Structural and genetic basis for the serological differentiation of Pasteurella multocida Heddleston serotypes 2 and 5

Abstract

Pasteurella multocida is classified into 16 serotypes according to the Heddleston typing scheme. As part of a comprehensive study to define the structural and genetic basis of this scheme, we have determined the structure of the lipopolysaccharide (LPS) produced by P. multocida strains M1404 (B:2) and P1702 (E:5), the type strains for serotypes 2 and 5, respectively. The only difference between the LPS structures made by these two strains was the absence of a phosphoethanolamine (PEtn) moiety at the 3 position of the second heptose (Hep II) in M1404. Analysis of the lpt-3 gene, required for the addition of this PEtn residue, revealed that the gene was intact in P1702 but contained a nonsense mutation in M1404. Expression of an intact copy of lpt-3 in M1404 resulted in the attachment of a PEtn residue to the 3 position of the Hep II residue, generating an LPS structure identical to that produced by P1702. We identified and characterized each of the glycosyltransferase genes required for assembly of the serotype 2 and 5 LPS outer core. Monoclonal antibodies raised against serotype 2 LPS recognized the serotype 2/5-specific outer core LPS structure, but recognition of this structure was inhibited by the PEtn residue on Hep II. These data indicate that the serological classification of strains into Heddleston serotypes 2 and 5 is dependent on the presence or absence of PEtn on Hep II.

FIG. 1.

Schematic representation of the LPS structure produced by the Heddleston M1404 and P1702 strains (A), the genetic organization of the genes encoding the transferases responsible for synthesis of the outer core section of the LPS molecules (B), and the LPS structure produced by the P. multocida VP161 gatA mutant (C). Transferases known to be involved in the addition of each sugar are shown below the appropriate linkages. The PEtn residue attached to Hep II (boxed) is only observed in LPS derived from strain P1702.

FIG. 2.

Anomeric region of the ¹H-NMR two-dimensional TOCSY spectra (excluding Hep II) of the purified core oligosaccharide from the Heddleston serotype 2 strain M1404. Residues and resonances are as indicated. The spectrum was obtained at 25°C and is referenced relative to the HOD (deuterated water) signal at 4.78 ppm.

FIG. 3.

Anomeric region of the ¹H-NMR two-dimensional NOESY spectra (excluding Hep II) of the purified core oligosaccharide from the Heddleston serotype 5 strain P1702. Residues and inter-NOE connectivities for residues in the extension from Hep I are as indicated. The spectrum was obtained at 25°C and is referenced relative to the HOD (deuterated water) signal at 4.78 ppm.

FIG. 4.

The region of the ¹H-NMR two-dimensional TOCSY spectra for the spin systems from the Hep II residues of the purified core oligosaccharide from the Heddleston serotype 5 strain P1702 (a) and the Heddleston serotype 2 strain M1404 (b). Residues and resonances are as indicated. The spectra were obtained at 25°C and are referenced relative to the HOD (deuterated water) signal at 4.78 ppm.

FIG. 5.

CE-ES-MS/MS spectrum of core oligosaccharide from P. multocida strain AL1131. The positively charged ion at m/z 1017²⁺ was selectively hit in an MS/MS experiment, revealing diagnostic ions for the Heddleston 2 outer core oligosaccharide of Hex-HexNAc-Hep. PCho was identified as a terminal moiety attached to the Hex residue.

FIG. 6.

Immunoblot analysis of the P. multocida strains examined in this study using MAbs T1C6 (A) and T6B2 (B). WCLs were derived from P. multocida strain VP161 (serotype A:1); the VP161 gatA mutant (AL725); AL725 containing pAL99 (AL807); AL725 expressing HptF (AL1086); AL725 expressing HptF and NctA (AL1088); AL725 expressing HptF, NctA, and GatD (AL1131); P. multocida strain P1702 (serotype E:5); P. multocida strain M1404 (serotype B:2); M1404 containing pAL99 (AL1100); and M1404 expressing Lpt-3 (AL1101).