The capsular polysaccharides of Pasteurella multocida serotypes B and E: Structural, genetic and serological comparisons.

We describe the structural characterization of the capsular polysaccharides (CPSs) of Pasteurella multocida serotypes B and E. CPS was isolated following organic solvent precipitation of the supernatant from flask grown cells. Structural analysis utilizing nuclear magnetic resonance spectroscopy enabled the determination of the CPS structures and revealed significant structural similarities between the two serotypes, but also provided an explanation for the serological distinction. This observation was extended by the development of polyclonal sera to the glycoconjugate of serotype B CPS that corroborated the structural likenesses and differences. Finally, identification of these structures enabled a more comprehensive interrogation of the genetic loci and prediction of roles for some of the encoded proteins in repeat unit biosynthesis.

Ultrastructural changes in endothelial cells of buffaloes following in-vitro exposure to Pasteurella multocida B:2.

BACKGROUND: Pasteurella multocida B:2 causes haemorrhagic septicaemia in cattle and buffaloes. However, buffaloes are found to be more susceptible to the infection than cattle. Upon infection, the pathogen rapidly spread from the respiratory tract to the blood circulation within 16-72 h, causing septicaemia. So far, limited study has been conducted to evaluate the response of endothelial cells of buffalo towards P. multocida B:2 and its lipopolysaccharide (LPS). This study aimed to evaluate the ultrastructural changes in the aortic endothelium of buffaloes (BAEC) following exposure to P. multocida B:2 and its endotoxin. The endothelial cells were harvested from the aorta of healthy buffaloes and were prepared as monolayer cell cultures. The cultures were divided into 3 groups before Group 1 was inoculated with 107 cfu/ml of whole cell P. multocida B:2, Group 2 with LPS, which was extracted earlier from 107 cfu/ml of P. multocida B:2 and Group 3 with sterile cell culture medium. The cells were harvested at 0, 6, 12, 18, 24, 36, and 48 h post-inoculation for assessment of cellular changes using transmission electron microscopy. RESULTS: The BAEC of Groups 1 and 2 demonstrated moderate to severe endothelial lysis, suggestive of acute cellular injury. In general, severity of the ultrastructural changes increased with the time of incubation but no significant difference (p > 0.05) in the severity of the cellular changes between Groups 1 and 2 was observed in the first 18 h. The severity of lesions became significant (p < 0.05) thereafter. Both treated Groups 1 and 2 showed significantly (p < 0.05) more severe cellular changes compared to the control Group 3 from 6 h post-inoculation. The severity reached peak at the end of the study period with score 3 for Group 1 and score 2.8 for Group 2. CONCLUSIONS: This study revealed that both whole cells P. multocida B:2 and LPS endotoxin showed similar moderate to severe cellular damage, but whole-cell P. multocida B:2 appeared to be more potent in causing much severe damage than LPS alone.

Cytosolic Delivery of Multidomain Cargos by the N Terminus of Pasteurella multocida Toxin.

The zoonotic pathogen Pasteurella multocida produces a 146-kDa modular toxin (PMT) that enters host cells and manipulates intracellular signaling through action on its Gα protein targets. The N terminus of PMT (PMT-N) mediates cellular uptake through receptor-mediated endocytosis, followed by the delivery of the C-terminal catalytic domain from acidic endosomes into the cytosol. The putative native cargo of PMT consists of a 710-residue polypeptide with three distinct modular subdomains (C1-C2-C3), where C1 contains a membrane localization domain (MLD), C2 has an as-yet-undefined function, and C3 catalyzes the deamidation of a specific active-site glutamine residue in Gα protein targets. However, whether the three cargo subdomains are delivered intact or undergo further proteolytic processing during or after translocation from the late endosome is unclear. Here, we demonstrate that PMT-N mediates the delivery of its native C-terminal cargo as a single polypeptide, corresponding to C1-C2-C3, including the MLD, with no evidence of cleavage between subdomains. We show that PMT-N also delivers nonnative green fluorescent protein (GFP) cargo into the cytosol, further supporting that the receptor-binding and translocation functions reside within PMT-N. Our findings further show that PMT-N can deliver C1-C2 alone but that the presence of C1-C2 is important for the cytosolic delivery of the catalytic C3 subdomain by PMT-N. In addition, we further refine the minimum C3 domain required for intracellular activity as comprising residues 1105 to 1278. These findings reinforce that PMT-N serves as the cytosolic delivery vehicle for C-terminal cargo and demonstrate that its native cargo is delivered intact as C1-C2-C3.

