Innovative Reversed-Phase Chromatography Platform Approach for the Fast and Accurate Characterization of Membrane Vesicles' Protein Patterns.

Outer membrane vesicles (OMVs) have been widely explored to develop vaccine candidates for bacterial pathogens due to their ability to combine adjuvant properties with immunogenic activity. OMV expresses a variety of proteins and carbohydrate antigens on their surfaces. For this reason, there is an analytical need to thoroughly characterize the species expressed at their surface: we here present a simple and accurate reversed-phase ultrahigh-performance liquid chromatography (RP-UPLC) method developed according to quality by design principles. This work provides an analytical alternative to the classical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) characterization. The higher selectivity and sensitivity of the RP-UHPLC assay allow for the identification of additional protein species with respect to SDS-PAGE and facilitate its precise relative abundance quantification. According to validation results, the assay showed high accuracy, linearity, precision, repeatability, and a limit of quantification of 1% for less abundant proteins. This performance paves the way for improved production campaign consistency while also being analytically simple (no sample pretreatment required), making it suitable for routine quality control testing. In addition, the applicability of the assay to a wider range of vesicle classes (GMMA) was demonstrated.

Evolution of Vaccines Formulation to Tackle the Challenge of Anti-Microbial Resistant Pathogens.

The increasing diffusion of antimicrobial resistance (AMR) across more and more bacterial species emphasizes the urgency of identifying innovative treatment strategies to counter its diffusion. Pathogen infection prevention is among the most effective strategies to prevent the spread of both disease and AMR. Since their discovery, vaccines have been the strongest prophylactic weapon against infectious diseases, with a multitude of different antigen types and formulative strategies developed over more than a century to protect populations from different pathogens. In this review, we review the main characteristics of vaccine formulations in use and under development against AMR pathogens, focusing on the importance of administering multiple antigens where possible, and the challenges associated with their development and production. The most relevant antigen classes and adjuvant systems are described, highlighting their mechanisms of action and presenting examples of their use in clinical trials against AMR. We also present an overview of the analytical and formulative strategies for multivalent vaccines, in which we discuss the complexities associated with mixing multiple components in a single formulation. This review emphasizes the importance of combining existing knowledge with advanced technologies within a Quality by Design development framework to efficiently develop vaccines against AMR pathogens.

Exploring the Role of GMMA Components in the Immunogenicity of a 4-Valent Vaccine against Shigella.

Shigellosis is the leading cause of diarrheal disease, especially in children of low- and middle-income countries, and is often associated with anti-microbial resistance. Currently, there are no licensed vaccines widely available against Shigella, but several candidates based on the O-antigen (OAg) portion of lipopolysaccharides are in development. We have proposed Generalized Modules for Membrane Antigens (GMMA) as an innovative delivery system for OAg, and a quadrivalent vaccine candidate containing GMMA from S. sonnei and three prevalent S. flexneri serotypes (1b, 2a and 3a) is moving to a phase II clinical trial, with the aim to elicit broad protection against Shigella. GMMA are able to induce anti-OAg-specific functional IgG responses in animal models and healthy adults. We have previously demonstrated that antibodies against protein antigens are also generated upon immunization with S. sonnei GMMA. In this work, we show that a quadrivalent Shigella GMMA-based vaccine is able to promote a humoral response against OAg and proteins of all GMMA types contained in the investigational vaccine. Proteins contained in GMMA provide T cell help as GMMA elicit a stronger anti-OAg IgG response in wild type than in T cell-deficient mice. Additionally, we observed that only the trigger of Toll-like Receptor (TLR) 4 and not of TLR2 contributed to GMMA immunogenicity. In conclusion, when tested in mice, GMMA of a quadrivalent Shigella vaccine candidate combine both adjuvant and carrier activities which allow an increase in the low immunogenic properties of carbohydrate antigens.

Dissecting the contribution of O-Antigen and proteins to the immunogenicity of Shigella sonnei generalized modules for membrane antigens (GMMA).

