Characterization of the lipopolysaccharide produced by Pasteurella multocida serovars 6, 7 and 16: identification of lipopolysaccharide genotypes L4 and L8.

Glycobiology Advance Access published October 7, 2014

1 Characterisation of the lipopolysaccharide produced by Pasteurella multocida serovars 6, 7 and 16; identification of lipopolysaccharide genotypes L4 and L8

Marina Harper1,2, Frank St. Michael3, Jason A. Steen2,5, Marietta John4, Amy Wright4, Lieke van Dorsten3,6, Evgeny Vinogradov3, Ben Adler2, Andrew D. Cox3,† and John D. Boyce2,4,† 2

Australian Research Council Centre of Excellence in Structural and Functional Microbial

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Vaccine Program, Human Health Therapeutics Portfolio, National Research Council,

Ottawa, ON, Canada. K1A 0R6 4

Department of Microbiology, Monash University, Clayton, Victoria 3800, Australia

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Current address: The University of Queensland, St. Lucia, Queensland, Australia 4067

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Current address: Eppendorf Nederland B.V., Nijmegen, Netherlands

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Corresponding Author: Dr. Marina Harper, TEL: +61 3 9902 9184, Department of

Microbiology, FAX: +61 3 9902 9222, Building 76, Monash University, E-mail: [email protected], Clayton, Victoria, Australia, 3800 †

Joint senior author

Keywords: Core oligosaccharide/ Genetics/ LPS/ Pasteurella multocida/ Structure.

© The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected]

Downloaded from http://glycob.oxfordjournals.org/ at Nipissing University on October 16, 2014

Genomics, Monash University, VIC 3800, Australia

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Abstract Pasteurella multocida is an important veterinary pathogen that produces a wide range of lipopolysaccharide (LPS) structures, many of which mimic host glycoproteins. In this study, we complete our analysis of the LPS produced by the P. multocida Heddleston serovars by reporting the LPS structure and the LPS outer core biosynthesis loci of the type strains representing Heddleston serovars 6, 7 and 16. Genetic analysis revealed that the type strains

have designated LPS genotype L4. Comparative bioinformatic analysis revealed that although the serovar 16 type strain contained a different LPS locus, L8, there was a significant degree of nucleotide identity between the L4 and L8 loci. Structural analysis revealed that the LPS glycoforms produced by the L4 and L8 strains all contained the highly conserved inner core produced by all other P. multocida strains examined to date. The residues within the LPS outer core produced by the L4 and L8 strains were either Gal or derivatives of Gal; unlike all other P. multocida Heddleston type strains examined there are no heptosyltransferases encoded in the L4 and L8 outer core biosynthesis loci. The structure of the L4 LPS outer core produced by the serovar 6 type strain consisted of -Gal-(1-3)-GalNAc-(1-4)--GalNAc3OAc-(1-4)--GalNAc3OAc-(1-3)--Gal, whereas the serovar 7 type strain produced a highly truncated LPS outer core containing only a single -Gal residue. The structure of the L8 LPS outer core produced by the serovar 16 type strain consisted of -Gal-(1-3)--GalNAc-(1-4)-(-GalNAc-(1-3)-)--GalNAc.


representing serovars 6 and 7 share the same LPS outer core biosynthesis locus which we

3 Introduction Pasteurella multocida is a Gram-negative bacterium that is the causative agent of many serious diseases including avian fowl cholera, bovine hemorrhagic septicemia, atrophic rhinitis in pigs and respiratory diseases in sheep, cattle and rabbits (Wilkie et al., 2012). P. multocida isolates have traditionally been classified into 5 serogroups, A, B, D, E and F, based on capsular antigens (Carter, 1955) and into 16 Heddleston serovars (1-16) based on lipopolysaccharide (LPS) antigens (Heddleston et al., 1972).