The Myriad Properties of Pasteurella multocida Lipopolysaccharide.

Pasteurella multocida is a heterogeneous species that is a primary pathogen of many different vertebrates. This Gram-negative bacterium can cause a range of diseases, including fowl cholera in birds, haemorrhagic septicaemia in ungulates, atrophic rhinitis in swine, and lower respiratory tract infections in cattle and pigs. One of the primary virulence factors of P. multocida is lipopolysaccharide (LPS). Recent work has shown that this crucial surface molecule shows significant structural variability across different P. multocida strains, with many producing LPS structures that are highly similar to the carbohydrate component of host glycoproteins. It is likely that this LPS mimicry of host molecules plays a major role in the survival of P. multocida in certain host niches. P. multocida LPS also plays a significant role in resisting the action of chicken cathelicidins, and is a strong stimulator of host immune responses. The inflammatory response to the endotoxic lipid A component is a major contributor to the pathogenesis of certain infections. Recent work has shown that vaccines containing killed bacteria give protection only against other strains with identical, or nearly identical, surface LPS structures. Conversely, live attenuated vaccines give protection that is broadly protective, and their efficacy is independent of LPS structure.

Covalent inhibitors of LgtC: A blueprint for the discovery of non-substrate-like inhibitors for bacterial glycosyltransferases.

Non-substrate-like inhibitors of glycosyltransferases are sought after as chemical tools and potential lead compounds for medicinal chemistry, chemical biology and drug discovery. Here, we describe the discovery of a novel small molecular inhibitor chemotype for LgtC, a retaining α-1,4-galactosyltransferase involved in bacterial lipooligosaccharide biosynthesis. The new inhibitors, which are structurally unrelated to both the donor and acceptor of LgtC, have low micromolar inhibitory activity, comparable to the best substrate-based inhibitors. We provide experimental evidence that these inhibitors react covalently with LgtC. Results from detailed enzymological experiments with wild-type and mutant LgtC suggest the non-catalytic active site residue Cys246 as a likely target residue for these inhibitors. Analysis of available sequence and structural data reveals that non-catalytic cysteines are a common motif in the active site of many bacterial glycosyltransferases. Our results can therefore serve as a blueprint for the rational design of non-substrate-like, covalent inhibitors against a broad range of other bacterial glycosyltransferases.

The ability of lipopolysaccharide (LPS) of Pasteurella multocida B:2 to induce clinical and pathological lesions in the nervous system of buffalo calves following experimental inoculation.

Lipopolysaccharide (LPS) of P. multocida B:2, a causative agent of haemorrhagic septicaemia (HS) in cattle and buffaloes, is considered as the main virulence factor and contribute in the pathogenesis of the disease. Recent studies provided evidences about the involvement of the nervous system in pathogenesis of HS. However, the role of P. multocida B:2 immunogens, especially the LPS is still uncovered. Therefore, this study was designed to investigate the role of P. multocida B:2 LPS to induce pathological changes in the nervous system. Nine eight-month-old, clinically healthy buffalo calves were used and distributed into three groups. Calves of Group 1 and 2 were inoculated orally and intravenously with 10 ml of LPS broth extract represent 1 × 1012 cfu/ml of P. multocida B:2, respectively, while calves of Group 3 were inoculated orally with 10 ml of phosphate buffer saline as a control. Significant differences were found in the mean scores for clinical signs, post mortem and histopathological changes especially in Group 2, which mainly affect different anatomic regions of the nervous system, mainly the brain. On the other hand, lower scores have been recorded for clinical signs, gross and histopathological changes in Group 1. These results provide for the first time strong evidence about the ability of P. multocida B:2 LPS to cross the blood brain barrier and induce pathological changes in the nervous system of the affected buffalo calves.

The membrane localization domains of two distinct bacterial toxins form a 4-helix-bundle in solution.

Membrane localization domain (MLD) was first proposed for a 4-helix-bundle motif in the crystal structure of the C1 domain of Pasteurella multocida toxin (PMT). This structure motif is also found in the crystal structures of several clostridial glycosylating toxins (TcdA, TcdB, TcsL, and TcnA). The Ras/Rap1-specific endopeptidase (RRSP) module of the multifunctional autoprocessing repeats-in-toxins (MARTX) toxin produced by Vibrio vulnificus has sequence homology to the C1-C2 domains of PMT, including a putative MLD. We have determined the solution structure for the MLDs in PMT and in RRSP using solution state NMR. We conclude that the MLDs in these two toxins assume a 4-helix-bundle structure in solution.

