Immunological characterisation of truncated lipooligosaccharide-outer membrane protein based conjugate vaccine against Moraxella catarrhalis and nontypeable Haemophilus influenzae.

Moraxella catarrhalis and nontypeable Haemophilus influenzae are important bacterial causes of otitis media in children and respiratory diseases in adults. Lipooligosaccharide (LOS) from M. catarrhalis and outer membrane protein 26 (OMP26) from NTHi are major surface antigens identified as potential vaccine components against these organisms. We previously constructed M. catarrhalis in which LOS is truncated, but contains a structure common to the three known serotypes of M. catarrhalis. OMP26 is known to enhance clearance of NTHi following vaccination in animal models, so was chosen as the carrier protein. In this study, we conjugated wild-type and truncated M. catarrhalis detoxified-LOS to a recombinant modified OMP26, rOMP26VTAL. Vaccination of mice with these conjugates resulted in a significant increase in anti-LOS and anti-rOMP26VTAL IgG levels. Importantly, mouse antisera showed complement-mediated bactericidal activity against all M. catarrhalis serotype A and B strains and a NTHi strain tested. Serotypes A & B make up more than 90% of isolates. These data suggest that the LOS and OMP based conjugate can be used as vaccine components and require further investigation in animal models.

Neisseria gonorrhoeae Becomes Susceptible to Polymyxin B and Colistin in the Presence of PBT2.

Neisseria gonorrhoeae (N. gonorrhoeae) causes the sexually transmitted disease gonorrhea, which has a global incidence of 106 million cases per year. No vaccine is available to prevent the disease, and the emergence of multidrug resistant (MDR) strains makes N. gonorrhoeae an immediate public health threat. Here, we show that an ionophore, PBT2, can reverse the intrinsic resistance of N. gonorrhoeae to polymyxin B and colistin. These antibiotics administered in combination with PBT2 may be an effective path to treat MDR gonococcal infections.

The role of lipooligosaccharide in the biological activity of Moraxella bovis strains Epp63, Mb25 and L183/2, and isolation of capsular polysaccharide from L183/2.

The Gram-negative bovine pathogen Moraxella bovis is a causative agent of Infectious bovine keratoconjunctivitis, 'pink-eye' that affects cattle. Here we report that strain L183/2 has the same capsular polysaccharide (CPS) of unsulfated chondroitin, as does strain Mb25, whereas strain Epp63 does not express CPS. NMR analysis of the oligosaccharides (OS) derived from the lipooligosaccharides (LOS) in these three strains by NMR has shown that strain Mb25 and Epp63 have the same OS structure with a terminal N-acetylgalactosamine ((1S)-GalaNAc) residue →4,6-linked. Strain L183/2 lacks the (1 S)-GalaNAc residue. The biological role of M. bovis LOS was assessed by comparing the LOS from strains Epp63, Mb25 and L183/2 and truncated Epp63 LOS variants. LOS truncation affected M. bovis growth rate, susceptibility to antibiotics, detergents, bovine serum bactericidal activity, endotoxicity and adherence to HeLa cells.

Identification and characterisation of a biosynthetic locus for Moraxella bovis lipo-oligosaccharide.

Moraxella bovis is a Gram-negative gammaproteobacterium and is one of the causative agents of infectious bovine keratoconjunctivitis. The structure of lipooligosaccharide (LOS) from strain Epp63 was recently elucidated. In the present study a genetic locus of seven encoding genes with high similarity to glycosyltransferases has been identified. Mutation of these putative glycosyltransferase genes resulted in M. bovis mutant bacteria that expressed truncated LOS structures. The structures of the oligosaccharide (OS) expressed by the mutant strains were elucidated and demonstrated the role of the glycosyltransferase enzymes in the LOS biosynthesis of M. bovis. The glycosyltransferase genes designated lgt1, lgt3, and lgt6 are highly similar to the genes in the related bacterium M. catarrhalis. In addition, there are syntenic similarities with the corresponding LOS biosynthesis locus in M. catarrhalis and other members of Moraxellaceae.

1H NMR spectroscopic studies establish that heparanase is a retaining glycosidase.

Heparanase is an endo-ß-glucuronidase that cleaves heparan sulfate side chains of proteoglycans in basement membranes and the extracellular matrix (ECM). Heparanase is implicated in several diverse pathological processes associated with ECM degradation such as metastasis, inflammation and angiogenesis and is thus an important target for anti-cancer and anti-inflammatory drug discovery. Heparanase has been classed as belonging to the clan A glycoside hydrolase family 79 based on sequence analysis, secondary structure predictions and mutagenic analysis, and thus it has been inferred that it is a retaining glycosidase. However, there has been no direct experimental evidence to support this conclusion. Herein we describe (1)H NMR spectroscopic studies of the hydrolysis of the pentasaccharide substrate fondaparinux by heparanase, and provide conclusive evidence that heparanase hydrolyses its substrate with retention of configuration and is thus established as a retaining glycosidase. Knowledge of the mechanism of hydrolysis may have implications for future design of inhibitors for this important drug target.