determined that P. multocida produces LPS that lacks a polymeric O-antigen but instead produces a highly variable outer core region (Harper et al., 2011a). Although most of the Heddleston type strains produce structurally distinct LPS outer core regions, a number of the strains share the same LPS outer core biosynthesis locus (Harper et al., 2014; Harper et al., 2011b; Harper et al., 2013a; Harper et al., 2013b; St Michael et al., 2009). The different LPS structures produced by strains with the same LPS genotype is usually the result of mutations within the LPS outer core biosynthesis locus resulting in the inactivation of key LPS assembly/biosynthesis genes. As a result, strains containing these mutations typically produce a LPS with a truncated outer core compared to the "parent" or full-length structure. In this study we complete our comprehensive LPS structural and genetic analysis of the P. multocida Heddleston serovars. We report the structure for the LPS core oligosaccharides produced by the type strains representing serovars 6, 7 and 16 and identify two new outer core biosynthesis loci, L4 and L8, which encode the glycosyltransferases required for the outer core assembly in these strains. The Heddleston serotyping system was established in 1972 and initially identified 15 antigenically distinct serovars. The last of these, serovar 16, was identified in 1978 and was represented by only a single turkey isolate, type strain P2723 (Brogden et al., 1978). We show through comparative bioinformatic


Our previous studies on the structure and genetics of P. multocida LPS have

4 analysis that the L8 locus on the genome of P2723 is related to the L4 locus and to the previously characterized L3 locus (Harper et al., 2013a), suggesting that a recombination event between L3 and L4 ancestral strains led to the genesis of the L8 LPS outer core biosynthesis locus.

Results LPS sugar analyses

(Glc), galactose (Gal) and L-glycero-D-manno-heptose (LD-Hep) in the approximate ratio of 2:1:3. For the serovar 6 type strain, P2192, sugar analysis of the purified LPS revealed glucose (Glc), galactose (Gal), galactosamine (GalN) and L-glycero-D-manno-heptose (LDHep) in the approximate ratio of 2:2:3:3. For serovar 16 type strain, P2723, sugar analysis of the purified LPS revealed glucose (Glc), galactose (Gal), galactosamine (GalN) and Lglycero-D-manno-heptose (LD-Hep) in the approximate ratio of 2:3:2:2 respectively. Small amounts of glucosamine (GlcN) were also identified in the purified LPS obtained from each serovar type strain and were presumably derived from the lipid A. Capillary electrophoresis-electrospray-mass spectrometry analyses Capillary electrophoresis-electrospray-mass spectrometry (CE-ES-MS) analyses of Odeacylated LPS (LPS-OH) from the serovar 6 type strain revealed a major triply charged ion at m/z 1028.23- which was assigned a composition of 3HexNAc, 4Hex, 3Hep, Kdo-P, Lipid A-OH (Table I). Smaller amounts of larger glycoforms (m/z 1069.23- and 1110.23-) consistent with the addition of phosphoethanolamine (PEtn) residues and glycoforms (m/z 1022.03- and 1062.83-) consistent with 2Kdo, 3Hex glycoforms were also observed. Similarly, mass spectrometry (MS) analysis on the non-fractionated core oligosaccharides (OS) suggested a composition of 3HexNAc, 4Hex, 3Hep, Kdo and 3HexNAc, 3Hex, 3Hep, Kdo as the major


Sugar analysis of the purified LPS from the serovar 7 type strain, P1997, revealed glucose

5 glycoforms consistent with the LPS-OH MS data (Table I). PEtn was observed in approximately 50% of the core OS glycoforms. Two O-acetyl groups were also identified in CE-MS analysis of the core OS (Table I). CE-ES-MS analyses of LPS-OH from serovar 7 type strain revealed a major doubly charged ion at m/z 1157.32- which was assigned a composition of 3Hex, 3Hep, Kdo-P, Lipid A-OH (Table I). Smaller amounts of larger glycoforms (m/z 1218.82- and 1280.32-) consistent with additional PEtn residues and glycoforms (m/z 1145.82-and 1207.82-) consistent with

