Ida Pernice

Plasminogen- and Fibronectin-binding Protein B Is Involved in the Adherence of Streptococcus pneumoniae to Human Epithelial Cells

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. The ability of this bacterium to adhere to epithelial cells is considered as an essential early step in colonization and infection. By screening a whole genome phage display library with sera from infected patients, we previously identified three antigenic fragments matching open reading frame spr0075 of the strain R6 genome. This locus encodes for an ∼120-kDa protein, herein referred to as plasminogen- and fibronectin-binding protein B (PfbB), which displays an LPXTG cell wall anchoring motif and six repetitive domains. In this study, by using isogenic pfbB-deleted mutants of the encapsulated D39 and of the unencapsulated DP1004 type 2 pneumococcal strains, we show that PfbB is involved in S. pneumoniae adherence to various epithelial respiratory tract cell lines. Our data suggest that PfbB directly mediates bacterial adhesion, because fluorescent beads coated with the recombinant PfbB sp17 fragment (encompassing one of the six repetitive domains and the C-terminal region) efficiently bound to epithelial cells. Mutants lacking PfbB bound to fibronectin and plasminogen considerably less efficiently than wild type bacteria, whereas sp17-coated beads specifically bound to both of these substrates. Taken together, our data suggest that, by directly interacting with fibronectin, PfbB significantly increases the ability of S. pneumoniae to adhere to human epithelial cells.

A multiplex PCR protocol for rapid identification of Candida glabrata and its phylogenetically related species Candida nivariensis and Candida bracarensis

We have developed a multiplex PCR protocol for the detection of Candida glabrata and its closely related species Candida nivariensis and Candida bracarensis. The method uses four PCR primers, targeting the ITS1 region and the 5.8S ribosomal RNA gene. The combination of these primers yielded unique results to all Candida species tested. The PCR assay we developed was found to be a rapid, specific and easy to perform method and it will be useful for characterizing large numbers of isolates for epidemiological studies.

Transmission of Trichosporon asahii oesophagitis by a contaminated endoscope

Summary.  Two cases of oesophageal trichosporonosis due to a suspected nosocomial infection are reported. Both the patients were immunocompetent and had undergone an endoscopic examination on the same day. Six strains of Trichosporon were isolated: three strains from the oesophageal biopsy of the first patient, one strain from the endoscopic forceps, one from the air in the endoscopy room, and one from the oesophageal biopsy of the second patient. The nosocomial nature of the infection and the role of the endoscopic forceps in transporting the micro‐organism was suspected, but the morphology and physiology of the isolated strains did not confirm such hypothesis. To elucidate the nature of the infection and the genetic similarities of the strains isolated, all strains were typed with RFLPs of the rDNA fragment and with RAPD. The results of RAPD using primer (GTG)5 (GACA)4, M13 core sequence, and the 15‐mer oligonucleotide GAGGGTGGXGGXTCT indicated the molecular identity of three strains supporting the hypothesis concerning a transport of the aetiological agent from the first patient to the second and that the carrier was the forceps of the endoscopic device.

Display libraries on bacteriophage lambda capsid

Phage display is an established technology that has been successfully applied, in the last fifteen years, to projects aimed at deciphering biological processes and/or at the isolation of molecules of practical value in several diverse applications. Bacteriophage lambda, representing a molecular cloning and expression tool widely utilized since decades, has also been exploited to develop vectors for the display of libraries on its capsid. In the last few years, lambda display approach has been consistently offering new enthralling perspectives of technological application, such as domain mapping, antigen discovery, and protein interaction studies or, more generally, in functional genomics.