GMMA are exosomes released from engineered Gram-negative bacteria resembling the composition of outer membranes. We applied the GMMA technology for the development of an O-Antigen (OAg) based vaccine against Shigella sonnei, the most epidemiologically relevant cause of shigellosis. S. sonnei OAg has been identified as a key antigen for protective immunity, and GMMA are able to induce anti-OAg-specific IgG response in animal models and healthy adults. The contribution of protein-specific antibodies induced upon vaccination with GMMA has never been fully elucidated. Anti-protein antibodies are induced in mice upon immunization with either OAg-negative and OAg-positive GMMA. Here we demonstrated that OAg chains shield the bacteria from anti-protein antibody binding and therefore anti-OAg antibodies were the main drivers of bactericidal activity against OAg-positive bacteria. Interestingly, antibodies that are not targeting the OAg are functional against OAg-negative bacteria. The immunodominant protein antigens were identified by proteomic analysis. Our study confirms a critical role of the OAg on the immune response induced by S. sonnei GMMA. However, little is known about OAg length and density regulation during infection and, therefore, protein exposure. Hence, the presence of protein antigens on S. sonnei GMMA represents an added value for GMMA vaccines compared to other OAg-based formulations.

NHBA is processed by kallikrein from human saliva.

Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein of Neisseria meningitidis and a component of the Bexsero vaccine. NHBA is characterized by the presence of a highly conserved Arg-rich region involved in binding to heparin and heparan sulphate proteoglycans present on the surface of host epithelial cells, suggesting a possible role of NHBA during N. meningitidis colonization. NHBA has been shown to be cleaved by the meningococcal protease NalP and by human lactoferrin (hLF), a host protease presents in different body fluids (saliva, breast milk and serum). Cleavage occurs upstream or downstream the Arg-rich region. Since the human nasopharynx is the only known reservoir of infection, we further investigated the susceptibility of NHBA to human proteases present in the saliva to assess whether proteolytic cleavage could happen during the initial steps of colonization. Here we show that human saliva proteolytically cleaves NHBA, and identified human kallikrein 1 (hK1), a serine protease, as responsible for this cleavage. Kallikrein-related peptidases (KLKs) have a distinct domain structure and exist as a family of 15 genes which are differentially expressed in many tissues and in the central nervous system. They are present in plasma, lymph, urine, saliva, pancreatic juices, and other body fluids where they catalyze the proteolysis of several human proteins. Here we report the characterization of NHBA cleavage by the tissue kallikrein, expressed in saliva and the identification of the cleavage site on NHBA both, as recombinant protein or as native protein, when expressed on live bacteria. Overall, these findings provide new insights on NHBA as target of host proteases, highlights thepotential role of NHBA in the Neisseria meningitidis nasopharyngeal colonization, and of kallikrein as a defensive agent against meningococcal infection.

Total hip arthroplasty: minimally invasive surgery or not? Meta-analysis of clinical trials.

BACKGROUND: There exist a relevant number of clinical trials comparing the minimally invasive surgery to the standard-invasive approach in total hip arthroplasty (THA). Up to date, there are still debates concerning the most effective approach in THA. AIM: The purpose of this study is to compare the clinical outcomes concerning patients undergoing primary THA performed via the minimally invasive versus standard-invasive surgery incision. MATERIAL AND METHODS: The search was performed in the main databases, evaluating both quantitative and qualitative results. All the randomised controlled trials (RCTs) and non-randomised controlled trials (nRCTs) comparing the minimally invasive versus the standard-invasive approach were enrolled in this study. We focused on the clinical and radiological outcomes and on the complication rate. Study methodological quality was assessed performing the PEDro critical appraisal scale. All meta-analyses were performed using the Review Manager software. To analyse the publication's bias, we performed the Funnel plot. RESULT: We enrolled in our study 4761 patients, undergoing to 4842 total hip arthroplasties. The mean follow-up was 22.26 months. In favour of the minimally invasive group, we reported less total estimated blood loss, shorter surgical duration, and a shorter length of stay. In favour of the standard-invasive group, we reported a higher value of the Harris hip score. Concerning the radiological outcomes, we did not report substantial differences across the two exposures. No difference was observed regarding the risk of femoral fractures, dislocation, and revision rates. We evidenced an increasing risk occurred in an iatrogenic nerve palsy during the minimally invasive approach. CONCLUSION: Based on currently available evidences concerning the outcomes following THA and the analysis of our results, we stated no remarkable benefits of the minimally invasive compared to the standard-invasive surgery.

