Screening of 71 P. multocida proteins for protective efficacy in a fowl cholera infection model and characterization of the protective antigen PlpE.

BACKGROUND: There is a strong need for a recombinant subunit vaccine against fowl cholera. We used a reverse vaccinology approach to identify putative secreted or cell surface associated P. multocida proteins that may represent potential vaccine candidate antigens. PRINCIPAL FINDINGS: A high-throughput cloning and expression protocol was used to express and purify 71 recombinant proteins for vaccine trials. Of the 71 proteins tested, only one, PlpE in denatured insoluble form, protected chickens against fowl cholera challenge. PlpE also elicited comparable levels of protection in mice. PlpE was localized by immunofluorescence to the bacterial cell surface, consistent with its ability to elicit a protective immune response. To explore the role of PlpE during infection and immunity, a plpE mutant was generated. The plpE mutant strain retained full virulence for mice. CONCLUSION: These studies show that PlpE is a surface exposed protein and was the only protein of 71 tested that was able to elicit a protective immune response. However, PlpE is not an essential virulence factor. This is the first report of a denatured recombinant protein stimulating protection against fowl cholera.

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.

Identification of novel glycosyltransferases required for assembly of the Pasteurella multocida A:1 lipopolysaccharide and their involvement in virulence.

We previously determined the structure of the Pasteurella multocida Heddleston type 1 lipopolysaccharide (LPS) molecule and characterized some of the transferases essential for LPS biosynthesis. We also showed that P. multocida strains expressing truncated LPS display reduced virulence. Here, we have identified all of the remaining glycosyltransferases required for synthesis of the oligosaccharide extension of the P. multocida Heddleston type 1 LPS, including a novel alpha-1,6 glucosyltransferase, a beta-1,4 glucosyltransferase, a putative bifunctional galactosyltransferase, and two heptosyltransferases. In addition, we identified a novel oligosaccharide extension expressed only in a heptosyltransferase (hptE) mutant background. All of the analyzed mutants expressing LPS with a truncated main oligosaccharide extension displayed reduced virulence, but those expressing LPS with an intact heptose side chain were able to persist for long periods in muscle tissue. The hptC mutant, which expressed LPS with the shortest oligosaccharide extension and no heptose side chain, was unable to persist on the muscle or cause any disease. Furthermore, all of the mutants displayed increased sensitivity to the chicken antimicrobial peptide fowlicidin 1, with mutants expressing highly truncated LPS being the most sensitive.

Pasteurella multocida expresses two lipopolysaccharide glycoforms simultaneously, but only a single form is required for virulence: identification of two acceptor-specific heptosyl I transferases.

Lipopolysaccharide (LPS) is a critical virulence determinant in Pasteurella multocida and a major antigen responsible for host protective immunity. In other mucosal pathogens, variation in LPS or lipooligosaccharide structure typically occurs in the outer core oligosaccharide regions due to phase variation. P. multocida elaborates a conserved oligosaccharide extension attached to two different, simultaneously expressed inner core structures, one containing a single phosphorylated 3-deoxy-D-manno-octulosonic acid (Kdo) residue and the other containing two Kdo residues. We demonstrate that two heptosyltransferases, HptA and HptB, add the first heptose molecule to the Kdo(1) residue and that each exclusively recognizes different acceptor molecules. HptA is specific for the glycoform containing a single, phosphorylated Kdo residue (glycoform A), while HptB is specific for the glycoform containing two Kdo residues (glycoform B). In addition, KdkA was identified as a Kdo kinase, required for phosphorylation of the first Kdo molecule. Importantly, virulence data obtained from infected chickens showed that while wild-type P. multocida expresses both LPS glycoforms in vivo, bacterial mutants that produced only glycoform B were fully virulent, demonstrating for the first time that expression of a single LPS form is sufficient for P. multocida survival in vivo. We conclude that the ability of P. multocida to elaborate alternative inner core LPS structures is due to the simultaneous expression of two different heptosyltransferases that add the first heptose residue to the nascent LPS molecule and to the expression of both a bifunctional Kdo transferase and a Kdo kinase, which results in the initial assembly of two inner core structures.

A heptosyltransferase mutant of Pasteurella multocida produces a truncated lipopolysaccharide structure and is attenuated in virulence.

Pasteurella multocida is the causative agent of fowl cholera in birds. In a previous study using signature-tagged 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.

Characterization of two lipoproteins in Pasteurella multocida.

An in vivo expression technology (IVET) system was previously developed and used to identify Pasteurella multocida genes, which are upregulated during infection of the host. Of the many genes identified, two encoded products which showed similarity to the Haemophilus influenzae lipoproteins, protein D and PCP, which have been shown to stimulate heterologous immunity against infection with H. influenzae. Therefore, the lipoprotein homologues in P. multocida, designated GlpQ and PCP, were investigated. GlpQ and PCP were shown to be lipoproteins by demonstrating that post-translational processing of the proteins was inhibited by globomycin. The P. multocida GlpQ homologue showed glycerophosphodiester phosphodiesterase enzyme activity, indicating that it is a functional homologue of other characterized GlpQ enzymes. Using surface immunoprecipitation, PCP was found to be surface exposed, but GlpQ was not. Non-lipidated forms of GlpQ and PCP were expressed and purified from Escherichia coli and used to vaccinate mice. However, mice were not protected from challenge with live P. multocida. The lipoproteins were then expressed in E. coli in the lipidated form and used to vaccinate mice and chickens. Protection against challenge with live P. multocida was not observed.

Signature-tagged mutagenesis of Pasteurella multocida identifies mutants displaying differential virulence characteristics in mice and chickens.

Pasteurella multocida is the causative agent of fowl cholera in birds. Signature-tagged mutagenesis (STM) was used to identify potential virulence factors in a mouse septicemia disease model and a chicken fowl cholera model. A library of P. multocida mutants was constructed with a modified Tn916 and screened for attenuation in both animal models. Mutants identified by the STM screening were confirmed as attenuated by competitive growth assays in both chickens and mice. Of the 15 mutants identified in the chicken model, only 5 were also attenuated in mice, showing for the first time the presence of host-specific virulence factors and indicating the importance of screening for attenuation in the natural host.