core OS suggested a composition of 3Hex, 3Hep, Kdo and 2Hex, 3Hep, Kdo as the major glycoforms consistent with the LPS-OH MS data (Table I). PEtn was observed in approximately 50% of the core OS glycoforms. LPS-OH from serovar 16 type strain was analysed by CE-ES-MS revealing a major triply charged ion at m/z 1055.83- corresponding to a composition of 5Hex, 2HexNAc, 3Hep, PEtn, Kdo-P, Lipid A-OH with smaller amounts of a glycoform with an additional PEtn and also a glycoform with no PEtn. Similarly, MS analysis on the non-fractionated core OS revealed a major doubly charged ion at m/z 1067.32- corresponding to a composition of 5Hex, 2HexNAc, 3Hep, PEtn, aKdo, with smaller amounts of a glycoform with only 4Hex residues. MS/MS studies were performed on the core OS from serovar 16 in positive ion mode on the ions at m/z 1069.32+, corresponding to the complete glycoform (Figure 1). A series of ions was sequentially lost from the molecular ion as indicated in Figure 1a. A series of ions was also observed building up from the singly charged ion at m/z 204.0+ that corresponds to a HexNAc residue (Figure 1b). The MS/MS data therefore indicated that the outer core was composed of the following sequence of sugar residues Hex-Hex-(HexNAc)-HexNAc-Hex extending from the first Glc residue.


2Kdo, 2Hex glycoforms were also observed. Similarly, MS analysis on the non-fractionated

6 Methylation analyses on fractionated core OS Methylation analysis was performed on the fractionated core OS [Fraction (Fr.) 18] from serovar 6 in order to determine the linkage pattern of the molecule; it revealed the presence of terminal Glc, terminal Gal, 4-substituted Glc, 3-substituted Gal, terminal LD-Hep, 4substituted GalNAc and 3,4,6-trisubsituted LD-Hep in approximately equimolar amounts. Smaller amounts of 2-substituted LD-Hep, 3,4 -disubstituted LD-Hep, 3,4,6-trisubsituted LDHep and 3-substituted GalNAc were also observed. Methylation analysis on the fractionated

substituted Glc and terminal LD-Hep in approximately equimolar amounts. Smaller amounts of 2-substituted LD-Hep, 3,4 -disubstituted LD-Hep and 3,4,6-trisubsituted LD-Hep were also observed. Methylation analysis was performed on the fractionated core OS (Frs. 13-15) from serovar 16, revealing the presence of terminal Glc, terminal Gal, 4-substituted Glc, 4substituted Gal, 3-substituted Gal and terminal LD-Hep in approximately equimolar amounts, with 2-substituted LD-Hep, 4, 6-disubstituted LD-Hep, 3,4,6-trisubsituted LD-Hep, terminal GalNAc as well as a 3,4-disubstituted GalNAc identified in lower amounts. Nuclear magnetic resonance studies; serovar 6 In order to elucidate the exact locations and linkage patterns of the LPS from serovar 6, nuclear magnetic resonance (NMR) studies were initially performed on completely deacylated LPS. The assignment of 1H resonances of the inner core residues for each serovar was achieved by correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY) and nuclear Overhauser effect spectroscopy (NOESY) experiments with reference to the published data for the structurally related oligosaccharides from previously analyzed P. multocida serovars (St Michael et al., 2009; St Michael et al., 2005a; St Michael et al., 2005b; St Michael et al., 2005c) and revealed that the conserved inner core structure (Hep IIII and Glc I-II) was present (Supplementary Table I). The non-stoichiometric


core OS (Fr. 15) from serovar 7 revealed the presence of terminal Glc, terminal Gal, 4-

7 phosphorylation at Hep II inferred from MS analyses was also confirmed from the identification of two sets of signals for the Hep II residue. Similarly, the assignment of 13C resonances of the samples was achieved by virtue of 13C-1H HSQC and 13C-1H HSQCTOCSY experiments (data not shown) (Supplementary Table I). The outer core residues of the oligosaccharide extension beyond the inner core in serovar 6 LPS were characterized and their resonances assigned by COSY, TOCSY and NOESY experiments (Supplementary Table I). In addition to the residues of the conserved inner core structure, two Gal and three

