A functional analysis of the Streptococcus pneumoniae genes involved in the synthesis of type1 and type 3 capsular polysaccharides.

GARCÍA E; ARRECUBIETA C; MUÑOZ R; MOLLERACH M; LÓPEZ R

Oeiras, Portugal

Workshop; Streptococcus pneumoniae. Molecular biology and Mechanisms of disease-Update for the1990s; 1996

European Team on the Molecular Biology of the Pneumococcus

Capsular polysaccharides protect bacteria from phagocytosis by polymorphonuclear leukocytes and the virulence of S. pneumoniae is determined by the size and the chemical composition of the capsule. Type 3 pneumococci produce a capsule composed of cellobiuronic acid units connected in a β(1→3) linkage. Cellobiuronic acid is a disaccharide consisting of D-glucuronic acid (GlcA) β(1→4) linked to D-glucose (Glc). The genes implicated in the biosynthesis of type 3 capsule have been cloned, expressed and biochemically characterized. The three-type 3-specific genes -designated as cap3ABC- are transcribed together. However, the two complete open reading frames located upstream of the cap3A are not transcribed and consequently are not required for capsule formation. The cap3C gene was able to complement the galU defect on E. coli mutant. Therefore Cap3C should be a UDP-Glc pyrophosforylase, i.e., the enzyme that converts Glc-1-P to UDP-Glc. All but one of the spontaneous, unencapsulated pneumococcal mutants isolated in the laboratory mapped in the cap3A gene and three of them were characterized at the nucleotide level. We also cloned and expressed the cap3A gene in E. coli JM109 (DE3) using plasmid pT7-7. IPTG induced extracts prepared from E. coli cells containing the recombinant plasmid pTVU1 (cap3A) showed an overexpressed 46-kDa protein when analyzed by SDS-PAGE and converted UDP-Glc to UDP-GlcA as determined by HPLC. Consequently Cap3A encodes an UDP-Glc dehydrogenase. On the other hand Cap3B is similar to several polysaccharide synthases and particularly to HasA, the hyaluronan synthase of S. pyogenes . When cap3B was cloned into E. coli BL21 (DE3) (pLysS) using a pT7-7 as an expression vector, the transformed cells synthesized a high molecular size polysaccharide that was immunologically indistinguishable from the type 3 pneumococcal polysaccharide. A significant amount of type 3 polysaccharide was found in the periplasmic space as revealed by cell fractionation experiments. In addition, cap3B was cloned into pLSE1 (an E. coli/S. pneumoniae shuttle vector) and expressed from the tet promoter. When the recombinant plasmid pLSE3B was introduced by transformation into encapsulated pneumococci of types 1,2,5 or 8, the erythromycin-resistant transformants displayed binary type of capsule, this is, they showed a type 3 capsule in addition to that of the recipient type. Unencapsulated (S2-) laboratory strains of S. pneumoniae also synthesized type 3 capsule when transformed with pLSE3B. Since UDP-Glc should be omnipresent as it plays a central role in the biosynthesis of most cellular polysaccharides, our results show that whether the recipient cell contains UDP-GlcA, the Cap3B synthase is sufficient to synthesize pneumococcal type 3 capsular polysaccharide. The capsular polysaccharide from S. pneumoniae type 1fas the structure →3)-α-Sug-(1→4)-α-D-GalA-(1→3)-α-D-GalA-(1→ , in which Sug denotes 2-acetamido-4-amino-2,4,6-trideoxy-D-galactopyranose and GalA stands for galacturonic acid. About 30% of the GalA residues appear to be acylated. Most of the knowledge on the genetics and biochemistry of type 1 capsule biosynthesis came from the pioneering work carried out on binary strains by Austrian, Bernheimer and coworkers. We have cloned and sequenced seven type 1-specific genes (designated as cap1A-G and there functions have been preliminary assigned based on sequences similarities. Restoration of type 3 capsulation was achieved when a cap3A mutant was transformed with a plasmid carrying cap1G indicating that this gene encodes the type 1 UDP-Glc dehydrogenase. The divergence between cap1G and cap3A surprisingly high (35%) which explains why these two genes do not recombine as previously reported.