCSs that are involved in diverse signalling cascades ranging from global virulence gene regulation (e.g. quorum sensing by the Agr system), the bacterial response to antimicrobial agents, cell wall metabolism, respiration and nutrient sensing. These regulatory circuits are often interconnected and affect each other's expression, thus fine-tuning staphylococcal gene regulation. This manuscript gives an overview of the current knowledge of staphylococcal environmental sensing by TCS and its influence on virulence gene expression and virulence itself. Understanding bacterial gene regulation by TCS can give major insights into staphylococcal pathogenicity and has important implications for knowledge-based drug design and vaccine formulation.

Staphylococcus aureus is the major cause of human septic arthritis and osteomyelitis, which deserve special attention due to their rapid evolution and resistance to treatment. The progression of the disease depends on both bacterial presence in situ and uncontrolled disruptive immune response, which is responsible for chronic disease. Articular and bone infections are often the result of blood bacteremia, with the knees and hips being the most frequently infected joints showing the worst clinical outcome. We report the development of a hematogenous model of septic arthritis in murine knees, which progresses from an acute to a chronic phase, similarly to what occurs in humans. Characterization of the local and systemic inflammatory and immune responses following bacterial infection brought to light specific signatures of disease. Immunization of mice with the vaccine formulation we have recently described (4C-Staph), induced a strong antibody response and specific CD4+ effector memory T cells, and resulted in reduced bacterial load in the knee joints, a milder general inflammatory state and protection against bacterial-mediated cellular toxicity. Possible correlates of protection are finally proposed, which might contribute to the development of an effective vaccine for human use., Competing Interests: This work was sponsored by Novartis Vaccines and Diagnostics Srl, now acquired by the GSK group of companies. All authors have declared the following interests: all authors were permanent employees of Novartis Vaccines at the time of the study. Following the acquisition of Novartis Vaccines by the GSK group of companies in March, 2015, all but A.C. and T.S. are now permanent employees of the GSK group of companies. E.D.G., F.B., A.S. and S.B. report ownership of GSK shares and/or restricted GSK shares. A.C., F.B., G.B. and E.C. are listed as inventors on patents owned by the GSK group of companies.

During bacterial pathogenesis extensive contacts between the human and the bacterial extracellular proteomes take place. The identification of novel host-pathogen interactions by standard methods using a case-by-case approach is laborious and time consuming. To overcome this limitation, we took advantage of large libraries of human and bacterial recombinant proteins. We applied a large-scale protein microarray-based screening on two important human pathogens using two different approaches: (I) 75 human extracellular proteins were tested on 159 spotted Staphylococcus aureus recombinant proteins and (II) Neisseria meningitidis adhesin (NadA), an important vaccine component against serogroup B meningococcus, was screened against ≈2300 spotted human recombinant proteins. The approach presented here allowed the identification of the interaction between the S. aureus immune evasion protein FLIPr (formyl-peptide receptor like-1 inhibitory protein) and the human complement component C1q, key players of the offense-defense fighting; and of the interaction between meningococcal NadA and human LOX-1 (low-density oxidized lipoprotein receptor), an endothelial receptor. The novel interactions between bacterial and human extracellular proteins here presented might provide a better understanding of the molecular events underlying S. aureus and N. meningitidis pathogenesis.

Staphylococcus aureus alpha-hemolysin (Hla) assembles into heptameric pores on the host cell membrane, causing lysis, apoptosis, and junction disruption. Herein, we present the design of a newly engineered S. aureus alpha-toxin, HlaPSGS, which lacks the predicted membrane-spanning stem domain. This protein is able to form heptamers in aqueous solution in the absence of lipophilic substrata, and its structure, obtained by transmission electron microscopy and single-particle reconstruction analysis, resembles the cap of the wild-type cytolytic Hla pore. HlaPSGS was found to be impaired in binding to host cells and to its receptor ADAM10 and to lack hemolytic and cytotoxic activity. Immunological studies using human sera as well as sera from mice convalescent from S. aureus infection suggested that the heptameric conformation of HlaPSGS mimics epitopes exposed by the cytolytic Hla pore during infection. Finally, immunization with this newly engineered Hla generated high protective immunity against staphylococcal infection in mice. Overall, this study provides unprecedented data on the natural immune response against Hla and suggests that the heptameric HlaPSGS is a highly valuable vaccine candidate against S. aureus., (Copyright © 2016 Fiaschi et al.)