1

H-resonances. Their linkages were assigned based on inter-NOE connectivities and found to

be consistent with the methylation analyses. Their 13C resonances were determined by virtue of 13C-1H HSQC and 13C-1H HSQC-TOCSY experiments (data not shown). Due to considerable overlap in the -anomeric region of the completely deacylated LPS NMR spectrum, coupled with the identification of O-acetyl residues in MS analyses of the core OS, we decided to also analyse the NMR spectra of the core OS from serovar 6 (Supplementary Table II) (Figure 2). These analyses confirmed and extended the NMR analyses performed on the completely deacylated LPS. O-acetyl groups were identified at 3positions of both the GalNAc I and GalNAc II residues by virtue of the large down field shift of the H-3 resonances from 3.91 to 5.39 ppm and from 3.68 to 5.27 ppm respectively when comparing the completely deacylated LPS to core OS chemical shifts (Figure 2). Nuclear magnetic resonance studies; serovar 7 NMR analyses of the core OS from the serovar 7 LPS revealed the same inner core oligosaccharide as identified in serovar 6 (Supplementary Table II). The only residue distal to this inner core in serovar 7 was a terminal Gal residue. An inter-NOE connectivity from the anomeric proton of this galactose residue was assigned as the proton at the 4-position of the Glc I residue (Supplementary Table II).


galactosamine (GalN) residues were identified from spin systems arising from their anomeric

8 Nuclear magnetic resonance studies; serovar 16 For serovar 16, NMR studies were performed on the core OS fraction that gave the most resolved and homogeneous spectrum (Fr. 13-15). The assignment of 1H resonances of the inner core oligosaccharide revealed that the conserved inner core structure (Hep I-III and Glc I-II) was present for serovar 16 (Supplementary Table III). Similarly, the assignment of 13C resonances of the samples was achieved by virtue of 13C-1H HSQC and 13C-1H HSQCTOCSY experiments (data not shown) (Supplementary Table III).

serovar 16 were characterized by COSY, TOCSY and NOESY experiments (Figure 3). In addition to the residues of the conserved inner core structure, three Gal and two N-acetyl galactosamine (GalNAc) residues were identified. Their linkages were assigned based on inter-NOE connectivities and found to be consistent with the methylation analyses. Their 13C resonances were determined by virtue of 13C-1H HSQC and 13C-1H HSQC-TOCSY experiments (data not shown). Confirmation of the 3-position of Hep II as the location of PEtn substitution for all serovars were obtained from 31P-1H-HSQC and 31P-1H-HSQC-TOCSY experiments on the core oligosaccharides (data not shown). The intensity of resonances for the anomeric resonances of the Hep II residues indicated that in all serovars, consistent with the MS data, the PEtn residues were present non-stoichiometrically. Analyses of the L4 outer core biosynthesis locus in the serovars 6 and 7 type strains. Nucleotide sequencing and bioinformatic analysis of the L4 LPS outer core biosynthesis locus in the serovar 6 type strain P2192, revealed that the locus contained five open reading frames predicted to be involved in L4 LPS assembly that were named gatL, latB, gatK, natD and natE (Figure 4). The latB gene encodes a protein belonging to the acyltransferase 3 superfamily and shares 36% amino acid identity with LatA, a predicted acetyltranferase


The outer core residues of the oligosaccharide extension beyond the inner core in

9 encoded within the P. multocida L5 LPS outer core biosynthesis locus (Harper et al., 2012). LatB also shares 34% identity with the Haemophilus influenzae O-acetylase, OafA, which is responsible for the addition of an O-acetyl group to the distal heptose of the inner-core LPS (Fox et al., 2005). We therefore predict that LatB is responsible for the O-acetylation of two of the three GalNAc residues in the L4 LPS outer core (Figure 4, Tables I and III). The gatK gene encodes a family 25 glycosyltransferase with 58% identity to the β-1,4 galactosyltransferase, LbgA, from Haemophilus ducreyi and 48% identity to the β-1,4

Glc or Gal to the 4 position of Glc I (Deadman et al., 2009; Post et al., 2007). As the LPS inner cores produced by P. multocida and Haemophilus spp. are highly similar (Melaugh et al., 1992; Phillips et al., 1992)(Figure 4) we predict that GatK is a β-1,4 galactosyltransferase required for the addition of Gal I to the 4 position of Glc I in the L4 LPS molecule. Nucleotide sequencing of the LPS outer core biosynthesis loci in the serovar 6 and 7 type strains revealed that they were identical with the exception of just two nucleotide changes in the serovar 7 LPS locus. The first nucleotide change was located in natD (A362G) and resulted in a single amino acid substitution (N121S). The second nucleotide change was located in natE (C223T) and resulted in an immediate stop codon and early termination of translation. Together with the structural data that showed that the L7 LPS outer core consisted of a single 1,4 linked β-Gal residue (Figure 4), these nucleotide changes indicate that the glycosyltransferase required for the addition of one or both O-acetylated N-acetylgalactosamine residues was either NatE or NatD. NatD shares identity with β-1,4-Nacetylgalactosaminyltransferases (82/CgtA superfamily) including the CgtA glycosyltransferases from Helicobacter pylori and Campylobacter jejuni (39% and 38% identity respectively) (Gilbert et al., 2000). In contrast, bioinformatic analysis of NatE revealed only a partial glycosyltransferase group 1 domain at the C-terminal end (amino acids


