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 α-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 α-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 α-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.

Group BStreptococcusPullulanase Crystal Structures in the Context of a Novel Strategy for Vaccine Development

“…SAG_1216), corresponding to residues 41 to 1214, was amplified, cloned, and expressed in bacterial cells as described in Materials and Methods. As previously reported, two expression forms are evident following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with two bands visible at 98 kDa and 139 kDa, corresponding to a N-terminally truncated form (SAP-L) and a full-length form (SAP-H), respectively (31). SAP-L accounts for 80 to 90% of the total expression and arises due to a second translation start site at Val346 (GTG), a phenomenon also observed for the type I pullulanase from Anaerobranca gottschalkii (6,31).…”

“…We have recently reported that incubation of GBS in the presence of sera from animals immunized with SAP impaired the capacity of the bacterium to degrade alpha-glucans in vitro (31). Based on these data and the evidence reported in this paper that recombinant SAP binds to host cells, we propose to use SAP as a potential vaccine component.…”

“…We recently identified a novel surface-exposed ␣-glucandegrading enzyme, named SAP, belonging to the streptococcal family of pullulanases (31). The sap gene is highly conserved among group B streptococcus (GBS) strains; homologous genes, such as those for PulA and SpuA, are present in other pathogenic streptococci.…”

“…The sap gene is highly conserved among group B streptococcus (GBS) strains; homologous genes, such as those for PulA and SpuA, are present in other pathogenic streptococci. SAP is a member of the class 13 glycoside hydrolase (GH13; ␣-amylase) family and is a type I pullulanase; in vitro studies have shown that recombinant SAP can degrade ␣-glucans such as pullulan, glycogen, and starch (31). Furthermore, fluorescence-activated scanning analysis and confocal imaging studies performed on whole bacteria indicate that the presence of ␣-glucan polysaccharides in culture medium upregulates the expression of SAP on the bacterial surface (31).…”

“…SAP is a member of the class 13 glycoside hydrolase (GH13; ␣-amylase) family and is a type I pullulanase; in vitro studies have shown that recombinant SAP can degrade ␣-glucans such as pullulan, glycogen, and starch (31). Furthermore, fluorescence-activated scanning analysis and confocal imaging studies performed on whole bacteria indicate that the presence of ␣-glucan polysaccharides in culture medium upregulates the expression of SAP on the bacterial surface (31). As reported for other streptococcal pullulanases, we found specific anti-SAP antibodies in human sera from healthy volunteers.…”