A Heptosyltransferase Mutant of Pasteurella multocida Produces a Truncated Lipopolysaccharide Structure and Is Attenuated in Virulence

Abstract

Pasteurella multocida is the causative agent of fowl cholera in birds. In a previous study using signaturetagged mutagenesis, we identified a mutant, AL251, which was attenuated for virulence in mice and in the natural chicken host. Sequence analysis indicated that AL251 had an insertional inactivation of the gene waaQ PM , encoding a putative heptosyl transferase, required for the addition of heptose to lipopolysaccharide (LPS) (M. Harper, J. D. Boyce, I. W. Wilkie, and B. Adler, Infect. Immun. 71:5440-5446, 2003). In the present study, using mass spectrometry and nuclear magnetic resonance, we have confirmed the identity of the enzyme encoded by waaQ PM as a heptosyl transferase III and demonstrated that the predominant LPS glycoforms isolated from this mutant are severely truncated. Complementation experiments demonstrated that providing a functional waaQ PM gene in trans can restore both the LPS to its full length and growth in mice to wild-type levels. Furthermore, we have shown that mutant AL251 is unable to cause fowl cholera in chickens and that the attenuation observed is not due to increased serum sensitivity.Pasteurella multocida is an encapsulated, gram-negative coccobacillus and is the causative agent of a wide range of animal diseases including avian fowl cholera. Several P. multocida virulence factors have previously been identified, including the capsule in serogroups A and B (4, 6), PMT toxin in strains causing atrophic rhinitis in pigs (18), putative filamentous hemagglutinins PfhB1 and PfhB2 (19), and several iron acquisition proteins such as TonB, ExbD and ExbB (3,19,27).During infections by gram-negative bacteria, the presence of lipopolysaccharide (LPS) stimulates the innate immune system whereby the inflammatory response plays a critical role in helping to clear the bacteria and prevent infection. This initial response to gram-negative bacteria can be elicited by a number of bacterial components, the most potent being lipid A, a component of the core structure of LPS. If the inflammatory response in the host is unable to clear the bacteria and the infection is allowed to proceed, the presence of large amounts of systemic LPS can result in endotoxic shock, in which an overproduction of inflammatory mediators causes damage to tissues, septic shock, organ failure, and death (31).LPS is considered to play an important role in the pathogenesis of disease due to P. multocida. Recently it has been shown that LPS from P. multocida assists in adhesion to neutrophils and transmigration through endothelial cells (20). However, there are conflicting reports about the endotoxic properties of LPS isolated from P. multocida. LPS isolated from a serotype B:2 strain was shown to be endotoxic, and intravenously administered LPS could reproduce clinical signs of hemorrhagic septicemia in buffalo (24). However, turkey poults were found to be relatively resistant to the lethal effects of LPS isolated from serogroup A strains of P. multocida, although the inflammatory response and microscopic hepatic lesions we...

Unique Properties of the Chicken TLR4/MD-2 Complex: Selective Lipopolysaccharide Activation of the MyD88-Dependent Pathway

“…Comparison of the potency of different LPS derivatives in activating the chTLR4/MD-2/NF-B signaling pathway revealed virtually no differences in biological activity between LPS derived from S. Gallinarum, S. Enteritidis, P. multocida, and its harmless waaQ-negative derivate (59), despite that these microbes display large diversity in virulence. These data suggest that, at least for these bacterial species, LPS recognition by chTLR4/chMD-2 per se is not correlated with disease.…”

Development of a Rapid Multiplex PCR Assay To Genotype Pasteurella multocida Strains by Use of the Lipopolysaccharide Outer Core Biosynthesis Locus

“…We have designated these genetic loci L1 through to L8. The type strains of Heddleston serovars 1,2,3,5,6,8,9,12, and 16 express full-length or "parent" LPS structures, and the type strains of Heddleston serovars 4, 7, 10, 11, 13, 14, and 15 express truncated LPS-the result of mutations within the LPS outer core biosynthesis loci. The partial differentiation of P. multocida strains on the basis of LPS biosynthesis genes has been reported previously (17).…”

“…multocida LPS is an immunodominant antigen critical for homologous protection stimulated by bacterin (killed-cell) vaccines (4). Furthermore, in the P. multocida strain VP161, a fulllength LPS molecule is essential for the ability to cause acute disease (5,6). Heddleston serotyping is currently the only method used to differentiate P. multocida strains on the basis of LPS type.…”

Structural and Genetic Basis for the Serological Differentiation of Pasteurella multocida Heddleston Serotypes 2 and 5

“…3). This resonance was subsequently assigned as the proton at the 6 position of the Hep IV residue by virtue of 13 C-1 H heteronuclear single quantum coherence (HSQC) and 13 C-1 H HSQC-TOCSY spectroscopy experiments (data not shown). This assignment is also consistent with the methylation analysis data which identified a 6-linked LD-Hep residue.…”

“…Lipopolysaccharide (LPS) is also an important virulence factor in P. multocida (13) and can be used for the identification of strains, with two main somatic typing systems reported (14,17). The Namioka system is based on a tube agglutination test and is able to recognize 11 serotypes (17), whereas the Heddleston system uses a gel diffusion precipitation test and can recognize 16 serotypes; the Heddleston system is currently the preferred method (14).…”