hexosyltransferase, Lex2B, from H. influenzae. In Haemophilus, LbgA and Lex2B add either

10 650-1188). The gatL gene encoded a family 2 glycosyltransferase with 48% amino acid identity to NatB, the 1,3 N-acetyl galactosyltransferase responsible for the transfer of β-GalNAc I to the -Gal II in L3 LPS assembly (Figure 4)(Harper et al., 2013a). Analyses of the L8 outer core biosynthesis locus in serovar 16 type strain. Nucleotide sequencing and bioinformatic analysis of the L8 LPS outer core biosynthesis locus in the serovar 16 type strain revealed five predicted LPS glycosyltransferase genes, which we have named hetB, natF, natG, gatK and gatM. BLAST analysis comparing the L4

and gatK, shared 87% nucleotide identity with the L4 locus (Figure 4). The gatK glycosyltransferase gene was the most highly conserved, sharing 96% nucleotide identity with the L4 gatK. We therefore predict that GatK performs the same function in both L4 and L8 LPS assembly, namely the addition of Gal I to the 4 position of Glc I. The L8 transferases NatF and NatG share 61% amino acid identity with each other and 61% and 72% amino acid identity respectively with the NatD in the L4 LPS locus, which we predict is a β-1,4-N-acetylgalactosamine transferase. Given the shared similarity of NatF and NatG to NatD, we propose that they may also encode 1,4-N-acetylgalactosamine transferases, required for the addition of the two α-GalNAc residues to the L8 LPS outer core. The 3' end (nts 464-1197) of hetB shared 94% identity with the equivalent region of natE in the L4 locus, but shared only 67% identity at the 5' end (nts 1-465); translated this resulted in a significant difference in the amino acid sequence at the N terminal end of HetB indicating that HetB may perform a different function in LPS assembly. Comparative bioinformatic analysis using BLAST indicated that HetB has a complete GTB_type superfamily domain as well as a partial glycosyltransferase group 1 domain at the C-terminal end.


and L8 loci revealed that a large region (4.7kb) of the L8 locus, containing hetB, natF, natG

11 The final L8 glycosyltransferase gene, gatM, encodes a protein belonging to the glycosyltransferase 8/GTA type superfamily and is located within the unique region of the L8 locus (Figure 4). We predict that GatM is a galactosyltransferase as it contains a complete RfaJ domain and shares 61% identity (77% similarity) with GatG, required for the addition of Gal II to the 4 position of Gal I in the outer core of the L3 LPS molecule (Harper et al., 2013a). Interestingly, the region of the L8 locus containing gatM shares significant nucleotide identity (> 69%) with the region of the L3 locus that includes gatG and natB, suggesting that

loci (Figure 4).

Discussion In this study we have determined the structure of the core oligosaccharides derived from the LPS of the P. multocida Heddleston type strains representing serovars 6, 7 and 16. All strains produce LPS with the conserved inner core, containing a tri-heptose side chain, that has been consistently present in all previously analysed P. multocida LPS as well as in LPS produced by Actinobacillus pleuropneumoniae and Mannheimia haemolytica (Brisson et al., 2002; Logan et al., 2006; St Michael et al., 2004). Interestingly, a Gal residue is attached to the 4 position of the inner core Glc I in both the L4 and L8 LPS outer core. This is distinct from all other full-length LPS structures identified in P. multocida that contain a Hep residue at the 6 position of the inner core Glc I. Indeed, the L4 and L8 outer cores consist of only of Gal and GalNAc. Additionally, two of the GalNAc residues in the LPS elaborated by the locus 4/serovar 6 type strain are O-acetylated at the 3 position. Genetic analyses revealed that the serovar 6 and 7 type strains share a common LPS outer core assembly locus, L4, but that the serovar 7 locus contained two nucleotide changes compared to the serovar 6 locus. These mutations in the serovar 7 L4 locus correlate with the


a recombination event may have occurred between ancestral strains harbouring the L3 and L8

