Involvement of lipooligosaccharide heptose residues of Haemophilus parasuis SC096 strain in serum resistance, adhesion and invasion.

Haemophilus parasuis is the causative agent of Glässer's disease. To investigate the role of lipooligosaccharide (LOS) in H. parasuis infection, ΔopsX, ΔrfaF and ΔwaaQ mutants defective in expressing opsX, rfaF and waaQ heptosyltransferases were constructed by transformation. Compared to the wild-type SC096 strain, the ΔopsX and ΔrfaF mutants, but not the ΔwaaQ mutant, produced severely truncated LOS. The mutants exhibited various degrees of reduction in resistance to complement-mediated killing in porcine and rabbit sera. In addition, the ΔopsX and ΔrfaF mutant strains showed impaired ability to adhere to and invade porcine kidney epithelial cells (PK-15) and porcine umbilical vein endothelial cells, indicating roles for heptose I and II residues in the interaction with host cells. The ΔwaaQ mutant strain, with no obvious truncation of LOS structure, did not exhibit significant defects in adhesion to and invasion of host cells. This study provides insight into the contribution of the inner core oligosaccharide, especially heptose I and heptose II residues, to the virulence-associated properties of H. parasuis.

Selective detection of 3-deoxymannooctulosonic acid in intact lipopolysaccharides by spin-echo 13C NMR.

The 3-deoxy-D-mannooctulosonic acid (KDO) region of lipopolysaccharides (LPS) from the heptoseless mutant Salmonella minnesota R595 and inner core and heptoseless mutants derived from Escherichia coli K12 was studied by 13C NMR spectroscopy. A spin-echo spectral editing technique was employed for the selective detection of the quaternary anomeric carbon of ketosidically linked KDO. Only two quaternary carbon resonances attributable to KDO were detected in the anomeric carbon spectral region of each LPS from heptoseless mutants E. coli D31m4 (99.7 and 100.8 ppm) and S. minnesota R595 (100.0 and 100.9 ppm). Integrated signal intensities from fully relaxed normal 13C spectra showed that equivalent molar quantities of KDO and glucosamine (i.e. 2 mol of each) were present in each of these samples. Similarly, only two KDO anomeric carbon resonances were detected in the LPS from the inner core mutants E. coli D21f1 (100.8 and 101.2 ppm) and E. coli D21e7 (100.8 and 101.2 ppm). These data confirm the presence of a KDO disaccharide structure rather than a trisaccharide as determined by others using thiobarbituric acid-based assays. The LPS of E. coli D21 (complete inner core oligosaccharide) exhibited four quaternary anomeric carbon resonances (99.4, 100.7, 101.8, and 102.7 ppm). The unequal intensities of these resonances, however, demonstrated that significant heterogeneity exists with respect to KDO substitution in this LPS. A third KDO moiety present in substoichiometric amounts could be consistent with this observation. However, this possibility could not be distinguished from other modes of substitutional heterogeneity involving only 2 KDO residues.