The Group B capsular polysaccharide type IX was isolated and purified and the structure of its repeating unit was determined. of GBS neonatal disease (6). The most common serotypes among GBS infective isolates are Ia Ib II III and V although there are some geographical and historical variations (7). For instance the frequency of type V has increased in the last few years whereas serotype IV has recently emerged as a CB-184 cause of adult and neonatal invasive disease (8). Therefore accurately monitoring the variation of this important target antigen is essential for predicting vaccine coverage. The serotype IX was identified only in 2007 during the analysis of three human isolates that failed to react with antisera against the nine GBS types described at that time. Preparation of specific antisera allowed the identification of additional Rabbit polyclonal to PPP1R10. type IX strains among formerly non-typeable isolates (9). The identity of GBS type IX was also confirmed by a multiplex PCR assay containing a mix of primers specific for the 10 different allelic variants of the genes (10). Interestingly type IX strains can also cause neonatal infections (11 12 The precise chemical structures of GBS type Ia Ib CB-184 II III IV V VI VII and VIII CPSs are well described (13 -20). They are composed of repeating units of four to seven monosaccharides with a backbone and one or two side chains. Four monosaccharides (Glcgene sequences indicated that their differential evolution proceeded through en bloc replacement of individual glycosyltransferase genes with DNA sequences encoding enzymes with new linkage specificities (22). The chemical structure of CPS type IX and the genetic composition of its operon have not yet been delineated. Here we report the isolation purification structural characterization and immunochemical analysis of type IX CPS and the genetic analysis of the genes encoding the enzymes responsible for its synthesis. EXPERIMENTAL PROCEDURES Bacterial Strains GBS type IX strains IT-NI-016 (isolated from a neonatal early onset disease case) IT-PW-62 and IT-PW-64 (from colonized pregnant women) were kindly provided by Alberto Berardi (Policlinico di Modena Italy) Roberta Creti Lucilla Baldassarri and Graziella Orefici (Istituto Superiore di Sanità Rome CB-184 Italy). The three strains as well as CZ-PW-119 (VI) CZ-PW-045 (VII) and CZ-NI-016 (serotype IV) were isolated and typed by latex and molecular approaches in the frame of the DEVANI study (10 11 23 The capsular genotype of type IX isolates was confirmed by genome analysis (see below). Strains 2603 V/R (serotype V) COH1 (serotype III) 18 (serotype II) and JM9130013 (serotype VIII) were obtained from Dennis Kasper (Harvard Medical School Boston MA). Strains 515 (serotype Ia) and H36B (serotype Ib) were obtained from Carol Baker (Baylor College of Medicine Houston TX). Isolation and Purification of the Type IX Capsular Polysaccharide The GBS strain IT-NI-016 was used for preparation of CPS IX from 1 liter of bacterial culture grown to exponential phase in Todd Hewitt broth. The purification process was based on previously described procedures (24). Briefly the bacterial pellet was recovered by centrifugation at 4 0 rpm for 20 min and incubated with 0.8 n NaOH at 37 °C for 36 h. After centrifugation at 4 0 rpm for 20 min 1 m Tris buffer (1:9 v/v) was added to the supernatant and diluted with 1:1 (v/v) HCl to reach a neutral pH. To further purify type IX CPS 2 m CaCl2 (0.1 m final concentration) and ethanol (30% (v/v) final concentration) were added to CB-184 the solution. After centrifugation at 4 0 × for 20 min the supernatant was subjected to tangential flow filtration with a 10 0 weight cutoff (Hydrosart Sartorius; 0.1-m2 surface) against 14 volumes of 50 mm Tris 500 mm NaCl pH 8.8 and 7 volumes of 10 mm sodium phosphate pH 7.2. To reconstitute full for 20 min to remove any minor particles. The supernatant was subjected to filtration with a 10 0 weight cutoff (Vivaspin Sartorius; 20-ml device) against 300 mm Na2CO3 300 mm NaCl pH 8.8. A 1:1 diluted solution of 4.15 μl/ml acetic anhydride in ethanol was added and the reaction was incubated at room temperature for 2 h. The sample was subjected to filtration with a 10 0 weight cutoff (Vivaspin Sartorius; 20-ml device) against Milli-Q water CaCl2 (0.1 m final concentration) and ethanol (30% (v/v) final concentration) were added and the sample was centrifuged at 4 0 × for 20 min. The.