Protective Activity of Streptococcus pneumoniae Spr1875 Protein Fragments Identified Using a Phage Displayed Genomic Library

There is considerable interest in pneumococcal protein antigens capable of inducing serotype-independent immunoprotection and of improving, thereby, existing vaccines. We report here on the immunogenic properties of a novel surface antigen encoded by ORF spr1875 in the R6 strain genome. An antigenic fragment encoded by spr1875, designated R4, was identified using a Streptococcus pneumoniae phage displayed genomic library after selection with a human convalescent serum. Immunofluorescence analysis with anti-R4 antisera showed that Spr1875 was expressed on the surface of strains belonging to different serotypes. Moreover, the gene was present with little sequence variability in 27 different pneumococcal strains isolated worldwide. A mutant lacking Spr1875 was considerably less virulent than the wild type D39 strain in an intravenous mouse model of infection. Moreover, immunization with the R4 recombinant fragment, but not with the whole Spr1875 protein, induced significant protection against sepsis in mice. Lack of protection after immunization with the whole protein was related to the presence of immunodominant, non-protective epitopes located outside of the R4 fragment. In conclusion, our data indicate that Spr1875 has a role in pneumococcal virulence and is immunogenic. As the R4 fragment conferred immunoprotection from experimental sepsis, selected antigenic fragments of Spr1875 may be useful for the development of a pneumococcal protein-based vaccine

The α-Cyclodextrin/Moringin Complex: A New Promising Antimicrobial Agent against Staphylococcus aureus

Antimicrobial resistance is one of the major clinical concerns, making the discovery of new antimicrobial drugs desirable. Moringin (MOR), the major isothiocyanate produced from Moringa oleifera seeds, could represent an alternative therapeutic strategy to commonly used antibiotics. The aim of our study was to investigate the antimicrobial effect of MOR conjugated with α-cyclodextrin (MOR/α-CD), a complex with an improved solubility and stability in aqueous solutions. Our data demonstrated that MOR/α-CD was able to exert antimicrobial activity against the S. aureus reference strains (ATCC 25923, ATCC 6538, and ATCC BAA-977). Moreover, MOR/α-CD showed bacteriostatic effects (MIC = minimum inhibitory concentration = 0.5 mg/mL) and bactericidal properties (MBC = minimum bactericidal concentration = 1 mg/mL) against the overall assessed strains. In addition, MOR/α-CD showed bactericidal activity against the S. aureus strain ATCC BAA-977 after treatment with erythromycin (Ery), which induced clindamycin-resistance on the erm (A) gene. This evidence led us to assume that MOR/α-CD could be a promising antimicrobial agent against strains with the clindamycin-resistant phenotype (CC-resistant)

Treatment of Periodontal Ligament Stem Cells with MOR and CBD Promotes Cell Survival and Neuronal Differentiation via the PI3K/Akt/mTOR Pathway

Periodontal ligament mesenchymal stem cells (hPDLSCs), as well as all mesenchymal stem cells, show self-renewal, clonogenicity, and multi-tissue differentiation proprieties and can represent a valid support for regenerative medicine. We treated hPDLSCs with a combination of Moringin (MOR) and Cannabidiol (CBD), in order to understand if treatment could improve their survival and their in vitro differentiation capacity. Stem cells survival is fundamental to achieve a successful therapy outcome in the re-implanted tissue of patients. Through NGS transcriptome analysis, we found that combined treatment increased hPDLSCs survival, by inhibition of apoptosis as demonstrated by enhanced expression of anti-apoptotic genes and reduction of pro-apoptotic ones. Moreover, we investigated the possible involvement of PI3K/Akt/mTOR pathway, emphasizing a differential gene expression between treated and untreated cells. Furthermore, hPDLSCs were cultured for 48 h in the presence or absence of CBD and MOR and, after confirming the cellular viability through MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide) assay, we examined the presence of neuronal markers, through immunofluorescence analysis. We found an increased expression of Nestin and GAP43 (growth associated protein 43) in treated cells. In conclusion, hPDLSCs treated with Moringin and Cannabidiol showed an improved survival capacity and neuronal differentiation potential

Protective activity of a protein fragment identified by screening of a phage display genomic library of Streptococcus pneumoniae

Objective: Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. The pathogenicity of pneumococci has been attributed to various virulence factors, most of which are located on its surface. The present study was undertaken to identify novel virulence factors and antigens with immunoprotective activity by using a phage display genomic library of Streptococcus pneumoniae. Methods. A lambda phage display library from the D39 S. pneumoniae strain was selected using sera from patients convalescing from invasive pneumococcal disease. For virulence studies, adult BALB/c mice (Charles River) were inoculated i.v. with the wild type D39 strain or with an equal number of CFUs from a mutant lacking spr1875 (Δspr1875). Results. Using one a convalescent serum, we selected from the phage display library an insert encoding for a fragment designated as R4, 161 amino acid in length, whose sequence matched ORF spr1875 D39 genome. This ORF encoded for a putative surface protein with a signal peptide and a LysM domain, which is found in a number of important virulence factors. To evaluate whether the spr1875 protein is involved in pneumococcal pathogenicity we constructed the Δspr1875. Using 7 x 106 CFUs for challenge, all mice inoculated with D39 died in six days, while 80% of the mice challenged with Δspr1875 survived. The R4 fragment was capable of inducing significant, albeit partial, protection in mice inoculated with a wide variety of virulent streptococcal strains. Conclusion. We have identified a novel virulence factor of S. pneumoniae that showed protective activity in a mouse model of systemic pneumococcal disease. Further studies are needed to characterize the biological activities of this antigen

