Streptococcus iniae capsule impairs phagocytic clearance and contributes to virulence in fish.

Surface capsular polysaccharides play a critical role in protecting several pathogenic microbes against innate host defenses during infection. Little is known about virulence mechanisms of the fish pathogen Streptococcus iniae, though indirect evidence suggests that capsule could represent an important factor. The putative S. iniae capsule operon contains a homologue of the cpsD gene, which is required for capsule polymerization and export in group B Streptococcus and Streptococcus pneumoniae. To elucidate the role of capsule in the S. iniae infectious process, we deleted cpsD from the genomes of two virulent S. iniae strains by allelic exchange mutagenesis to generate the isogenic capsule-deficient DeltacpsD strains. Compared to wild-type S. iniae, the DeltacpsD mutants had a predicted reduction in buoyancy and cell surface negative charge. Transmission electron microscopy confirmed a decrease in the abundance of extracellular capsular polysaccharide. Gas-liquid chromatography-mass spectrometry analysis of the S. iniae extracellular polysaccharides showed the presence of l-fucose, d-mannose, d-galactose, d-glucose, d-glucuronic acid, N-acetyl-d-galactosamine, and N-acetyl-d-glucosamine, and all except mannose were reduced in concentration in the isogenic mutant. The DeltacpsD mutants were highly attenuated in vivo in a hybrid striped bass infection challenge despite being more adherent and invasive to fish epithelial cells and more resistant to cationic antimicrobial peptides than wild-type S. iniae. Increased susceptibility of the S. iniae DeltacpsD mutants to phagocytic killing in whole fish blood and by a fish macrophage cell line confirmed the role of capsule in virulence and highlighted its antiphagocytic function. In summary, we report a genetically defined study on the role of capsule in S. iniae virulence and provide preliminary analysis of S. iniae capsular polysaccharide sugar components.

Multiplex PCR assay that identifies the major lipooligosaccharide serotype expressed by Moraxella catarrhalis clinical isolates.

A heterologous cluster of glycosyltransferase genes was identified in the three Moraxella catarrhalis LOS serotype strains. Multiple PCR primers designed to this region amplified products that differentiate between the serotypes more rapidly and efficiently than previously described serological analyses. This assay will be valuable for clinical and research-based studies.

Translocation and surface expression of lipidated serogroup B capsular Polysaccharide in Neisseria meningitidis.

The capsule of N. meningitidis serogroup B, (alpha2-->8)-linked polysialic acid and the capsules of other meningococcal serogroups and of other gram-negative bacterial pathogens are anchored in the outer membrane through a 1,2-diacylglycerol moiety. Previous work on the meningococcal cps complex in Escherichia coli K-12 indicated that deletion of genes designated lipA and lipB caused intracellular accumulation of hyperelongated capsule polymers lacking the phospholipid substitution. To better understand the role of lip and lipB in capsule expression in a meningococcal background, the location, sequence, and relationship to related bacterial capsule genes were defined and specific mutations in lipA and lipB were generated in the serogroup B meningococcal strain NMB. The lipA and lipB genes are located on the 3' end of the ctr operon and are most likely transcribed independently. Inactivation of lipA, lipB, and both resulted in the same total levels of capsular polymer production as in the parental controls; however, these mutants were as sensitive as an unencapsulated mutant to killing by normal human serum. Immunogold electron microscopy and flow cytometric analyses revealed intracellular inclusions of capsular polymers in lipA, lipB, and lipA lipB mutants. Capsular polymers purified from lipA, lipB, and lipA lipB mutants were lipidated. The phospholipid anchor was shown by gas chromatography-mass spectroscopy analysis to be a phosphodiester-linked 1,2-dipalmitoyl (C16:0) glycerol moiety and was identical in structure to that found on the wild-type meningococcal capsule polymers. Thus, lipA and lipB do not encode proteins responsible for diacylglycerophosphatidic acid substitution of the meningococcal capsule polymer; rather, they are required for proper translocation and surface expression of the lipidated polymer.

The Neisseria meningitidis serogroup A capsular polysaccharide O-3 and O-4 acetyltransferase.

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.