Evaluaton Of The Role Of LPS Outer Core Of Pasteurella Multocida In Response To Environmental Stresses And In Bacrerial Virulence In Ducks

Pasteurella multocida is a kind of Gram-negative bacterium that can cause contact infectious diseases in a varity of animal species,such as avian cholera in poultry.The lipopolysaccharide(LPS)of Pasteurella multocida(P.multocida)consists of two regions,a lipid A domain and an inner and outer core oligosaccharide,in which the outer core is variable and determines the classification of LPS serovars.The complete outer core has been previously proven to be essential for serovar 1 to display full virulence in chicken,however,the function of the outer core in bacterial virulence has not been fully elucidated.In the study,we investigated the ability of serovar 1 strains with outer core structures of different lengths to resist various environmental stresses and their virulence in ducks.1 Construction and characterization of Pasteurella multocida mutant strainsThree essential transferase genes pcgD(encoding the phosphocholine transferase),gatA(the galactosyltransferase),hptE(the heptosyltransferase)were deleted in wild-type(WT)strain.Three mutant strains of Pasteurella multocida,PMZ1(?pcg D),PMZ2(?gat A),PMZ3(?hptE),were constructed by suicide plasmid-mediated homologous recombination.At the same time,the corresponding complemented strains PMZ1(p CZ54),PMZ2(p CZ55)and PMZ3(p CZ56)were constructed based on the shuttle plasmid p MC-express.Silver staining confirmed that all the mutant strains produced truncated LPS profiles,and the?hpt E with the shortest oligosaccharide extension migrated furthest than the?pcg D and?gat A,and the?pcg D only without two p Cho and one galactose residues migrated close behind the?gat A.At the same time,the LPS phenotype of each complemented strain was similar to the WT strain.2 The role of outer core oligosaccharides in the stress response of Pasteurella multocidaEnvironmental stress sensitivity tests showed that compared to the WT strain,three mutant strains exhibited increased sensitivity to acid,alkali,high osmolarity and hydrogen peroxide rather than duck serum and polymycin B.The strains?gat A and?hpt E also showed reduced survival inside macrophages,but strikingly,all the mutant strains were more resistant to phagocytosis by macrophages,indicating a positive role of LPS outer core in phagocytic uptake.The loss of genes related to the outer core oligosaccharide synthesis significantly reduced the tolerance of bacteria to the four extreme environments of acid environment,alkaline environment,high osmotic pressure and hydrogen peroxide,indicating the presence of LPS outer core oligosaccharides helps the bacteria survive in these three extreme environments.3 The role of outer core oligosaccharides in bacterial virulence in ducksDucks were infected with the WT strain or the mutant strain orally or intramuscularly,and then the colonization ability and virulence of each strain in vivo were examined.The results showed that all the mutant strains displayed decreased colonization of duck tissues and were attenuated in ducks;however,the ?gatA showed the biggest drop in virulence,and is avirulent with the LD₅₀ at least 10⁴ fold higher than that of the WT strain.The pathological lesions in the liver and spleen showed that no pathological damage was found in PMZ2(?gat A)challenged group,and slight pathological damage such as local cell swelling and necrosis were observed in PMZ1(?pcg D)and PMZ3(?hpt E)groups.In conclusion,our study demonstrated that the LPS outer core of P.multocida has a beneficial effect on bacterial resistance to some environmental stresses,and is required for the bacteria to display full virulence in ducks,but the effect on virulence dose not correlate with the length of outer core oligosaccharide.