Analysis of the Streptococcus agalactiae exoproteome

International audienceThe two-component regulatory system CovRS is the main regulator of virulence gene expression in Group B Streptococcus (GBS), the leading cause of invasive infections in neonates. In this study we analyzed by mass spectrometry the GBS extracellular protein complex (i.e. the exoproteome) of NEM316 wild-type (WT) strain and its isogenic covRS deletion mutant (ΔcovRS). A total of 53 proteins, 49 of which had classical secretion signals, were identified: 12 were released by both strains while 21 and 20 were released exclusively by WT and ΔcovRS strains, respectively. In addition to known surface proteins, we detected here unstudied cell-wall associated proteins and/or orthologs of putative virulence factors present in other pathogenic streptococci. While the functional role of these proteins remains to be elucidated, our data suggest that the analysis of the exoproteome of bacterial pathogens under different gene expression conditions may be a powerful tool for the rapid identification of novel virulence factors and vaccine candidates

IDENTIFICATION OF FIBRINOGEN BINDING PROTEIN FRAGMENTS USING GENOMIC PHAGE DISPLAY LIBRARIES OF GROUP B STREPTOCOCCUS

Obiective: The ability to bind fibrinogen (Fng) is a virulence determinant for a diverse group of bacterial pathogens. Group B streptococcus (GBS) is an important cause of sepsis and invasive infections. The objective of the present work was to obtain novel insights regarding the mechanism of Fng binding by GBS. Methods: We constructed two genomic libraries of GBS using, respectively, the COH-1 and 2603 R/V strains. These libraries were selected using Fng-coated beads as bait. For virulence studies neonatal (24-hour-old) BALB/c mice (Charles River) were inoculated s.c. with a LD90 (60 CFU/pup) of the wild type serotype III GBS strain or with a fbsA defective mutant. Results: After one round of selection of the COH-1 library approximately 10% of the clones strongly reacted against Fng by immunoblot of phage plaques. The percentage of Fng-binding clones rose to 100% after 3 rounds of selection. All reactive clones contained 2-5 repeats of fibrinogen binding protein A (FbsA), a previously described factor shown to mediate adherence and invasion of human epithelial cells. We further show here that FbsA is an important virulence factor, as evidenced by a decreased ability of the fbsA mutant to cause infection. Experiments are underway to characterize the protective activities of FbsA after active immunization with the protein fragments identified by phage display libraries. Moreover we have developed an anti-FbsA neutralizing monoclonal antibody that is being tested for its ability to passively protect neonatal mice from GBS infection. Conclusion:Our data confirm and extend previous studies indicating that FbsA is an important Fng binding factor. Moreover we show that FbsA is an essential virulence factor in vivo

Rapid profiling of the antigen regions recognized by serum antibodies using massively parallel sequencing of antigen-specific libraries

There is a need for techniques capable of identifying the antigenic epitopes targeted by polyclonal antibody responses during deliberate or natural immunization. Although successful, traditional phage library screening is laborious and can map only some of the epitopes. To accelerate and improve epitope identification, we have employed massive sequencing of phage-displayed antigen-specific libraries using the Illumina MiSeq platform. This enabled us to precisely identify the regions of a model antigen, the meningococcal NadA virulence factor, targeted by serum antibodies in vaccinated individuals and to rank hundreds of antigenic fragments according to their immunoreactivity. We found that next generation sequencing can significantly empower the analysis of antigen-specific libraries by allowing simultaneous processing of dozens of library/serum combinations in less than two days, including the time required for antibody-mediated library selection. Moreover, compared with traditional plaque picking, the new technology (named Phage-based Representation OF Immuno-Ligand Epitope Repertoire or PROFILER) provides superior resolution in epitope identification. PROFILER seems ideally suited to streamline and guide rational antigen design, adjuvant selection, and quality control of newly produced vaccines. Furthermore, this method is also susceptible to find important applications in other fields covered by traditional quantitative serology

Occurrence of cumulative amino acid positions in NadA fragments obtained after two rounds of selection, as determined by traditional random picking of 50 plaques followed by Sanger sequencing of individual clones.

<p>Occurrence of cumulative amino acid positions in NadA fragments obtained after two rounds of selection, as determined by traditional random picking of 50 plaques followed by Sanger sequencing of individual clones.</p

Properties of the antigen-specific phage library before and after selection with a pool of serum samples from volunteers immunized with the Bexero anti-MenB vaccine.

<p>A–C, abundance of “natural frame” <i>nadA</i> fragments in the library before (A) and after the first and second rounds of selection (B and C, respectively). Each point represents the number of unique fragments (vertical axis) displaying the number of copies indicated in the horizontal axis; D–F, <i>nadA</i> fragment length distribution before (D) and after the first and second rounds of selection (E and F, respectively).</p

Schematic outline of the epitope mapping approach.

<p>The gene encoding the antigen is fragmented by DNAse digestion and the gene fragments are inserted into lambda phage vectors. The phage library is mixed with immune serum and phage particles binding to immunoglobulins are separated using Protein-G coated magnetic beads. The inserts of the phage population obtained after selection are massively sequenced and compared with those of the original unselected library using an <i>ad hoc</i> developed software which identifies the region(s) of the antigen targeted by serum antibodies.</p