CHARACTERIZATION OF CPSF AND ITS PRODUCT CMP-N-ACETYLNEURAMINIC ACID SYNTHETASE, A GROUP-B STREPTOCOCCAL ENZYME THAT CAN FUNCTION IN K1 CAPSULAR POLYSACCHARIDE BIOSYNTHESIS IN ESCHERICHIA-COLI

Group a Streptococcus (GBS) is the foremost cause of neonatal sepsis a nd meningitis in the United States, A major virulence factor for GBS i s its capsular polysaccharide, a high molecular weight polymer of bran ched oligosaccharide subunits. N-acetylneuraminic acid (Neu5Ac or sial ic acid), at the end of the polysaccharide side chains, is critical to the virulence function of the capsular polysaccharide. Neu5Ac must be activated by CMP-Neu5Ac synthetase before it is incorporated into the polymer. We showed previously that a transposon mutant of a serotype III GBS strain which had no detectable capsular Neu5Ac was deficient i n CMP-Neu5Ac-synthetase activity (Wessels et al., 1992). In this paper , we report the identification and characterization of cpsF, a gene in terrupted by transposon insertion in the previously described Neu5Ac-d eficient mutant, The predicted amino acid sequence of the cpsF gene pr oduct shares 57% similarity and 37% identity with CMP-Neu5Ac synthetas e encoded by the Escherichia coli K1 gene, neuA, The enzymatic functio n of the protein encoded by cpsF was established by cloning the gene i n E. coli under the control of the T7 polymerase/promoter. Lysates of E. coli in which the cpsF gene product was expressed, catalysed the co ndensation of CTP with Neu5Ac to form CMP-Neu5Ac, In addition, when a CMP-Neu5Ac synthetase-deficient mutant of E. coli K1 was transformed w ith cpsF, K1 antigen expression was restored. We conclude that cpsF en codes CMP-Neu5Ac synthetase in type III GBS, and that the GBS enzyme c an function in the capsule-synthesis of a heterologous bacterial speci es.