Neisseria Heparin Binding Antigen is targeted by the human alternative pathway C3-convertase.

Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein specific for Neisseria and constitutes one of the three main protein antigens of the Bexsero vaccine. Meningococcal and human proteases, cleave NHBA protein upstream or downstream of a conserved Arg-rich region, respectively. The cleavage results in the release of the C-terminal portion of the protein. The C-terminal fragment originating from the processing of meningococcal proteases, referred to as C2 fragment, exerts a toxic effect on endothelial cells altering the endothelial permeability. In this work, we reported that recombinant C2 fragment has no influence on the integrity of human airway epithelial cell monolayers, consistent with previous findings showing that Neisseria meningitidis traverses the epithelial barrier without disrupting the junctional structures. We showed that epithelial cells constantly secrete proteases responsible for a rapid processing of C2 fragment, generating a new fragment that does not contain the Arg-rich region, a putative docking domain reported to be essential for C2-mediated toxic effect. Moreover, we found that the C3-convertase of the alternative complement pathway is one of the proteases responsible for this processing. Overall, our data provide new insights on the cleavage of NHBA protein during meningococcal infection. NHBA cleavage may occur at different stages of the infection, and it likely has a different role depending on the environment the bacterium is interacting with.

Outer Membrane Vesicles (OMV)-based and Proteomics-driven Antigen Selection Identifies Novel Factors Contributing to Bordetella pertussis Adhesion to Epithelial Cells.

Despite high vaccination coverage world-wide, whooping cough, a highly contagious disease caused by Bordetella pertussis, is recently increasing in occurrence suggesting that novel vaccine formulations targeted at the prevention of colonization and transmission should be investigated. To identify new candidates for inclusion in the acellular formulation, we used spontaneously released outer membrane vesicles (OMV)1 as a potential source of key adhesins. The enrichment of Bvg+ OMV with adhesins and the ability of anti-OMV serum to inhibit the adhesion of B. pertussis to lung epithelial cells in vitro were demonstrated. We employed a proteomic approach to identify the differentially expressed proteins in OMV purified from bacteria in the Bvg+ and Bvg- virulence phases, thus comparing the outer membrane protein pattern of this pathogen in its virulent or avirulent state. Six of the most abundant outer membrane proteins were selected as candidates to be evaluated for their adhesive properties and vaccine potential. We generated E. coli strains singularly expressing the selected proteins and assessed their ability to adhere to lung epithelial cells in vitro Four out of the selected proteins conferred adhesive ability to E. coli Three of the candidates were specifically detected by anti-OMV mouse serum suggesting that these proteins are immunogenic antigens able to elicit an antibody response when displayed on the OMV. Anti-OMV serum was able to inhibit only BrkA-expressing E. coli adhesion to lung epithelial cells. Finally, stand-alone immunization of mice with recombinant BrkA resulted in significant protection against infection of the lower respiratory tract after challenge with B. pertussis Taken together, these data support the inclusion of BrkA and possibly further adhesins to the current acellular pertussis vaccines to improve the impact of vaccination on the bacterial clearance.

Surface and Exoproteomes of Gram-Positive Pathogens for Vaccine Discovery.

Reverse vaccinology has been very successful in the discovery of vaccine candidates against many pathogenic bacteria by integrating genome and proteome mining. This great achievement was facilitated by the complementarity of the in silico prediction of antigens and the empirical data on protein localization, expression, and immunogenicity obtained through different techniques based on electrophoresis, immunoblotting and mass spectrometry. An iterative process between information provided by DNA sequence analysis and proteomic data has been established leading to precise antigen identification. In this review, we report how the identification of surface and exoproteomes of Gram-positive pathogens have contributed to the selection of vaccine candidates. Moreover, we show how quantitative mass spectrometry is now paving the way for identifying protective antigens that play key roles during infection and represent the most promising vaccine targets.