Pasteurella multocida toxin: Targeting mast cell secretory granules during kiss-and-run secretion.

Pasteurella multocida toxin (PMT), a virulence factor of the pathogenic Gram-negative bacterium P. multocida, is a 146 kDa protein belonging to the A-B class of toxins. Once inside a target cell, the A domain deamidates the α-subunit of heterotrimeric G-proteins, thereby activating downstream signaling cascades. However, little is known about how PMT selects and enters its cellular targets. We therefore studied PMT binding and uptake in porcine cultured intestinal mucosal explants to identify susceptible cells in the epithelium and underlying lamina propria. In comparison with Vibrio cholera B-subunit, a well-known enterotoxin taken up by receptor-mediated endocytosis, PMT binding to the epithelial brush border was scarce, and no uptake into enterocytes was detected by 2h, implying that none of the glycolipids in the brush border are a functional receptor for PMT. However, in the lamina propria, PMT distinctly accumulated in the secretory granules of mast cells. This also occurred at 4 °C, ruling out endocytosis, but suggestive of uptake via pores that connect the granules to the cell surface. Mast cell granules are known to secrete their contents by a "kiss-and-run" mechanism, and we propose that PMT may exploit this secretory mechanism to gain entry into this particular cell type.

Structural analysis of lipopolysaccharide produced by Heddleston serovars 10, 11, 12 and 15 and the identification of a new Pasteurella multocida lipopolysaccharide outer core biosynthesis locus, L6.

Pasteurella multocida is a Gram-negative bacterial pathogen classified into 16 serovars based on lipopolysaccharide (LPS) antigens. Previously, we have characterized the LPS outer core biosynthesis loci L1, L2, L3, L5 and L7, and have elucidated the full range of LPS structures associated with each. In this study, we have determined the LPS structures produced by the type strains representing the serovars 10, 11, 12 and 15 and characterized a new LPS outer core biosynthesis locus, L6, common to all. The L6 outer core biosynthesis locus shares significant synteny with the L3 locus but due to nucleotide divergence, gene duplication and gene redundancy, the L6 and L3 LPS outer cores are structurally distinct. Using LPS structural and genetic differences identified in each L6 strain, we have predicted a role for most of the L6 glycosyltransferases in LPS assembly. Importantly, we have identified two glycosyltransferases, GctD and GatB, that differ by one amino acid, A162T, but use different donor sugars [uridine diphosphate (UDP)-Glc and UDP-Gal, respectively]. The longest outer core oligosaccharide, produced by the serovar 12 type strain, contained a terminal region consisting of ß-Gal-(1,4)-ß-GlcNAc-(1,3)-ß-Gal-(1,4)-ß-Glc that was identical in structure to the vertebrate glycosphingolipid, paragloboside. Mimicry of host glycosphingolipids has been observed previously in P. multocida strains belonging to L3 LPS genotype, which produce LPS similar in structure to the globo-series of glycosphingolipids. The expression of a paragloboside-like oligosaccharide on the LPS produced by the serovar 12 type strain indicates that strains belonging to the L6 LPS genotype may also engage in molecular mimicry.

Production methods for heparosan, a precursor of heparin and heparan sulfate.

Heparin and heparan sulfate belong to the glycosaminoglycan family. Heparin which is known as a powerful anticoagulant has been also described to have potential in therapeutic applications such as in the treatment against cancer and prevention of virus infections. Heparan sulfate, an analog of heparin, which is not used for medical purposes yet, was reported to have the same pharmaceutical potential as heparin. Both heparin and heparan sulfate share a common precursor molecule known as heparosan. Heparosan determines the polymer chain length and the sugar unit backbone composition, which are determinant structural parameters for the biological activity of heparin and heparan sulfate. In this review we give an overview of the different methods used to synthesize heparosan, and we highlight the pro and cons of each method in respect to the synthesis of bioengineered heparin-like molecules. Advancements in the field of the synthesis of bioengineered heparin are also reported.

Structure and biosynthetic locus of the lipopolysaccharide outer core produced by Pasteurella multocida serovars 8 and 13 and the identification of a novel phospho-glycero moiety.