12 production of a truncated LPS indicating that LPS outer core assembly had arrested after the addition of Gal I. One mutation resulted in a single amino acid change in NatD (N121S), whether this change affects the function of this transferase remains unknown. The remaining mutation was located in natE, and resulted in early termination of translation. Although it is not possible to determine transferase function based on bioinformatics analysis alone, NatD is most likely to be required for the addition of at least one GalNAc residue to the L4 LPS molecule as this protein belongs to the 82/CgtA superfamily which consists solely of β-1,4-

GalNAc residues occurs before or after O-acetylation, predicted to be performed by LatB. The serovar 16 type strain contained the LPS outer core biosynthesis locus, L8, a hybrid locus that shares significant nucleotide identity at the 3' end with the L4 locus and at the 5' end with the previously characterized L3 locus. The high level of nucleotide identity between the L4 gene, natD, and two of the L8 genes, natF and natG, suggests that both a recombination event and a gene duplication event has occurred between the L4 and L8 loci. It is probable that the L8 locus was generated from sequential recombination events involving P. multocida ancestral strains belonging to the L4 and L3 genotypes. In summary, we have elucidated the composition and structure of the LPS produced by serovars 6,7 and 16 and thus completed our structural and genetic analysis of the LPS produced by the 16 Heddleston serovars. Through LPS structural analyses and comparative genetic analyses of the LPS outer core biosynthesis loci we were able to assign predicted functions for some of the L4 and L8 glycosyltransferases and show that the L8 locus shared significant similarity with both the L4 and L3 loci.


N-acetyl-galactosamine transferases. It is not known whether the addition of the first two

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Materials and methods Media and growth conditions The bacterial strains and oligonucleotides used in this study are listed in Supplementary Table IV. For LPS structural analysis, all P. multocida strains were grown at 37°C, with shaking at 200 rpm for 16 h in 2L of brain heart infusion (BHI) medium. The cells were killed by addition of phenol (2% v/v final concentration). For genetic manipulations, P.

1.5% agar (w/v). Structural analysis of LPS LPS, isolated and purified from each serovar type strain, was O-deacylated, completely deacylated and core oligosaccharides (OS) prepared as described previously (St Michael et al., 2005b). Sugars were determined as their alditol acetate derivatives and linkage analysis determined following methylation analysis by GLC-MS as described previously (St Michael et al., 2005b). ES-MS and NMR experiments were performed as described previously (St Michael et al., 2005b). All sugars were identified with pyranose rings from NMR data unless otherwise stated. Absolute configurations were determined by GLC analyses of butyl glycoside derivatives (St Michael et al., 2004) and unless otherwise stated were identified as the D-isomers.

Genetic analyses Genomic DNA was prepared from each strain using the cetyltrimethyl ammonium bromide (CTAB) extraction method (Ausubel et al., 1995). Sequencing of the outer core biosynthetic loci was performed using PCR amplicons as template. Nucleotide sequencing of the LPS outer core biosynthesis locus in the type strains representing serovars 6, 7 and 16 was initially conducted using primers located in the conserved genes, priA and fpg, that flanked each locus


multocida was grown in brain heart infusion. Solid media were obtained by the addition of

14 and within the conserved ribosomal gene, rpl31_2 (Supplementary Table IV). Additional primers were designed and used for amplification and sequencing of each locus as required. The GenBank accession numbers for the nucleotide sequence of the LPS outer core biosynthesis loci L4 (serovar 6 type strain P2192) and L8 (serovar 16 type strain P2723) are KM670447 and KM670448 respectively.

Acknowledgements

thank our NRC-IBS colleagues, Perry Fleming (core Bacterial Culture Facility) for large scale biomass production and Jacek Stupak for CE-MS.