Expression of factor H binding protein in meningococcal strains can vary at least 15-fold and is genetically determined.

Factor H binding protein (fHbp) is a lipoprotein of Neisseria meningitidis important for the survival of the bacterium in human blood and a component of two recently licensed vaccines against serogroup B meningococcus (MenB). Based on 866 different amino acid sequences this protein is divided into three variants or two families. Quantification of the protein is done by immunoassays such as ELISA or FACS that are susceptible to the sequence variation and expression level of the protein. Here, selected reaction monitoring mass spectrometry was used for the absolute quantification of fHbp in a large panel of strains representative of the population diversity of MenB. The analysis revealed that the level of fHbp expression can vary at least 15-fold and that variant 1 strains express significantly more protein than variant 2 or variant 3 strains. The susceptibility to complement-mediated killing correlated with the amount of protein expressed by the different meningococcal strains and this could be predicted from the nucleotide sequence of the promoter region. Finally, the absolute quantification allowed the calculation of the number of fHbp molecules per cell and to propose a mechanistic model of the engagement of C1q, the recognition component of the complement cascade.

The Human Pathogen Streptococcus pyogenes Releases Lipoproteins as Lipoprotein-rich Membrane Vesicles.

Bacterial lipoproteins are attractive vaccine candidates because they represent a major class of cell surface-exposed proteins in many bacteria and are considered as potential pathogen-associated molecular patterns sensed by Toll-like receptors with built-in adjuvanticity. Although Gram-negative lipoproteins have been extensively characterized, little is known about Gram-positive lipoproteins. We isolated from Streptococcus pyogenes a large amount of lipoproteins organized in vesicles. These vesicles were obtained by weakening the bacterial cell wall with a sublethal concentration of penicillin. Lipid and proteomic analysis of the vesicles revealed that they were enriched in phosphatidylglycerol and almost exclusively composed of lipoproteins. In association with lipoproteins, a few hypothetical proteins, penicillin-binding proteins, and several members of the ExPortal, a membrane microdomain responsible for the maturation of secreted proteins, were identified. The typical lipidic moiety was apparently not necessary for lipoprotein insertion in the vesicle bilayer because they were also recovered from the isogenic diacylglyceryl transferase deletion mutant. The vesicles were not able to activate specific Toll-like receptor 2, indicating that lipoproteins organized in these vesicular structures do not act as pathogen-associated molecular patterns. In light of these findings, we propose to name these new structures Lipoprotein-rich Membrane Vesicles.

NAD-dependent ADP-ribosylation of the human antimicrobial and immune-modulatory peptide LL-37 by ADP-ribosyltransferase-1.

LL-37 is a cationic peptide belonging to the cathelicidin family that has antimicrobial and immune-modulatory properties. Here we show that the mammalian mono-ADP-ribosyltransferase-1 (ART1), which selectively transfers the ADP-ribose moiety from NAD to arginine residues, ADP-ribosylates LL-37 in vitro. The incorporation of ADP-ribose was first observed by Western blot analysis and then confirmed by MALDI-TOF. Mass-spectrometry showed that up to four of the five arginine residues present in LL-37 could be ADP-ribosylated on the same peptide when incubated at a high NAD concentration in the presence of ART1. The attachment of negatively charged ADP-ribose moieties considerably alters the positive charge of the arginine residues thus reducing the cationicity of LL-37. The cationic nature of LL-37 is key for its ability to interact with cell membranes or negatively charged biomolecules, such as DNA, RNA, F-actin and glycosaminoglycans. Thus, the ADP-ribosylation of LL-37 is expected to have the potential to modulate LL-37 biological activities in several physiological and pathological settings.

Structure and protective efficacy of the Staphylococcus aureus autocleaving protease EpiP.

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.

Quantification by LC-MS(E) of outer membrane vesicle proteins of the Bexsero® vaccine.