Pasteurella multocida strains are classified into 16 Heddleston serovars on the basis of the lipopolysaccharide (LPS) antigens expressed on the surface of the bacteria. The LPS structure and the corresponding LPS outer core biosynthesis loci of strains belonging to serovars 1, 2, 3, 5, 9 and 14 have been characterized, revealing a clear structural basis for serovar classification. However, several of these serovars are genetically related, sharing the same LPS outer core biosynthesis locus, but producing different LPS molecules as a result of mutations within LPS assembly genes. In this article, we report that the P. multocida type strains belonging to serovars 8 and 13 share the same LPS outer core biosynthesis locus and produce structurally related LPS molecules. Structural analysis of the serovar 8 LPS revealed an inner core that is conserved among P. multocida strains and the following outer core structure: X-(1-6)-(1S)GalaNAC-(1-4-6)-α-Gal-(1-3)-ß-Gal(PEtn)-(1-4)-L,D-α-Hep-(1-6) where X is a unique phospho-glycero moiety, 1-((4-aminobutyl)amino)-3-hydroxy-1-oxopropan-2-yl hydrogen phosphate, attached to the sixth position of (1S)GalaNAc. For serovar 13, the LPS structure is the same except for the absence of the terminal phospho-glycero moiety. Analysis of the common outer core biosynthesis locus from the serovar 8 and 13 type strains identified three genes that we predict are involved in the biosynthesis of this terminal moiety. Furthermore, bioinformatic comparisons with the characterized LPS outer core glycosyltransferases from Actinobacillus pleuropneumoniae serovar 1, strain 4074, allowed us to assign a function for each of the glycosyltransferases encoded within the serovar 8/13 LPS outer core biosynthesis locus.

Inhibition of protein synthesis on the ribosome by tildipirosin compared with other veterinary macrolides.

Tildipirosin is a 16-membered-ring macrolide developed to treat bacterial pathogens, including Mannheimia haemolytica and Pasteurella multocida, that cause respiratory tract infections in cattle and swine. Here we evaluated the efficacy of tildipirosin at inhibiting protein synthesis on the ribosome (50% inhibitory concentration [IC(50)], 0.23 ± 0.01 µM) and compared it with the established veterinary macrolides tylosin, tilmicosin, and tulathromycin. Mutation and methylation at key rRNA nucleotides revealed differences in the interactions of these macrolides within their common ribosomal binding site.

Predicting the outer membrane proteome of Pasteurella multocida based on consensus prediction enhanced by results integration and manual confirmation.

BACKGROUND: Outer membrane proteins (OMPs) of Pasteurella multocida have various functions related to virulence and pathogenesis and represent important targets for vaccine development. Various bioinformatic algorithms can predict outer membrane localization and discriminate OMPs by structure or function. The designation of a confident prediction framework by integrating different predictors followed by consensus prediction, results integration and manual confirmation will improve the prediction of the outer membrane proteome. RESULTS: In the present study, we used 10 different predictors classified into three groups (subcellular localization, transmembrane ß-barrel protein and lipoprotein predictors) to identify putative OMPs from two available P. multocida genomes: those of avian strain Pm70 and porcine non-toxigenic strain 3480. Predicted proteins in each group were filtered by optimized criteria for consensus prediction: at least two positive predictions for the subcellular localization predictors, three for the transmembrane ß-barrel protein predictors and one for the lipoprotein predictors. The consensus predicted proteins were integrated from each group into a single list of proteins. We further incorporated a manual confirmation step including a public database search against PubMed and sequence analyses, e.g. sequence and structural homology, conserved motifs/domains, functional prediction, and protein-protein interactions to enhance the confidence of prediction. As a result, we were able to confidently predict 98 putative OMPs from the avian strain genome and 107 OMPs from the porcine strain genome with 83% overlap between the two genomes. CONCLUSIONS: The bioinformatic framework developed in this study has increased the number of putative OMPs identified in P. multocida and allowed these OMPs to be identified with a higher degree of confidence. Our approach can be applied to investigate the outer membrane proteomes of other Gram-negative bacteria.

Pasteurella multocida CMP-sialic acid synthetase and mutants of Neisseria meningitidis CMP-sialic acid synthetase with improved substrate promiscuity.

Cytidine 5'-monophosphate (CMP)-sialic acid synthetases (CSSs) catalyze the formation of CMP-sialic acid from CTP and sialic acid, a key step for sialyltransferase-catalyzed biosynthesis of sialic acid-containing oligosaccharides and glycoconjugates. More than 50 different sialic acid forms have been identified in nature. To facilitate the enzymatic synthesis of sialosides with diverse naturally occurring sialic acid forms and their non-natural derivatives, CMP-sialic acid synthetases with promiscuous substrate specificity are needed. Herein we report the cloning, characterization, and substrate specificity studies of a new CSS from Pasteurella multocida strain P-1059 (PmCSS) and a CSS from Haemophillus ducreyi (HdCSS). Based on protein sequence alignment and substrate specificity studies of these two CSSs and a Neisseria meningitidis CSS (NmCSS), as well as crystal structure modeling and analysis of NmCSS, NmCSS mutants (NmCSS_S81R and NmCSS_Q163A) with improved substrate promiscuity were generated. The strategy of combining substrate specificity studies of enzymes from different sources and protein crystal structure studies can be a general approach for designing enzyme mutants with improved activity and substrate promiscuity.

