0 Results3.1 Morphology and growth rateThe colony morphology of 2951lgt1/4?mutant showed no obvious differences on BHI agar plates compared with 2952 and3292 wild-type strains. Growth rates in BHI broth were similar between 2971,3292 wild-type, but the 2951lgt1/4? mutant showed a slightly slower growth ratein logarithmic phase (Fig. 2).3.
2 Biological activity of the LOS truncatedmutantThe identify if inactivation ofrespective glycosyltransferase genes had any effect on the permeability of themutant strain, susceptibility of the mutant strain to a broad range ofhydrophobic agents and a hydrophilic glycopeptide was assessed. As shown inTable 1 2951lgt1/4? mutant showed more susceptibility towards hydrophobicantibodies and vancomycin than observed by the wild-type strains. In a LAL assay, whole-cell suspensions(OD600 = 0.1) gave 3.7 x 10³ endotoxin units (EU)/ mL for 2951 wild-type, 3.
9 x103 EU/mL for 3292 wild-type and 9.1 x 102 EU/mL for 2951lgt1/4?mutant, a fourfold reduction. In a bactericidal assay with normal human serum, bothwild-type strains survived at the same rate at all concentrations of humanserum.
However, truncation of LOS affected serum sensitivity for the2951lgt1/4? mutant, with only 35% survival of the OS truncated mutant cells in25% bovine serum (p<0.01) and 20% of the mutant cells died at 2.5% (p<0.001,Fig. 3). The survival rate for the 2951lgt1/4? mutant bacteria decreased, asthe concentration of bovine serum increased. These results suggest that OSchain truncation contributes to the serum bactericidal sensitivity.
The adherence ability of the OStruncated mutant to human epithelial cell lines Chang and HeLa were expressedas a percentage of initial inoculum (Table 2). The adherence of wild-typestrains 2951 to Chang and Hela epithelia were 43.5 ± 4.2%and 49.
0 ± 8.1%, 3292 were 49.1 ± 5.3% and 56.5 ± 9.4% respectively.
The mutantshowed significantly lower adherence to Chang and Hela cell lines, 22.2 ± 3.6% and28.
1 ± 6.4%, in comparison to the wild-types.3.
3 Characterisation of LOS, dLOSs, OMP26, AH-dLOS, and conjugatesThe LOS of M. catarrhalis was isolated using hot water/ phenol method,resulting in a LOS yield of 17 mg/L for 2951 wild-type, 27.2 mg/L for 3292wild-type and 22.4 mg/L for the 2951lgt1/4? mutant. Due to M. catarrhalis LOS being too toxic for mammalian cell antigens, aprocedure was employed to detoxify the LOSs.
After the treatment, the dLOSendotoxin reactivity was compared to the crude LOS by the LAL assay, where thewild-type strains 2951 LOS showed levels of 36,000 EU/µg, 3292 LOS was 38,600 and2951lgt1/4? LOS was 16,700 EU/µg, respectively. But the corresponding dLOS showed only 1.89, 1.26 and <0.10EU/ µg, accounting to a reduction of toxicity by 19,047, 30,634, 386,000-fold,respectively. OMP26 was analysed using SDS-PAGE (Fig.
4) with OMP26 band identifiedat around 26 kDa. Purified OMP26 sample revealed 0.3 mg/mL concentrationdetermined by BSA kit.
The molar ratio of AH to dLOSs in AH-dLOS was ~0.40,and a yield of ~83.8%,based on carbohydrate content. Talk about 1HNMR for dLOS and AH-dLOS.
After coupling of each dLOS with a carrier protein OMP26 toform dLOS-OMP26, a similar yield wasobserved with different molar ratio of 105:1 for 2951wild-type, 122:1 for 3292 wild-type and 149:1 for the 2951lgt1/4? mutant.