title: Functional characterization of a newly identified group B Streptococcus pullulanase eliciting antibodies able to prevent alpha-glucans degradation.
abstract: Streptococcal pullulanases have been recently proposed as key components of the metabolic machinery involved in bacterial adaptation to host niches. By sequence analysis of the Group B Streptococcus (GBS) genome we found a novel putative surface exposed protein with pullulanase activity. We named such a protein SAP. The sap gene is highly conserved among GBS strains and homologous genes, such as PulA and SpuA, have been described in other pathogenic streptococci. The SAP protein contains two N-terminal carbohydrate-binding motifs, followed by a catalytic domain and a C-terminal LPXTG cell wall-anchoring domain. In vitro analysis revealed that the recombinant form of SAP is able to degrade alpha-glucan polysaccharides, such as pullulan, glycogen and starch. Moreover, NMR analysis showed that SAP acts as a type I pullulanase. Studies performed on whole bacteria indicated that the presence of alpha-glucan polysaccharides in culture medium up-regulated the expression of SAP on bacterial surface as confirmed by FACS analysis and confocal imaging. Deletion of the sap gene resulted in a reduced capacity of bacteria to grow in medium containing pullulan or glycogen, but not glucose or maltose, confirming the pivotal role of SAP in GBS metabolism of alpha-glucans. As reported for other streptococcal pullulanases, we found specific anti-SAP antibodies in human sera from healthy volunteers. Investigation of the functional role of anti-SAP antibodies revealed that incubation of GBS in the presence of sera from animals immunized with SAP reduced the capacity of the bacterium to degrade pullulan. Of interest, anti-SAP sera, although to a lower extent, also inhibited Group A Streptococcus pullulanase activity. These data open new perspectives on the possibility to use SAP as a potential vaccine component inducing functional cross-reacting antibodies interfering with streptococcal infections.
authors.affiliation: Novartis Vaccines and Diagnostics Srl, Siena, Italy.
authors.forename: Isabella
authors.initials: I
authors.lastname: Santi
authors.forename: Alfredo
authors.initials: A
authors.lastname: Pezzicoli
authors.forename: Mattia
authors.initials: M
authors.lastname: Bosello
authors.forename: Francesco
authors.initials: F
authors.lastname: Berti
authors.forename: Massimo
authors.initials: M
authors.lastname: Mariani
authors.forename: John L
authors.initials: JL
authors.lastname: Telford
authors.forename: Guido
authors.initials: G
authors.lastname: Grandi
authors.forename: Marco
authors.initials: M
authors.lastname: Soriani
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis* / Q000097:blood; D000918:Antibody Specificity; D001483:Base Sequence; D003429:Cross Reactions; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D005936:Glucans / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013292:Streptococcus agalactiae / Q000201:enzymology* / Q000235:genetics / Q000276:immunology*; D014312:Trisaccharides / Q000378:metabolism
chemical_list: D000907:Antibodies, Bacterial; D017931:DNA Primers; D004269:DNA, Bacterial; D005936:Glucans; D011994:Recombinant Proteins; D014312:Trisaccharides; C008317:maltotriose; D006026:Glycoside Hydrolases; C100181:pullulanase