Cesira Galeotti

Preventing Bacterial Infections with Pilus-Based Vaccines: the Group B Streptococcus Paradigm

We recently described the presence of 3 pilus variants in the human pathogen group B streptococcus (GBS; also known as Streptococcus agalactiae), each encoded by a distinct pathogenicity island, as well as the ability of pilus components to elicit protection in mice against homologous challenge. To determine whether a vaccine containing a combination of proteins from the 3 pilus types could provide broad protection, we analyzed pili distribution and conservation in 289 clinical isolates. We found that pilus sequences in each island are conserved, all strains carried at least 1 of the 3 islands, and a combination of the 3 pilus components conferred protection against all tested GBS challenge strains. These data are the first to indicate that a vaccine exclusively constituted by pilus components can be effective in preventing infections caused by GBS, and they pave the way for the use of a similar approach against other pathogenic streptococci.

Specific Involvement of Pilus Type 2a in Biofilm Formation in Group B Streptococcus

Streptococcus agalactiae is the primary colonizer of the anogenital mucosa of up to 30% of healthy women and can infect newborns during delivery and cause severe sepsis and meningitis. Persistent colonization usually involves the formation of biofilm and increasing evidences indicate that in pathogenic streptococci biofilm formation is mediated by pili. Recently, we have characterized pili distribution and conservation in 289 GBS clinical isolates and we have shown that GBS has three pilus types, 1, 2a and 2b encoded by three corresponding pilus islands, and that each strain carries one or two islands. Here we have investigated the capacity of these strains to form biofilms. We have found that most of the biofilm-formers carry pilus 2a, and using insertion and deletion mutants we have confirmed that pilus type 2a, but not pilus types 1 and 2b, confers biofilm-forming phenotype. We also show that deletion of the major ancillary protein of type 2a did not impair biofilm formation while the inactivation of the other ancillary protein and of the backbone protein completely abolished this phenotype. Furthermore, antibodies raised against pilus components inhibited bacterial adherence to solid surfaces, offering new strategies to prevent GBS infection by targeting bacteria during their initial attachment to host epithelial cells.

Phosphorylation of the Synthetic Hexasaccharide Repeating Unit Is Essential for the Induction of Antibodies to Clostridium difficile PSII Cell Wall Polysaccharide

Clostridium difficile is emerging worldwide as a major cause of nosocomial infections. The negatively charged PSII polysaccharide has been found in different strains of C. difficile and, thereby, represents an important target molecule for a possible carbohydrate-based vaccine. In order to identify a synthetic fragment that after conjugation to a protein carrier could be able to induce anti-PSII antibodies, we exploited a combination of chemical synthesis with immunochemistry, confocal immunofluorescence microscopy, and solid state NMR. We demonstrate that the phosphate group is crucial in synthetic glycans to mimic the native PSII polysaccharide; both native PSII and a phosphorylated synthetic hexasaccharide repeating unit conjugated to CRM(197) elicit comparable immunogenic responses in mice. This finding can aid design and selection of carbohydrate antigens to be explored as vaccine candidates.

A Helicobacter pylori Vacuolating Toxin Mutant That Fails To Oligomerize Has a Dominant Negative Phenotype

ABSTRACT Most Helicobacter pylori strains secrete a toxin (VacA) that causes massive vacuolization of target cells and which is a major virulence factor of H. pylori. The VacA amino-terminal region is required for the induction of vacuolization. The aim of the present study was a deeper understanding of the critical role of the N-terminal regions that are protected from proteolysis when VacA interacts with artificial membranes. Using a counterselection system, we constructed an H. pylori strain, SPM 326-Δ49-57, that produces a mutant toxin with a deletion of eight amino acids in one of these protected regions. VacA Δ49-57 was correctly secreted by H. pylori but failed to oligomerize and did not have any detectable vacuolating cytotoxic activity. However, the mutant toxin was internalized normally and stained the perinuclear region of HeLa cells. Moreover, the mutant toxin exhibited a dominant negative effect, completely inhibiting the vacuolating activity of wild-type VacA. This loss of activity was correlated with the disappearance of oligomers in electron microscopy. These findings indicate that the deletion in VacA Δ49-57 disrupts the intermolecular interactions required for the oligomerization of the toxin.