A rapidly acting, single dose vaccine against Staphylococcus aureus would be highly beneficial for patients scheduled for major surgeries or in intensive care units. Here we show that one immunization with a multicomponent S. aureus candidate vaccine, 4C-Staph, formulated with a novel TLR7-dependent adjuvant, T7-alum, readily protected mice from death and from bacterial dissemination, both in kidney abscess and peritonitis models, outperforming alum-formulated vaccine. This increased efficacy was paralleled by higher vaccine-specific and α-hemolysin-neutralizing antibody titers and Th1/Th17 cell responses. Antibodies played a crucial protective role, as shown by the lack of protection of 4C-Staph/T7-alum vaccine in B-cell-deficient mice and by serum transfer experiments. Depletion of effector CD4+ T cells not only reduced survival but also increased S. aureus load in kidneys of mice immunized with 4C-Staph/T7-alum. The role of IL-17A in the control of bacterial dissemination in 4C-Staph/T7-alum vaccinated mice was indicated by in vivo neutralization experiments. We conclude that single dose 4C-Staph/T7-alum vaccine promptly and efficiently protected mice against S. aureus through the combined actions of antibodies, CD4+ effector T cells, and IL-17A. These data suggest that inclusion of an adjuvant that induces not only fast antibody responses but also IL-17-producing cell-mediated effector responses could efficaciously protect patients scheduled for major surgeries or in intensive care units.

Staphylococcal leukotoxins are a family of β-barrel, bicomponent, pore-forming toxins with membrane-damaging functions. These bacterial exotoxins share sequence and structural homology and target several host-cell types. Leukotoxin ED (LukED) is one of these bicomponent pore-forming toxins that Staphylococcus aureus produces in order to suppress the ability of the host to contain the infection. The recent delineation of the important role that LukED plays in S. aureus pathogenesis and the identification of its protein receptors, combined with its presence in S. aureus methicillin-resistant epidemic strains, establish this leukocidin as a possible target for the development of novel therapeutics. Here, the crystal structures of the water-soluble LukE and LukD components of LukED have been determined. The two structures illustrate the tertiary-structural variability with respect to the other leukotoxins while retaining the conservation of the residues involved in the interaction of the protomers in the bipartite leukotoxin in the pore complex.

