The polysaccharide capsules of Streptococcus pneumoniae represent the single most important virulence factor of this organism. Structurally and antigenically diverse, 90 serotypes have been recognized. Biosynthesis of these polysaccharides involves at least two distinct mechanisms. One, observed for the type 3 polysaccharide (-3)-beta-D-GlcUA-(1,4)-beta-D-Glc-(1-), involves a single processive enzyme (the type 3 synthase) that is responsible for generating both glycosidic linkages and for transporting the polymer out of the cell. The type 3 synthase is a member of a family of glycosyltransferases that includes the streptococcal and eukaryotic hyaluronan synthases, chitin synthases, and cellulose synthases. Our work with the type 3 synthase has shown that synthesis initiates on a lipid primer. Synthesis occurs by a processive mechanism under high substrate concentrations. Under low substrate concentrations, however, the synthase functions as a nonprocessive enzyme. When only a single substrate is present, chain termination and release of the polysaccharide result. Synthase stability in S. pneumoniae appears to be dependent on substrate levels, thus suggesting another means of controlling synthase function. The mechanism of synthesis of most other S. pneumoniae capsular polysaccharides is similar to that of some LPS O-antigens, involving initiation and transport of subunits across the membrane on a lipid carrier, followed by polymerization. Homologous proteins (CpsABCD) involved in this type of biosynthesis occur in S. pneumoniae, Rhizobium, other streptococci, and staphylococci. We have demonstrated an interaction among the S. pneumoniae proteins, and we hypothesize that they function to enhance polymerase activity. The goals of the proposed research are to characterize the S. pneumoniae polymerases and the factors that directly influence their functions. We will address these goals by: 1) identifying the primer for type 3 synthesis and reconstituting the system; 2) identifying functional domains/residues of the type 3 synthase; 3) characterizing synthase functions and polysaccharide release in vivo; and 4) characterizing functions common to the synthesis of non-type 3 capsular polysaccharides. The results of these studies will provide insights into mechanisms of polysaccharide synthesis that are shared among many prokaryotic and eukaryotic systems.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM053017-06
Application #
6625950
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Program Officer
Marino, Pamela
Project Start
1995-07-01
Project End
2006-03-31
Budget Start
2003-04-01
Budget End
2004-03-31
Support Year
6
Fiscal Year
2003
Total Cost
$294,175
Indirect Cost
Name
University of Alabama Birmingham
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
063690705
City
Birmingham
State
AL
Country
United States
Zip Code
35294
Yother, Janet (2011) Capsules of Streptococcus pneumoniae and other bacteria: paradigms for polysaccharide biosynthesis and regulation. Annu Rev Microbiol 65:563-81
Forsee, W Thomas; Cartee, Robert T; Yother, Janet (2009) A kinetic model for chain length modulation of Streptococcus pneumoniae cellubiuronan capsular polysaccharide by nucleotide sugar donor concentrations. J Biol Chem 284:11836-44
Forsee, W Thomas; Cartee, Robert T; Yother, Janet (2009) Characterization of the lipid linkage region and chain length of the cellubiuronic acid capsule of Streptococcus pneumoniae. J Biol Chem 284:11826-35
Ventura, Christy L; Cartee, Robert T; Forsee, W Thomas et al. (2006) Control of capsular polysaccharide chain length by UDP-sugar substrate concentrations in Streptococcus pneumoniae. Mol Microbiol 61:723-33
Forsee, W Thomas; Cartee, Robert T; Yother, Janet (2006) Role of the carbohydrate binding site of the Streptococcus pneumoniae capsular polysaccharide type 3 synthase in the transition from oligosaccharide to polysaccharide synthesis. J Biol Chem 281:6283-9
Cartee, Robert T; Forsee, W Thomas; Yother, Janet (2005) Initiation and synthesis of the Streptococcus pneumoniae type 3 capsule on a phosphatidylglycerol membrane anchor. J Bacteriol 187:4470-9
Cartee, Robert T; Forsee, W Thomas; Bender, Matthew H et al. (2005) CpsE from type 2 Streptococcus pneumoniae catalyzes the reversible addition of glucose-1-phosphate to a polyprenyl phosphate acceptor, initiating type 2 capsule repeat unit formation. J Bacteriol 187:7425-33
Bender, Matthew H; Cartee, Robert T; Yother, Janet (2003) Positive correlation between tyrosine phosphorylation of CpsD and capsular polysaccharide production in Streptococcus pneumoniae. J Bacteriol 185:6057-66
Bender, M H; Yother, J (2001) CpsB is a modulator of capsule-associated tyrosine kinase activity in Streptococcus pneumoniae. J Biol Chem 276:47966-74
Cartee, R T; Forsee, W T; Jensen, J W et al. (2001) Expression of the Streptococcus pneumoniae type 3 synthase in Escherichia coli. Assembly of type 3 polysaccharide on a lipid primer. J Biol Chem 276:48831-9

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