The Capsule Polymerase CslB of Neisseria meningitidis Serogroup L Catalyzes the Synthesis of a Complex Trimeric Repeating Unit Comprising Glycosidic and Phosphodiester Linkages

Neisseria rneningitidis is a human pathogen causing bacterial meningitis and sepsis. The capsular polysaccharide surrounding N. meningitidis is a major virulence factor. The capsular polysaccharide consists of polyhexosarnine phosphates in N. meningitidis serogroups A and X. The capsule polymerases (CPs) of these serogroups are members of the Stealth protein family comprising o-hexose-1-phosphate transferases from bacterial and protozoan pathogens. CsIA, one of two putative CPs of the pathophysiologically less relevant N. meningitidis serogroup is one of the smallest known Stealth proteins and caught our attention for structure-function analyses. Because the N. meningitidis serogroup I. capsule polymer consists of a trimeric repeating unit ([-> 3)-beta-D-GUNAc-(1 -> 3)-beta-D-GlcNAc-(1 -> 3)-alpha-D-GIcNAc-(10P03Hn), we speculated that the two predicted CPs (CsIA and Cs113) work together in polymer production. Consequently, both enzymes were cloned, overexpressed, and purified as recombinant proteins. Contrary to our expectation, enzymatic testing identified CsIB to be sufficient to catalyze the synthesis of the complex trimeric N rneningitidis serogroup I. capsule polymer repeating unit. No polymerase activity was detected for CsIA, although the enzyme facilitated the hydrolysis of UDP-GIcNAc. Bioinformatics analyses identified two glycosyltransferase (GT) domains in Cs113. The N-terminal domain modeled with 100% confidence onto a number of GT-A folded proteins, whereas the C-terminal domain modeled with 100% confidence onto TagF, a GT-B folded teichoic acid polymerase from Staphylococcus epiderrnidis. Amino acid positions known to have critical catalytic functions in the template pro- teins were conserved in Cs1B, and their point mutation abolished enzyme activity. Cs113 represents an enzyme of so far unique complexity regarding both the catalyzed reaction and enzyme architecture.