Abbreviations BHI, brain heart infusion; CE-ES-MS, capillary electrophoresis-electrospray-mass spectrometry; COSY, correlation spectroscopy; Gal, galactose; GalNAc, Nacetylgalactosamine; GalNAc3OAc, 3-O-acetylated N-acetyl-galactosamine; Glc, glucose; Hep, heptose; hex, hexose; HSQC, heteronuclear single-quantum coherence; LD-Hep, Lglycero-D-manno-heptose; Kdo, 3-Deoxy-D-manno-oct-2-ulosonic acid; aKdo, anhydro-3deoxy-D-manno-oct-2-ulosonic acid : LPS, lipopolysaccharide; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; OH, O-deacylated; OS, oligosaccharides; PCR, polymerase chain reaction; PEtn, phosphoethanolamine; TOCSY, total correlation spectroscopy.


This work was funded in part by the Australian Research Council, Canberra, Australia. We

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16 structures but share the same lipopolysaccharide biosynthesis outer core locus. Vet Microbiol. 150:289-296. Harper, M, St Michael, F, John, M, Vinogradov, E, Steen, J A, van Dorsten, L, Steen, J A, Turni, C, Blackall, P J, Adler, B, Cox, A D, Boyce, J D. 2013a. Pasteurella multocida Heddleston serovar 3 and 4 strains share a common lipopolysaccharide biosynthesis locus but display both inter- and intrastrain lipopolysaccharide heterogeneity. J Bacteriol. 195:48544864.

Harper, M, St Michael, F, Vinogradov, E, John, M, Steen, J A, van Dorsten, L, Boyce, J D, Adler, B, Cox, A D. 2013b. 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. Glycobiology. 23:286-294. Heddleston, K L, Gallagher, J E, Rebers, P A. 1972. Fowl cholera: gel diffusion precipitin test for serotyping Pasteurella multocida from avian species. Avian Dis. 16:925-936. Logan, S M, Chen, W, Aubry, A, Gidney, M A, Lacelle, S, St Michael, F, Kuolee, R, Higgins, M, Neufeld, S, Cox, A D. 2006. Production of a D-glycero-D-mannoheptosyltransferase mutant of Mannheimia haemolytica displaying a veterinary pathogen specific conserved LPS structure; development and functionality of antibodies to this LPS structure. Vet Microbiol. 116:175-186. Melaugh, W, Phillips, N J, Campagnari, A A, Karalus, R, Gibson, B W. 1992. Partial characterization of the major lipooligosaccharide from a strain of Haemophilus ducreyi, the causative agent of chancroid, a genital ulcer disease. J Biol Chem. 267:13434-13439. Phillips, N J, Apicella, M A, Griffiss, J M, Gibson, B W. 1992. Structural characterization of the cell surface lipooligosaccharides from a nontypable strain of Haemophilus influenzae. Biochemistry. 31:4515-4526. Post, D M, Munson, R S, Jr., Baker, B, Zhong, H, Bozue, J A, Gibson, B W. 2007. Identification of genes involved in the expression of atypical lipooligosaccharide structures from a second class of Haemophilus ducreyi. Infect Immun. 75:113-121. St Michael, F, Harper, M, Parnas, H, John, M, Stupak, J, Vinogradov, E, Adler, B, Boyce, J D, Cox, A D. 2009. Structural and genetic basis for the serological differentiation of Pasteurella multocida Heddleston serotypes 2 and 5. J Bacteriol. 191:6950-6959. St Michael, F, Li, J, Cox, A D. 2005a. Structural analysis of the core oligosaccharide from Pasteurella multocida strain X73. Carbohydr Res. 340:1253-1257.


Harper, M, St Michael, F, Vinogradov, E, John, M, Boyce, J D, Adler, B, Cox, A D. 2012. Characterization of the lipopolysaccharide from Pasteurella multocida Heddleston serovar 9: identification of a proposed bi-functional dTDP-3-acetamido-3,6-dideoxy-alpha-D-glucose biosynthesis enzyme. Glycobiology. 22:332-344.

