Biochemical Activities of Streptococcus pneumoniae Serotype 2 Capsular Glycosyltransferases and Significance of Suppressor Mutations Affecting the Initiating Glycosyltransferase Cps2E

Abstract

The capsular polysaccharide (CPS) is essential for Streptococcus pneumoniae virulence. Its synthesis requires multiple enzymes, and defects that block completion of the pathway can be lethal in the absence of secondary suppressor mutations. In this study, we examined the functions of three capsular glycosyltransferases (Cps2F, Cps2G, and Cps2I) involved in serotype 2 CPS synthesis, whose deletions select for secondary mutations. We demonstrate that Cps2F is a rhamnosyltransferase that catalyzes addition of the third and fourth sugars in the capsule repeat unit, while Cps2G adds the fifth sugar (glucose). Addition of the terminal residue (glucuronic acid) could not be detected; however, activities of the other glycosyltransferases together with bioinformatic analyses suggest that this step is mediated by Cps2I. Most of the secondary suppressor mutations resulting from loss of these enzymes occur in cps2E, the gene encoding the initiating glycosyltransferase. Examination of the 69 S. pneumoniae serotypes containing Cps2E homologues yielded a consensus amino acid sequence for this protein and demonstrated that there is a highly significant association between the residues that are 100% conserved and those altered by suppressor mutations. Cps2E contains an extracytoplasmic loop whose function is unknown. Among our collection of mutants, six contained missense mutations affecting amino acids in the extracytoplasmic loop. These residues are highly conserved among S. pneumoniae Cps2E homologues, and mutations therein severely reduced CPS synthesis and Cps2E activity. The critical functions of these amino acids suggest a role for the Cps2E extracytoplasmic loop in initiation, and possibly regulation, of capsule synthesis.

Mutations in Pneumococcal cpsE Generated via In Vitro Serial Passaging Reveal a Potential Mechanism of Reduced Encapsulation Utilized by a Conjunctival Isolate

“…However, it is still incompletely understood how this is accomplished: a phosphorelay system comprised of CpsB, CpsC, and CpsD and transcriptional regulation of capsule genes comprise two levels of control of capsule levels (33-35, 62, 63). This study and work by others supports the notion that there exists another potential layer of control, involving mutations in the gene encoding the initiating glycosyltransferase, cpsE (39,40). Our experiments using serotype-matched acapsular and encapsulated conjunctival isolates support the idea that this level of control occurs in nature.…”

“…The two colony types were genetically stable, yielding high and low capsule expression, respectively, for the opaque and transparent types (data not shown). Since mutations in the cpsE gene, which encodes the initiating glycosyltransferase (38), were reported to result in a reduction of capsule expression (39,41,42), we sequenced this gene in both transparent and opaque variants. These data revealed that, while the opaque strain had a wild-type cps4E sequence, the transparent variant possessed a duplication of a 6-nucleotide sequence, GCGATT, which results in the addition of an alanine and isoleucine after amino acid 45 ( Fig.…”

“…This suggests that this two-amino-acid-duplication mutant retains some glycosyltransferase activity. A thorough analysis of cpsE in D39 (serotype 2) identified several residues in the extracytoplasmic loop (amino acids 117 to 264) and cytoplasmic tail (amino acids 284 to 455) that were critical for glycosyltransferase activity (39). In D39 and most other serogroups, including 19A, CpsE is a large protein, approximately 455 amino acids, while TIGR4 is unique in coding for a smaller CpsE protein, of 211 amino acids.…”

“…Importantly, cpsE is the first serotype-specific gene and encodes the initiating glycosyltransferase that catalyzes the linkage of the sugar-phosphate to the lipid acceptor on the cytoplasmic face of the cell membrane (36)(37)(38). Mutations in cpsE, including simple sequence repeats within this gene, have been shown to reduce levels of encapsulation and may potentially represent another layer of complexity in the scheme of capsular polysaccharide regulation (36,(39)(40)(41)(42).…”

Pneumococcal Capsules and Their Types: Past, Present, and Future

“…Paired with genetic studies, studies on capsular synthesis have shown that the synthetic machinery is fairly sensitive to mutation inasmuch as mutations resulting in incomplete or dramatically altered repeat units are not compatible with the flippase/polymerase and are lethal to the organism, presumably due to the sequestration of undecaprenyl phosphate acceptor (79,80).…”