Natural selection in the chicken host identifies 3-deoxy-D-manno-octulosonic acid kinase residues essential for phosphorylation of Pasteurella multocida lipopolysaccharide.
Pasteurella multocida is the causative agent of a number of diseases in animals, including fowl cholera. P. multocida strains simultaneously express two lipopolysaccharide (LPS) glycoforms (glycoforms A and B) that differ only in their inner core structure. Glycoform A contains a single 3-deoxy-d-manno-octulosonic acid (Kdo) residue that is phosphorylated by the Kdo kinase, KdkA, whereas glycoform B contains two unphosphorylated Kdo residues. We have previously shown that P. multocida mutants lacking the heptosyltransferase, HptA, produce full-length glycoform B LPS and a large amount of truncated glycoform A LPS, as they cannot add heptose to the glycoform A inner core. These hptA mutants were attenuated in chickens because the truncated LPS made them vulnerable to host defense mechanisms, including antimicrobial peptides. However, here we show that birds inoculated with high doses of the hptA mutant developed fowl cholera and the P. multocida isolates recovered from diseased birds no longer expressed truncated LPS. Sequencing analysis revealed that the in vivo-derived isolates had mutations in kdkA, thereby suppressing the production of glycoform A LPS. Interestingly, a number of the spontaneous KdkA mutant strains produced KdkA with a single amino acid substitution (A112V, R123P, H168Y, or D193N). LPS structural analysis showed that complementation of a P. multocida kdkA mutant with wild-type kdkA restored expression of glycoform A to wild-type levels, whereas complementation with any of the mutated kdkA genes did not. We conclude that in P. multocida KdkA, the amino acids A112, R123, H168, and D193 are critical for Kdo kinase function and therefore for glycoform A LPS assembly.
Harper
Marina
M
Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, VIC 3800, Australia.
Cox
Andrew D
AD
St Michael
Frank
F
Ford
Mark
M
Wilkie
Ian W
IW
Adler
Ben
B
Boyce
John D
JD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002645:Chickens / Q000382:microbiology*; D002771:Cholera / Q000382:microbiology / Q000662:veterinary; D016695:Glycosyltransferases / Q000235:genetics / Q000502:physiology; D008070:Lipopolysaccharides / Q000737:chemistry*; D008969:Molecular Sequence Data; D009154:Mutation; D016979:Pasteurella multocida / Q000472:pathogenicity*; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000502:physiology; D013329:Structure-Activity Relationship
D008070:Lipopolysaccharides; D016695:Glycosyltransferases; C086735:heptosyltransferase; C400526:3-deoxy-D-manno-octulosonic acid kinase; D017853:Phosphotransferases (Alcohol Group Acceptor)