Meningococcal disease is a major cause of morbidity and mortality worldwide. Its epidemiology is currently dominated by five capsular serogroups (A, B, C, W, and Y). While effective vaccines already exist for serogroups A, C, W and Y, except for under clonal outbreaks, no vaccine was available against serogroup B. Recently, a four component vaccine, Bexsero(®), designed to prevent infection caused by this serogroup, has been approved in Europe and other Countries for use in individuals from two months of age and older. The active components of this vaccine are three recombinant proteins identified by reverse vaccinology combined with detergent extracted outer membrane vesicles (DOMV) prepared from a New Zealand epidemic strain. Considering their intrinsic complexity, we performed additional characterization of DOMVs on top of the standard quality control testing carried out for batch release. We applied the Hi3 label-free LC-MS(E) methodology to qualitatively and quantitatively characterize the DOMV protein content. We first, successfully investigated the robustness and the accuracy of the methodology for the DOMV characterization and we then applied it to compare six DOMV production lots. Around 100 proteins were quantified from each preparation. When classified according to their predicted cellular localization, about 90% of the total protein amount belongs consistently to the outer membrane compartment. Using nonparametric hypothesis testing and complementary log-log linear regression, the quantifications of a subset of 21 proteins common to all lots and including approximately 90% (85-92%) of the total protein amount quantified in any DOMV lot were found consistent across lots. The relevance of these results is two-fold, showing that the Hi3 quantification methodology is robust for a broad range of proteins and indicating that the manufacturing process currently used for the production of the Bexsero(®) DOMV components is highly reproducible and consistent.

Identification of a novel zinc metalloprotease through a global analysis of Clostridium difficile extracellular proteins.

Clostridium difficile is a major cause of infectious diarrhea worldwide. Although the cell surface proteins are recognized to be important in clostridial pathogenesis, biological functions of only a few are known. Also, apart from the toxins, proteins exported by C. difficile into the extracellular milieu have been poorly studied. In order to identify novel extracellular factors of C. difficile, we analyzed bacterial culture supernatants prepared from clinical isolates, 630 and R20291, using liquid chromatography-tandem mass spectrometry. The majority of the proteins identified were non-canonical extracellular proteins. These could be largely classified into proteins associated to the cell wall (including CWPs and extracellular hydrolases), transporters and flagellar proteins. Seven unknown hypothetical proteins were also identified. One of these proteins, CD630_28300, shared sequence similarity with the anthrax lethal factor, a known zinc metallopeptidase. We demonstrated that CD630_28300 (named Zmp1) binds zinc and is able to cleave fibronectin and fibrinogen in vitro in a zinc-dependent manner. Using site-directed mutagenesis, we identified residues important in zinc binding and enzymatic activity. Furthermore, we demonstrated that Zmp1 destabilizes the fibronectin network produced by human fibroblasts. Thus, by analyzing the exoproteome of C. difficile, we identified a novel extracellular metalloprotease that may be important in key steps of clostridial pathogenesis.

Protective activity of the CnaBE3 domain conserved among Staphylococcus aureus Sdr proteins.

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.

Auto ADP-ribosylation of NarE, a Neisseria meningitidis ADP-ribosyltransferase, regulates its catalytic activities.

NarE is an arginine-specific mono-ADP-ribosyltransferase identified in Neisseria meningitidis that requires the presence of iron in a structured cluster for its enzymatic activities. In this study, we show that NarE can perform auto-ADP-ribosylation. This automodification occurred in a time- and NAD-concentration-dependent manner; was inhibited by novobiocin, an ADP-ribosyltransferase inhibitor; and did not occur when NarE was heat inactivated. No reduction in incorporation was evidenced in the presence of high concentrations of ATP, GTP, ADP-ribose, or nicotinamide, which inhibits NAD-glycohydrolase, impeding the formation of free ADP-ribose. Based on the electrophoretic profile of NarE on auto-ADP-ribosylation and on the results of mutagenesis and mass spectrometry analysis, the auto-ADP-ribosylation appeared to be restricted to the addition of a single ADP-ribose. Chemical stability experiments showed that the ADP-ribosyl linkage was sensitive to hydroxylamine, which breaks ADP-ribose-arginine bonds. Site-directed mutagenesis suggested that the auto-ADP-ribosylation site occurred preferentially on the R(7) residue, which is located in the region I of the ADP-ribosyltransferase family. After auto-ADP-ribosylation, NarE showed a reduction in ADP-ribosyltransferase activity, while NAD-glycohydrolase activity was increased. Overall, our findings provide evidence for a novel intramolecular mechanism used by NarE to regulate its enzymatic activities.