Lack of known mechanisms of protection against Staphylococcus aureus in humans is hindering development of efficacious vaccines. Preclinical as well as clinical data suggest that antibodies play an important role against S. aureus. For instance, certain hypogammaglobulinaemic patients are at increased risk of staphylococcal infections. However, development of effective humoral response may be dampened by converging immune-evasion mechanisms of S. aureus. We hypothesize that B-cell proliferation induced by staphylococcal protein A (SpA) and continuous antigen exposure, without the proper T-cell help and cytokine stimuli, leads to antigen-activated B-cell deletion and anergy. Recent findings suggest an important role of type I neutrophils (PMN-I) and conventionally activated macrophages (M1) against S. aureus, while alternatively activated macrophages (M2) favour biofilm persistence and sepsis. In addition, neutrophil-macrophage cooperation promotes extravasation and activation of neutrophils as well as clearance of bacteria ensnared in neutrophil extracellular traps. Activation of these processes is modulated by cytokines and T cells. Indeed, low CD4(+) T-cell counts represent an important risk factor for skin infections and bacteraemia in patients. Altogether, these observations could lead to the identification of predictive correlates of protection and ways for shifting the balance of the response to the benefit of the host through vaccination., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Staphylococcus aureus is a human bacterial pathogen causing a variety of diseases. The occurrence of multidrug-resistant strains of Staphylococcus aureus underlines the need for a vaccine. Defining immune correlates of protection may support the design of an effective vaccine. We used a murine Staphylococcus aureus infection model, in which bacteria were inoculated in an air pouch generated on the back of the animal. Analysis of the air-pouch content in mice immunized or not with an adjuvanted multiantigen vaccine formulation, four-component S. aureus vaccine (4C-Staph), prior to infection allowed us to measure bacteria, cytokines, and 4C-Staph-specific antibodies and to analyze host immune cells recruited to the infection site. Immunization with 4C-Staph resulted in accumulation of antigen-specific antibodies in the pouch and mitigated the infection. Neutrophils were the most abundant cells in the pouch, and they showed the upregulation of Fcγ receptor (FcγR) following immunization with 4C-Staph. Reduction of the infection was also obtained in mice immunized with 4C-Staph and depleted of neutrophils; these mice showed an increase in monocytes and macrophages. Upregulation of the FcγR and the presence of antigen-specific antibodies induced by immunization with 4C-Staph may contribute to increase bacterial opsonophagocytosis. Protection in neutropenic mice indicated that an effective vaccine could activate alternative protection mechanisms compensating for neutropenia, a condition often occurring in S. aureus-infected patients., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The opportunistic pathogen Staphylococcus aureus is one of the major causes of health care-associated infections. S. aureus is primarily an extracellular pathogen, but it was recently reported to invade and replicate in several host cell types. The ability of S. aureus to persist within cells has been implicated in resistance to antimicrobials and recurrent infections. However, few staphylococcal proteins that mediate intracellular survival have been identified. Here we examine if EsxA and EsxB, substrates of the ESAT-6-like secretion system (Ess), are important during intracellular S. aureus infection. The Esx proteins are required for staphylococcal virulence, but their functions during infection are unclear. While isogenic S. aureus esxA and esxB mutants were not defective for epithelial cell invasion in vitro, a significant increase in early/late apoptosis was observed in esxA mutant-infected cells compared to wild-type-infected cells. Impeding secretion of EsxA by deleting C-terminal residues of the protein also resulted in a significant increase of epithelial cell apoptosis. Furthermore, cells transfected with esxA showed an increased protection from apoptotic cell death. A double mutant lacking both EsxA and EsxB also induced increased apoptosis but, remarkably, was unable to escape from cells as efficiently as the single mutants or the wild type. Thus, using in vitro models of intracellular staphylococcal infection, we demonstrate that EsxA interferes with host cell apoptotic pathways and, together with EsxB, mediates the release of S. aureus from the host cell., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Despite the global medical needs associated with Staphylococcus aureus infections, no licensed vaccines are currently available. We identified and characterized a protein annotated as an epidermin leader peptide processing serine protease (EpiP), as a novel S. aureus vaccine candidate. In addition, we determined the structure of the recombinant protein (rEpiP) by X-ray crystallography. The crystal structure revealed that rEpiP was cleaved somewhere between residues 95 and 100, and we found that the cleavage occurs through an autocatalytic intramolecular mechanism. The protein expressed by S. aureus cells also appeared to undergo a similar processing event. To determine whether the protein acts as a serine protease, we mutated the hypothesized catalytic serine 393 residue to alanine, generating rEpiP-S393A. The crystal structure of this mutant protein showed that the polypeptide chain was not cleaved and was not interacting stably with the active site. Indeed, rEpiP-S393A was shown to be impaired in its protease activity. Mice vaccinated with rEpiP were protected from S. aureus infection (34% survival, P=0.0054). Moreover, the protective efficacy generated by rEpiP and rEpiP-S393A was comparable, implying that the noncleaving mutant could be used for vaccination purposes.