Peptide Mimics of the Group B Meningococcal Capsule Induce Bactericidal and Protective Antibodies after Immunization

Neisseria meningitidis serogroup B (MenB) is a leading cause of sepsis and meningitis in children. No vaccine is available for the prevention of these infections because the group B capsular polysaccharide (CP) (MenB CP) is unable to stimulate an immune response, due to its similarity with human polysialic acid. Because the MenB CP bears both human cross-reactive and non-cross-reactive determinants, we developed immunogenic peptide mimics of the latter epitopes. Peptides were selected from phage display libraries for their ability to bind to a protective anti-MenB CP mAb. One of these peptides (designated 9M) induced marked elevations in serum bactericidal activity, but not polysialic acid cross-reacting Abs, after gene priming followed by carrier-conjugate boosting. Moreover, the occurrence of bacteremia was prevented in infant rats by administration of immune sera before MenB challenge. 9M is a promising lead candidate for the development of an effective and affordable anti-MenB vaccine.

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.

Molecular subtyping of clinical and environmental strains of Cryptococcus neoformans variety neoformans serotype A isolated from southern Italy

Summary. Analysis of ribosomal DNA (rDNA) restriction fragment‐length polymorphism (RFLP) and random amplification of polymorphic DNA (RAPD) was used to investigate the genetic variability and biogeographic distribution of clinical and environmental strains of Cryptococcus neoformans isolated from a limited area of southern Italy, where the selection of a predomimant cryptococcal genotype could be expected. All isolates belonged to the species Cr. neoformans variety neoformans serotype A. RFLP analysis of a specific rDNA fragment allowed the distinction of strains of Cr. neoformans from closely related fungal reference species, but neither intraspecies nor intravarieties polymorphism was detected. On the contrary, RAPD fingerprints produced by priming with four different primers [(GTG)5, (GACA)4, M13 core sequence and the 8‐mer oligonucleotide (GCG‐GACGG)] were able to characterize the isolates up to the individual level, indicating the presence of marked heterogeneity among Cr. neoformans serotype A strains in southern Italy.

Analysis of the Streptococcus agalactiae exoproteome.

UNLABELLED The 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. BIOLOGICAL SIGNIFICANCE We believe that this manuscript will be of interest to Journal of Proteomics readers since the paper describes the identification of several putative virulence factors and vaccine candidates of the group B streptococcus, an important pathogen, using a simple proteomics strategy involving LC-MS analysis of culture supernatants obtained from two strains with divergent gene expression patterns. This technique provided the most comprehensive inventory of extracellular proteins obtained from a single streptococcal species thus far. The approach described has the added benefit of being easily applicable to a large number of different strains, making it ideal for the identification of conserved vaccine candidates.

Microsatellite-based genotyping of Candida parapsilosis sensu stricto isolates reveals dominance and persistence of a particular epidemiological clone among neonatal intensive care unit patients.

In this study, using multilocus microsatellite analysis, we report the genetic characterization of 27 Candida parapsilosis isolates recovered in two different periods of time (2007-2009 and 2011-2012) from infants hospitalized in the neonatal intensive care unit of a hospital in Messina, Italy. The results revealed the persistence and dominance of a particular infectious genotype among NICU patients and highlight the power of the used microsatellite markers in clarifying epidemiologic associations, detect micro-evolutionary variations and facilitating the recognition of outbreaks.

Interaction of organometallic cationic complex ions containing terpyridine ligands with nucleic acids: an investigation on aggregative phenomena

Organometallic cationic complex ions of the type [Pt(R-terpy)R′]Cl (R  H, Ph; R′  Me, Ph) are soluble and stable in aqueous solution, where they self-aggregate to form large supramolecular assemblies. The interaction of these aggregates with calf thymus DNA was investigated by UV-Vis and circular dichroism spectroscopy, resonance light scattering technique, thermal denaturation and gel electrophoresis mobility assays. Comparative studies were carried out on the complex [Pt(terpy)Me]Cl using poly(5′-A) and poly(vinylsulfonate). At high r1 ([complex]/[DNA]) ratios the complexes form extended aggregates on the surface of the polyanions, but at lower r1 ratios evidence was obtained for intercalation, at least in the case of the less hindered complex [Pt(terpy)Me]Cl. The relevance of steric effects on the binding process is discussed.