Group B Streptococcus pilus sortase regulation: a single mutation in the lid region induces pilin protein polymerization in vitro.

Gram-positive bacteria build pili on their cell surface via a class C sortase-catalyzed transpeptidation mechanism from pilin protein substrates. Despite the availability of several crystal structures, pilus-related C sortases remain poorly characterized to date, and their mechanisms of transpeptidation and regulation need to be further investigated. The available 3-dimensional structures of these enzymes reveal a typical sortase fold, except for the presence of a unique feature represented by an N-terminal highly flexible loop known as the "lid." This region interacts with the residues composing the catalytic triad and covers the active site, thus maintaining the enzyme in an autoinhibited state and preventing the accessibility to the substrate. It is believed that enzyme activation may occur only after lid displacement from the catalytic domain. In this work, we provide the first direct evidence of the regulatory role of the lid, demonstrating that it is possible to obtain in vitro an efficient polymerization of pilin subunits using an active C sortase lid mutant carrying a single residue mutation in the lid region. Moreover, biochemical analyses of this recombinant mutant reveal that the lid confers thermodynamic and proteolytic stability to the enzyme.

Arginine-specific mono ADP-ribosylation in vitro of antimicrobial peptides by ADP-ribosylating toxins.

Among the several toxins used by pathogenic bacteria to target eukaryotic host cells, proteins that exert ADP-ribosylation activity represent a large and studied family of dangerous and potentially lethal toxins. These proteins alter cell physiology catalyzing the transfer of the ADP-ribose unit from NAD to cellular proteins involved in key metabolic pathways. In the present study, we tested the capability of four of these toxins, to ADP-ribosylate α- and ß- defensins. Cholera toxin (CT) from Vibrio cholerae and heat labile enterotoxin (LT) from Escherichia coli both modified the human α-defensin (HNP-1) and ß- defensin-1 (HBD1), as efficiently as the mammalian mono-ADP-ribosyltransferase-1. Pseudomonas aeruginosa exoenzyme S was inactive on both HNP-1 and HBD1. Neisseria meningitidis NarE poorly recognized HNP-1 as a substrate but it was completely inactive on HBD1. On the other hand, HNP-1 strongly influenced NarE inhibiting its transferase activity while enhancing auto-ADP-ribosylation. We conclude that only some arginine-specific ADP-ribosylating toxins recognize defensins as substrates in vitro. Modifications that alter the biological activities of antimicrobial peptides may be relevant for the innate immune response. In particular, ADP-ribosylation of antimicrobial peptides may represent a novel escape mechanism adopted by pathogens to facilitate colonization of host tissues.

Multi high-throughput approach for highly selective identification of vaccine candidates: the Group A Streptococcus case.

We propose an experimental strategy for highly accurate selection of candidates for bacterial vaccines without using in vitro and/or in vivo protection assays. Starting from the observation that efficacious vaccines are constituted by conserved, surface-associated and/or secreted components, the strategy contemplates the parallel application of three high throughput technologies, i.e. mass spectrometry-based proteomics, protein array, and flow-cytometry analysis, to identify this category of proteins, and is based on the assumption that the antigens identified by all three technologies are the protective ones. When we tested this strategy for Group A Streptococcus, we selected a total of 40 proteins, of which only six identified by all three approaches. When the 40 proteins were tested in a mouse model, only six were found to be protective and five of these belonged to the group of antigens in common to the three technologies. Finally, a combination of three protective antigens conferred broad protection against a panel of four different Group A Streptococcus strains. This approach may find general application as an accelerated and highly accurate path to bacterial vaccine discovery.