In the human pathogen Staphylococcus aureus, there exists an enormous diversity of proteins containing DUFs (domains of unknown function). In the present study, we characterized the family of conserved staphylococcal antigens (Csa) classified as DUF576 and taxonomically restricted to Staphylococci. The 18 Csa paralogues in S. aureus Newman are highly similar at the sequence level, yet were found to be expressed in multiple cellular locations. Extracellular Csa1A was shown to be post-translationally processed and released. Molecular interaction studies revealed that Csa1A interacts with other Csa paralogues, suggesting that these proteins are involved in the same cellular process. The structures of Csa1A and Csa1B were determined by X-ray crystallography, unveiling a peculiar structure with limited structural similarity to other known proteins. Our results provide the first detailed biological characterization of this family and confirm the uniqueness of this family also at the structural level. We also provide evidence that Csa family members elicit protective immunity in in vivo animal models of staphylococcal infections, indicating a possible important role for these proteins in S. aureus biology and pathogenesis. These findings identify the Csa family as new potential vaccine candidates, and underline the importance of mining the bacterial unknown proteome to identify new targets for preventive vaccines.

Staphylococcus aureus is an opportunistic pathogen, commensal of the human skin and nares, but also responsible for invasive nosocomial as well as community acquired infections. Staphylococcus aureus adheres to the host tissues by means of surface adhesins, such as SdrC, SdrD, and SdrE proteins. The Sdr family of proteins together with a functional A domain, contain respectively two, three or five repeated sequences called B motifs which comprise the CnaB domains. SdrD and SdrE proteins were reported to be protective in animal models against invasive diseases or lethal challenge with human clinical S. aureus isolates. In this study we identified a 126 amino acid sequence containing a CnaB domain, conserved among the three Sdr proteins. The three fragments defined here as CnaBC2, D5 and E3 domains even though belonging to phylogenetically distinct strains, displayed high sequence similarity. Based on the sequence conservation data, we selected the CnaBE3 domain for further analysis and characterization. Polyclonal antibodies raised against the recombinant CnaBE3 domain recognized SdrE, SdrC and SdrD proteins of different S. aureus lineages. Moreover, we demonstrated that the CnaBE3 domain was expressed in vivo during S. aureus infections, and that immunization of this domain alone significantly reduces the bacterial load in mice challenged with S. aureus. Furthermore, we show that the reduction of bacteria by CnaBE3 vaccination is due to functional antibodies. Finally, we demonstrated that the region of the SdrE protein containing the CnaBE3 domain was resistant to trypsin digestion, a characteristic often associated with the presence of an isopeptide bond.

Staphylococcus aureus is a human pathogen causing globally significant morbidity and mortality. The development of antibiotic resistance in S. aureus highlights the need for a preventive vaccine. In the present paper we explore the structure and function of FhuD2 (ferric-hydroxamate uptake D2), a staphylococcal surface lipoprotein mediating iron uptake during invasive infection, recently described as a promising vaccine candidate. Differential scanning fluorimetry and calorimetry studies revealed that FhuD2 is stabilized by hydroxamate siderophores. The FhuD2-ferrichrome interaction was of nanomolar affinity in surface plasmon resonance experiments and fully iron(III)-dependent. We determined the X-ray crystallographic structure of ligand-bound FhuD2 at 1.9 Å (1 Å=0.1 nm) resolution, revealing the bilobate fold of class III SBPs (solute-binding proteins). The ligand, ferrichrome, occupies a cleft between the FhuD2 N- and C-terminal lobes. Many FhuD2-siderophore interactions enable the specific recognition of ferrichrome. Biochemical data suggest that FhuD2 does not undergo significant conformational changes upon siderophore binding, supporting the hypothesis that the ligand-bound complex is essential for receptor engagement and uptake. Finally, immunizations with FhuD2 alone or FhuD2 formulated with hydroxamate siderophores were equally protective in a murine staphylococcal infection model, confirming the suitability and efficacy of apo-FhuD2 as a protective antigen, and suggesting that other class III SBPs might also be exploited as vaccine candidates.

