Initiation and synthesis of the Streptococcus pneumoniae type 3 capsule on a phosphatidylglycerol membrane anchor.

The capsule of the gram-positive pathogen Streptococcus pneumoniae is essential for its virulence (4, 16, 18a). Capsule serves to prevent opsonophagocytosis by interfering with the recognition of complement bound to the bacterial surface by phagocytic receptors on immune cells (7, 40, 41) and, to a lesser extent, by reducing the amount of complement bound (1). Production of a capsule is also critical for colonization of the nasopharynx of mice (26). S. pneumoniae capsules demonstrate a high level of diversity, with 90 distinct serotypes that differ in their monosaccharide composition and/or their glycosidic linkages (19, 37). Thus far, two different mechanisms have been described for capsule synthesis in S. pneumoniae (10, 12, 17, 23). The majority of capsules are synthesized by a mechanism analogous to that of Wzy-dependent O-antigen synthesis in gram-negative bacteria, where the repeating-unit structure of the capsule is assembled on a lipid acceptor that is subsequently transported across the cytoplasmic membrane and polymerized into polysaccharide (39). At some point during polymerization, polysaccharide is transferred onto the cell wall (33). The transfer is size independent, and polymer ranging from very small to very large molecular size is detectable on both membrane and cell wall fractions (5). Generally, polysaccharides synthesized by this mechanism require a separate glycosyltransferase to form each glycosidic linkage of the repeat unit, as well as an enzyme to polymerize the repeat units. The second mechanism of synthesis, utilized in the production of type 3 and 37 capsules, requires only a single enzyme for formation of all the glycosidic linkages and polymerization (2, 12, 25). The type 3 synthase alternately adds Glc and GlcUA onto the nonreducing end of the polysaccharide chain by a processive mechanism whereby the growing polysaccharide remains tightly associated with a carbohydrate binding site on the synthase (10). A similar mechanism of synthesis is used by the enzymes involved in forming hyaluronan, chitin, and cellulose (24, 28), although hyaluronan from Streptococcus pyogenes and Streptococcus equisimilis has recently been shown to grow from the reducing end (34). These enzymes are classified in glycosyltransferase family 2 (8). The type 3 synthase, encoded by cps3S, is one of two gene products encoded in the type 3 capsule locus that is required for type 3 polysaccharide synthesis. The other, Cps3D, is a UDP-Glc dehydrogenase that oxidizes UDP-Glc to form UDP-GlcUA, the second substrate for type 3 polysaccharide formation (12). Both the type 3 synthase contained in S. pneumoniae membranes and the recombinant synthase contained in Escherichia coli membranes will rapidly assemble type 3 polysaccharide in vitro in the presence of both nucleotide sugar substrates (9, 10, 32). When one of these substrates becomes limiting or only a single nucleotide sugar substrate is present, synthesis is terminated. In E. coli membranes, the nascent polymer is ejected from the enzyme following termination and can no longer be extended, although it remains associated with the membrane. In addition, a single sugar is added onto a glycerophosphate lipid (9). In contrast, termination of synthesis in S. pneumoniae membranes results in only about 50% of the polymer being retained on the membrane and the remainder being released into solution (10, 15). In both systems, polysaccharide ejection is time, temperature, and substrate concentration dependent, indicating that it is an enzymatic process (9, 15). Although it has been suggested that processive glycosyltransferases do not require a primer for synthesis of their polymers (28), characterization of the type 3 synthase expressed in E. coli and of the cellulose synthase CesA demonstrated that these enzymes initiate synthesis of their respective polymers on lipid acceptors (9, 27). The formation of novel glycolipid products is also observed when glycosyltransferases from other gram-positive bacteria are expressed in E. coli. Jorasch et al. found, however, that these products are not synthesized in their native bacterial membranes (21). Here, we demonstrate that the type 3 synthase contained in S. pneumoniae membranes is able to synthesize glycolipids with properties similar to those observed for synthase-containing E. coli membranes and that initiation of type 3 polysaccharide synthesis utilizes phosphatidylglycerol.