Membrane interaction of Pasteurella multocida toxin involves sphingomyelin.

Pasteurella multocida toxin (PMT) is an AB toxin that causes pleiotropic effects in targeted host cells. The N-terminus of PMT (PMT-N) is considered to harbor the membrane receptor binding and translocation domains responsible for mediating cellular entry and delivery of the C-terminal catalytic domain into the host cytosol. Previous studies have implicated gangliosides as the host receptors for PMT binding. To gain further insight into the binding interactions involved in PMT binding to cell membranes, we explored the role of various membrane components in PMT binding, utilizing four different approaches: (a) TLC-overlay binding experiments with (125) I-labeled PMT, PMT-N or the C-terminus of PMT; (b) pull-down experiments using reconstituted membrane liposomes with full-length PMT; (c) surface plasmon resonance analysis of PMT-N binding to reconstituted membrane liposomes; (d) and surface plasmon resonance analysis of PMT-N binding to HEK-293T cell membranes without or with sphingomyelinase, phospholipase D or trypsin treatment. The results obtained revealed that, in our experimental system, full-length PMT and PMT-N did not bind to gangliosides, including monoasialogangliosides GM(1) , GM(2) or GM(3) , but instead bound to membrane phospholipids, primarily the abundant sphingophospholipid sphingomyelin or phosphatidylcholine with other lipid components. Collectively, these studies demonstrate the importance of sphingomyelin for PMT binding to membranes and suggest the involvement of a protein co-receptor.

Pasteurella multocida lipopolysaccharide: the long and the short of it.

Pasteurella multocida is a capsulated, gram-negative cocco-bacillus that can cause serious disease in a wide range of mammals and birds. P. multocida strains are classified into 16 serovars based on lipopolysaccharide (LPS) antigens. LPS is an essential virulence factor of P. multocida; mutants expressing severely truncated LPS are completely attenuated in chickens. LPS is also a major immunogen of P. multocida and protection against infections caused by P. multocida is generally considered to be serovar specific. In this review we summarize current knowledge of the structure and genetics of LPS assembly of P. multocida strains belonging to five different serovars. These include strains belonging to serovars 1 and 3, the most common serovars found in the poultry industry, and strains belonging serovars 2 and 5, the serovars associated with bovine haemorrhagic septicaemia outbreaks. A number of the serovars are genetically related; serovars 1 and 14 share the same LPS outer core biosynthesis locus, but due to a mutation within the phosphocholine biosynthesis gene, pcgA, the serovar 14 strain produces a truncated LPS structure. Similarly serovars 2 and 5 share an identical LPS outer core locus and express near-identical LPS structures. However, due to a single point mutation in the phosphoethanolamine (PEtn) transferase gene, lpt_3, the serovar 2 strain does not elaborate a PEtn residue on heptose II. Knowledge of the genetic basis for the LPS structures expressed by P. multocida will facilitate the development of rapid molecular methods for typing and diagnosis and will be essential for a rational approach to vaccine formulation.

A novel Erm monomethyltransferase in antibiotic-resistant isolates of Mannheimia haemolytica and Pasteurella multocida.

Mannheimia haemolytica and Pasteurella multocida are aetiological agents commonly associated with respiratory tract infections in cattle. Recent isolates of these pathogens have been shown to be resistant to macrolides and other ribosome-targeting antibiotics. Direct analysis of the 23S rRNAs by mass spectrometry revealed that nucleotide A2058 is monomethylated, consistent with a Type I erm phenotype conferring macrolide-lincosamide resistance. The erm resistance determinant was identified by full genome sequencing of isolates. The sequence of this resistance determinant, now termed erm(42), has diverged greatly from all previously characterized erm genes, explaining why it has remained undetected in PCR screening surveys. The sequence of erm(42) is, however, completely conserved in six independent M. haemolytica and P. multocida isolates, suggesting relatively recent gene transfer between these species. Furthermore, the composition of neighbouring chromosomal sequences indicates that erm(42) was acquired from other members of the Pasteurellaceae. Expression of recombinant erm(42) in Escherichia coli demonstrated that the enzyme retains its properties as a monomethyltransferase without any dimethyltransferase activity. Erm(42) is a novel addition to the Erm family: it is phylogenetically distant from the other Erm family members and it is unique in being a bona fide monomethyltransferase that is disseminated between bacterial pathogens.