Iron availability plays an essential role in staphylococcal pathogenesis. We selected FhuD2, a lipoprotein involved in iron-hydroxamate uptake, as a novel vaccine candidate against Staphylococcus aureus. Unprecedented for staphylococcal lipoproteins, the protein was demonstrated to have a discrete, punctate localization on the bacterial surface. FhuD2 vaccination generated protective immunity against diverse clinical S. aureus isolates in murine infection models. Protection appeared to be associated with functional antibodies that were shown to mediate opsonophagocytosis, to be effective in passive transfer experiments, and to potentially block FhuD2-mediated siderophore uptake. Furthermore, the protein was found to be up-regulated in infected tissues and was required for staphylococcal dissemination and abscess formation. Herein we show that the staphylococcal iron-hydroxamate uptake system is important in invasive infection and functions as an efficacious vaccine target.

Advances in high-throughput DNA sequencing technologies have determined an explosion in the number of sequenced bacterial genomes. Comparative sequence analysis frequently reveals evidences of homologous recombination occurring with different mechanisms and rates in different species, but the large-scale use of computational methods to identify recombination events is hampered by their high computational costs. Here, we propose a new method to identify recombination events in large datasets of whole genome sequences. Using a filtering procedure of the gene conservation profiles of a test genome against a panel of strains, this algorithm identifies sets of contiguous genes acquired by homologous recombination. The locations of the recombination breakpoints are determined using a statistical test that is able to account for the differences in the natural rate of evolution between different genes. The algorithm was tested on a dataset of 75 genomes of Staphylococcus aureus and 50 genomes comprising different streptococcal species, and was able to detect intra-species recombination events in S. aureus and in Streptococcus pneumoniae. Furthermore, we found evidences of an inter-species exchange of genetic material between S. pneumoniae and Streptococcus mitis, a closely related commensal species that colonizes the same ecological niche. The method has been implemented in an R package, Reco, which is freely available from supplementary material, and provides a rapid screening tool to investigate recombination on a genome-wide scale from sequence data.

Staphylococcal leukotoxins are a family of β-barrel, bicomponent, pore-forming toxins with membrane-damaging functions. These bacterial exotoxins share sequence and structural homology and target several host-cell types. Leukotoxin ED (LukED) is one of these bicomponent pore-forming toxins that Staphylococcus aureus Staphylococcus aureus produces in order to suppress the ability of the host to contain the infection. The recent delineation of the important role that LukED plays in S. aureus S. aureus pathogenesis and the identification of its protein receptors, combined with its presence in S. aureus S. aureus methicillin-resistant epidemic strains, establish this leukocidin as a possible target for the development of novel therapeutics. Here, the crystal structures of the water-soluble LukE and LukD components of LukED have been determined. The two structures illustrate the tertiary-structural variability with respect to the other leukotoxins while retaining the conservation of the residues involved in the interaction of the protomers in the bipartite leukotoxin in the pore complex. [ABSTRACT FROM AUTHOR]

Vaccine research has experienced a quantum leap after the beginning of the genomics era. High-throughput sequencing techniques, unlimited computing resources, as well as new bioinformatic algorithms are now changing the way we perform genomic studies. Whole genome sequencing will soon become the gold standard for phylogenetic and epidemiology studies and is already shedding new light on the dynamics of bacterial evolution. We believe that deep sequencing projects, together with structural studies on vaccine candidates, will allow targeting constant epitopes and avoid vaccine failure due to antigenic variability. Systems biology, which is expected to revolutionize vaccine research and clinical studies, greatly relies on high-throughput technologies such as RNA-seq. Furthermore, genomics is a key element to develop safer vaccines, and the accuracy of deep sequencing will allow monitoring vaccine coverage after their introduction on the market. Copyright © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]