Abstract
Neisseria meningitidis serogroup A capsular polysaccharide (CPS) is composed of a homopolymer of O-acetylated, alpha1-->6-linked ManNAc 1-phosphate that is distinct from the capsule structures of the other meningococcal disease-causing serogroups, B, C, Y, and W-135. The serogroup A capsule biosynthetic genetic cassette consists of four open reading frames, mynA-D (sacA-D), that are specific to serogroup A, but the functions of these genes have not been well characterized. mynC was found to encode an inner membrane-associated acetyltransferase that is responsible for the O-acetylation of the CPS of serogroup A. The wild-type CPS as revealed by 1H NMR had 60-70% O-acetylated ManNAc residues that contained acetyl groups at O-3, with some species acetylated at O-4 and at both O-3 and O-4. A non-polar mynC mutant generated by introducing an aphA-3 kanamycin resistance cassette produced CPS with no O-acetylation. A serogroup A capsule-specific monoclonal antibody was shown to recognize the wild-type O-acetylated CPS, but not the CPS of the mynC mutant, which lacked O-acetylation. MynC was C-terminally His-tagged and overexpressed in Escherichia coli to obtain the predicted approximately 26-kDa protein. The acetyltransferase activity of purified MynC was demonstrated in vitro using [14C]acetyl-CoA. MynC O-acetylated the O-acetylated CPS of the mynC mutant and further acetylated the wild-type CPS of serogroup A meningococci, but not the CPS of serogroup B or C meningococci. Genetic complementation of the mynC mutant confirmed the function of MynC as the serogroup A CPS O-3 and O-4 acetyltransferase. MynC represents a new subclass of O-acetyltransferases that utilize acetyl-CoA to decorate the D-mannosamine capsule of N. meningitidis serogroup A.
Highlights
Neisseria meningitidis serogroup A is responsible for the massive epidemics of meningococcal meningitis and septicemia that periodically affect sub-Saharan Africa, China, South America, and other parts of the world
The general genetic organization of CPS genes of N. meningitidis is similar to that of other bacterial systems such as Haemophilus influenzae, E. coli K1, etc., that are classified [9, 10] as group II capsules. It is usually composed of a unique biosynthetic genetic cassette and conserved genes involved in translocation of the CPS
Homology of MynC—A BLAST search performed with the deduced MynC amino acid sequence (247 amino acids) identified five proteins in the GenBankTM/EBI Data Bank with Ն25% sequence identity (Table II)
Summary
11 11 11 60 60 This study This study This study This study cross-linking of the capsule to the meningococcal cell surface [11]. We demonstrate that mynC (744 bp) encodes an O-acetyltransferase (247 amino acids) that transfers acetyl groups to the ManNAc residues of the serogroup A CPS
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