Crystal structure and immunogenicity of the class C acid phosphatase from Pasteurella multocida.

Pasteurella multocida is a pathogen of veterinary and medical importance. Here, we report the 1.85Å resolution crystal structure of the class C acid phosphatase from this organism (denoted rPmCCAP). The structure shows that rPmCCAP exhibits the same haloacid dehalogenase fold and dimeric assembly as the class C enzyme from Haemophilus influenzae. Formation of the dimer in solution is demonstrated using analytical ultracentrifugation. The active site is devoid of a magnesium ion due to the presence of citrate in the crystallization buffer. Absence of the metal ion minimally perturbs the active site structure, which suggests that the main role of the ion is to balance the negative charge of the substrate rather than stabilize the active site structure. The crystal lattice displays unusual crystal packing involving the C-terminal polyhistidine tag mimicking the substrate. Steady-state kinetic constants are determined for the substrates NMN, 5'-AMP, 3'-AMP, 2'-AMP, and p-nitrophenyl phosphate. The highest catalytic efficiency is observed with NMN. The production of polyclonal anti-rPmCCAP antibodies is demonstrated, and these antibodies are shown to cross-react with the H. influenzae class C phosphatase. The antibodies are used to detect PmCCAP in clinical P. multocida and Mannheimia haemolytica strains cultured from infected animals.

Pasteurella multocida Heddleston serovars 1 and 14 express different lipopolysaccharide structures but share the same lipopolysaccharide biosynthesis outer core locus.

Pasteurella multocida strains are classified using the Heddleston lipopolysaccharide (LPS) serotyping scheme into 16 serovars. Understanding the structural and genetic basis for this LPS typing scheme is important because protection against infections caused by P. multocida is generally considered to be serovar specific. Here we show that the serovar 14 type strain P2225 and the serovar 1 strains X73 and VP161 express similar LPS structures. However, the serovar 14 LPS lacks the terminal phosphocholine (PCho) residues present on the serovar 1 LPS and contains the 1,4-linked ß-galactose but not the 1,6-linked ß-galactose. Sequencing analysis of the LPS biosynthesis outer core loci of P2225 and the serovar 1 type strain X73 showed that they were nearly identical. However, the phosphocholine biosynthesis gene, pcgA of P2225 contained a 19bp nucleotide deletion. Complementation of P2225 with an intact pcgA resulted in an LPS structure identical to that expressed by serovar 1 strain VP161 and highly similar to that expressed by strain X73, with a 1,6-linked ß-galactose and both terminal PCho residues. This study has shown unequivocally that strains belonging to serovar 1 and 14 share a common LPS outer core locus and that minor changes within this locus can dramatically alter the LPS structure expressed on the surface of P. multocida, and thus has implications into our understanding of the potential to generate cross-protective vaccines.

[Expression and purification of an adhesive protein of rabbit Pasteurella multocida C51-3 and detection of its antigenicity].

The cp36 gene encoding an adhesive protein was amplified by PCR from genomic DNA of rabbit P. multocida C51-3 strain, and cloned into the pMD18-T vector and then sequenced. The mature adhesive protein without a signal peptide of cpm36 gene was amplified by PCR from the recombinant plasmid pMD18-cp36, then cloned into the prokaryotic expression vector pQE30 to provide a recombinant plasmid pQE30-cpm36. The recombinant protein of CPM36 was produced in Escherichia coli M15 harboring the recombinant plasmid pQE30-cpm36 by IPTG induction, and the recombinant protein purified by the affinity chromatography with Ni(2+)-NTA resin. The sequence analyses showed that the ORF of cp36 gene was 1032 bp in length, and DNA homology of the cp36 genes between the C51-3 strain and the previously reported different serotype strains of P. multocida in GenBank was 76.9 to 100%. The SDS-PAGE analyses revealed a single fusion protein band with a molecular weight of 37 kD, and the Western blotting analysis demonstrated that the recombinant protein CPM36 and native 36 kD protein of C51-3 were recognized specifically by an antiserum against the recombinant protein, suggesting that the recombinant protein is an antigenic protein.