Multilocus sequence typing (MLST) analysis reveals many novel genotypes and a high level of genetic diversity in Candida tropicalis isolates from Italy and Africa.

BACKGROUND: Candida tropicalis is a human pathogenic yeast frequently isolated in Latin America and Asian-Pacific regions, although recent studies showed that it is also becoming increasingly widespread throughout several African and south-European countries. Nevertheless, relatively little is known about its global patterns of genetic variation as most of existing multilocus sequence typing (MLST) data come from Asia and there are no genotyped African isolates. OBJECTIVES: We report detailed genotyping data from a large set of C. tropicalis isolates recovered from different clinical sources in Italy, Egypt and Cameroon in order to expand the allele/genotype library of MLST database (https://pubmlst.org/ctropicalis), and to explore the genetic diversity in this species. METHODS: A total of 103 C. tropicalis isolates were genotyped using the MLST scheme developed for this species. All isolates were also tested for in vitro susceptibility to various antifungals to assess whether certain genotypes were associated with drug-resistance. RESULTS AND CONCLUSIONS: A total of 104 different alleles were detected across the MLST-loci investigated. The allelic diversity found at these loci resulted in 51 unique MLST genotypes of which 36 (70.6%) were novel. Global optimal eBURST analysis identified 18 clonal complexes (CCs) and confirm the existence of a specific Italian-cluster (CC36). Three CCs were also statistically associated with fluconazole resistance, which was elevated in Cameroon and Egypt. Our data show high genetic diversity in our isolates suggesting that the global population structure of C. tropicalis is still poorly understood. Moreover, its clinical impact in Italy, Egypt and Cameroon appears to be relevant and should be carefully considered.

Yeast killer toxin-like candidacidal Ab6 antibodies elicited through the manipulation of the idiotypic cascade.

A mouse anti-anti-anti-idiotypic (Id) IgM monoclonal antibody (mAb K20, Ab4), functionally mimicking a Wyckerhamomyces anomalus (Pichia anomala) killer toxin (KT) characterized by fungicidal activity against yeasts presenting specific cell wall receptors (KTR) mainly constituted by ß-1,3-glucan, was produced from animals presenting anti-KT Abs (Ab3) following immunization with a rat IgM anti-Id KT-like mAb (mAb K10, Ab2). MAb K10 was produced by immunization with a KT-neutralizing mAb (mAb KT4, Ab1) bearing the internal image of KTR. MAb K20, likewise mAb K10, proved to be fungicidal in vitro against KT-sensitive Candida albicans cells, an activity neutralized by mAb KT4, and was capable of binding to ß-1,3-glucan. MAb K20 and mAb K10 competed with each other and with KT for binding to C. albicans KTR. MAb K20 was used to identify peptide mimics of KTR by the selection of phage clones from random peptide phage display libraries. Using this strategy, four peptides (TK 1-4) were selected and used as immunogen in mice in the form of either keyhole limpet hemocyanin (KLH) conjugates or peptide-encoding minigenes. Peptide and DNA immunization could induce serum Abs characterized by candidacidal activity, which was inhibited by laminarin, a soluble ß-1,3-glucan, but not by pustulan, a ß-1,6-glucan. These findings show that the idiotypic cascade can not only overcome the barrier of animal species but also the nature of immunogens and the type of technology adopted.

Analysis of the Streptococcus agalactiae exoproteome.

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.

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.

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 approximately 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.

Immunogenic mimics of Brucella lipopolysaccharide epitopes.

Brucella melitensis and Brucella abortus are responsible for brucellosis in bovine and ovine species and for Malta fever in humans. The lipopolysaccharide (LPS) of Brucella is an important virulence factor and can elicit protective antibodies. Because of their potential importance in vaccine design and in serological diagnosis, we developed peptides mimicking the antigenic properties of distinctive antigenic determinants of Brucella LPS. These peptides were selected from several phage display random peptide libraries for their ability to bind monoclonal antibodies directed against the A- or C-type epitopes of Brucella LPS. Plasmids encoding for two of the isolated peptides induced, after DNA immunization, LPS-specific antibody responses. Although these responses were only moderate in extent, these data further suggest the feasibility of using peptide mimics of carbohydrate epitopes as immunogens, a property which may be useful in the design of novel anti-Brucella vaccines.

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.

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.

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.

Peptides mimicking Vibrio cholerae O139 capsular polysaccharide elicit protective antibody response.

Vibrio cholerae is the etiological agent of cholera. V. cholerae serogroup O1 had been, until 1992, the only serogroup responsible for large epidemics and pandemics of cholera. In 1992, a new serotype of V. cholerae emerged in South-East Asia that caused a massive outbreak of cholera in India and neighboring countries. The new serotype was named V. cholerae O139. The main differences between V. cholerae O139 and O1 are that the former possesses a capsular polysaccharide and different lipopolysaccharide. Capsular polysaccharides are, in general, T-independent antigens giving rise to poor immune responses lacking immunological memory. In order to overcome this, monoclonal antibodies against the capsular polysaccharide of V. cholerae O139 were used to screen different phage-displayed random peptide libraries. Eight different phage clones were selected and characterized using enzyme immunoassay with the monoclonal antibodies, and then tested for specificity by competition with V. cholerae O139 capsular polysaccharide. Selected peptides were sequenced, synthesized and conjugated to bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH). The conjugated peptides were used to immunize mice. It is evident that the anti-peptide mouse antibodies bind to the V. cholerae O139 capsular polysaccharide. In addition, the anti-peptide antibodies are protective in a suckling mouse model. The protective efficacy is both specific and dose-dependent. A PCT (PCT/IT2003/000489) with the publication number WO 2004/056851 has been filed for the sequences of the eight peptides.