BibA Induces Opsonizing Antibodies Conferring In Vivo Protection against Group B Streptococcus

We recently characterized the function of BibA, an immunogenic surface-associated antigen expressed by group B Streptococcus (GBS) that is involved in virulence. In this study, we performed a bibA gene variability analysis on a panel of 72 clinical isolate strains. The bibA gene was present in all the strains analyzed, and 4 allelic variants, correlating with serotype, were identified. Moreover, although the BibA protein was expressed in all strains tested, only 54% of strains demonstrated surface exposure of the antigen in vitro. Importantly, expression of BibA on the bacterial surface was correlated with protection, because mice immunized with BibA were protected against challenge by a GBS strain with high levels of surface exposure of the antigen. In agreement with these findings, serum samples from mice immunized with recombinant BibA induced neutrophil-mediated in vitro opsonophagocytic killing of GBS. In conclusion, we propose BibA as a novel GBS vaccine candidate.

The LMW surface-layer proteins of Clostridium difficile PCR ribotypes 027 and 001 share common immunogenic properties.

The aim of this study was to investigate the S-layer proteins (SLPs) of the hypervirulent Clostridium difficile PCR ribotype 027 and compare them with those of PCR ribotype 001 and other PCR ribotypes involved in C. difficile infection and outbreaks, by molecular analysis and immunological assays. It has been demonstrated previously that PCR ribotype 027 SlpA is conserved in C. difficile strains belonging to this PCR ribotype and that it is a new variant, showing 88 % identity with SlpA of PCR ribotype 001. As the low-molecular-weight (LMW) SLPs of C. difficile are immunodominant antigens, attention was focused on this region of the genome. Sequencing of strains of different PCR ribotypes (001, 012, 014, 017, 027 and 078) showed that SlpA was conserved among strains belonging to the same PCR ribotype. Comparison of the LMW SLP region among these strains identified ten regions with sequence identity between PCR ribotypes 027 and 001, and low conservation with the other PCR ribotypes. In particular, two of these regions corresponded to areas predicted to be surface exposed. Three specific peptides, including those of the two surface-exposed regions, were recognized by human sera against PCR ribotypes 027 and 001 and by a rabbit polyclonal serum against the SLPs of PCR ribotype 027. In contrast, these peptides were not recognized by a polyclonal serum against the SLPs of PCR ribotype 012 used as a control. These results confirm the antigenic role of the LMW SLP and suggest that it may have a role in evasion of the host immune response.

Diversity of cwp loci in clinical isolates of Clostridium difficile.

An increased incidence of Clostridium difficile infection (CDI) is associated with the emergence of epidemic strains characterized by high genetic diversity. Among the factors that may have a role in CDI is a family of 29 paralogues, the cell-wall proteins (CWPs), which compose the outer layer of the bacterial cell and are likely to be involved in colonization. Previous studies have shown that 12 of the 29 cwp genes are clustered in the same region, named after slpA (cwp1), the slpA locus, whereas the remaining 17 paralogues are distributed throughout the genome. The variability of 14 of these 17 cwp paralogues was determined in 40 C. difficile clinical isolates belonging to six of the currently prevailing PCR ribotypes. Based on sequence conservation, these cwp genes were divided into two groups, one comprising nine cwp loci having highly conserved sequences in all isolates, and the other five loci showing low genetic conservation among isolates of the same PCR ribotype, as well as between different PCR ribotypes. Three conserved CWPs, Cwp16, Cwp18 and Cwp25, and two variable ones, Cwp26 and Cwp27, were characterized further by Western blot analysis of total cell extracts or surface-layer preparations of the C. difficile clinical isolates. Expression of genetically invariable CWPs was well conserved in all isolates, whilst genetically variable CWPs were not always expressed at comparable levels, even in strains containing identical sequences but belonging to different PCR ribotypes. This is the first report on the distribution and variability of a number of genes encoding CWPs in C. difficile.