17 St Michael, F, Li, J, Vinogradov, E, Larocque, S, Harper, M, Cox, A D. 2005b. Structural analysis of the lipopolysaccharide of Pasteurella multocida strain VP161: identification of both Kdo-P and Kdo-Kdo species in the lipopolysaccharide. Carbohydr Res. 340:59-68. St Michael, F, Vinogradov, E, Li, J, Cox, A D. 2005c. Structural analysis of the lipopolysaccharide from Pasteurella multocida genome strain Pm70 and identification of the putative lipopolysaccharide glycosyltransferases. Glycobiology. 15:323-333. St Michael, F S, Brisson, J R, Larocque, S, Monteiro, M, Li, J, Jacques, M, Perry, M B, Cox, A D. 2004. Structural analysis of the lipopolysaccharide derived core oligosaccharides of Actinobacillus pleuropneumoniae serotypes 1, 2, 5a and the genome strain 5b. Carbohydr Res. 339:1973-1984. Downloaded from http://glycob.oxfordjournals.org/ at Nipissing University on October 16, 2014

Wilkie, I W, Harper, M, Boyce, J D, Adler, B. 2012. Pasteurella multocida: Diseases and Pathogenesis. Curr Top Microbiol Immunol. 361:1-22.

18

Figure Legends Figure 1. Positive ion capillary electrophoresis-electrospray mass spectrum of P. multocida serovar 16 core OS Fr 13-15: a) MS/MS of m/z 1069.32+ detailing region m/z 900-2000; b) MS/MS of m/z 1069.32+ detailing region m/z 200-900. Inset are fragmentation patterns illustrating the fragment ions formed from the core OS. Ions marked with an asterisk in Figure 1b are from inner core fragmentation.

overlapped COSY (green-cyan), TOCSY (red) and NOESY (blue) spectra (all three panels) of the core oligosaccharide. Residues are labeled as indicated in Supplementary Table II.

Figure 3. NMR analyses of core OS from P. multocida Heddleston serovar 16. Regions of overlapped COSY (green-cyan), TOCSY (magenta) and NOESY (black) NMR spectra of the P. multocida serovar 16 core OS, showing correlations from anomeric protons. Residues are labeled as indicated in Supplementary Table III.

Figure 4. Schematic representation of the P. multocida serovar 6, serovar 7 and serovar 16 LPS outer core structures and the corresponding L4 (serovar 6 and 7) and L8 (serovar 16) LPS outer core biosynthesis loci. The previously reported P1059 (serovar 3) outer core structure and corresponding L3 LPS biosynthesis locus is shown for comparison. Percentage similarity at nucleotide level between the L3, L4 and L8 glycosyltransferase genes is shown in shaded regions and amino acid identity is shown underneath in brackets. The P. multocida glycosyltransferase genes known (L3) or predicted to be required for the assembly of each


Figure 2. NMR analyses of core OS from P. multocida Heddleston serovar 6. Regions of

19 LPS outer core are shown below each structural linkage. Mutations in the L4 locus, that are specific to the serovar 7 type strain (P1997) and the corresponding LPS outer core structure, are also shown. The conserved inner core structure, up to and including Glc I, is shown below (glycoform A only shown). Non-stoichiometric additions of PEtn are shown with a dotted line. Residues are Gal, galactose; GalNAc, N-acetyl-galactosamine; GalNAc3OAc, 3-Oacetylated N-acetyl-galactosamine; Glc, glucose; Hep, heptose; Kdo, 3-deoxy-D-mannooctulosonic acid; P, phosphate; PEtn, phosphoethanolamine. Downloaded from http://glycob.oxfordjournals.org/ at Nipissing University on October 16, 2014

20

Table I: Negative ion CE-ES-MS data and proposed compositions of O-deacylated LPS (LPS-OH) and core oligosaccharide (Core OS) from the Heddleston serovar 6, 7 and 16 type strains. Average mass units were used for calculation of molecular weight based on proposed composition as follows: Hep, 192.17; HexNAc, 203.19; Hex, 162.15; Kdo, 220.18; Lipid A-OH, 952.00; OAc, 42.00; P, 79.95; PEtn, 123.05. Serovar/Strain (Description)

Observed Ions (m/z) (M-4H)4- (M-3H)3-

(M-2H)2-

Serovar 6 (P2192) LPS-OH

771.0 801.8 832.8

1022.0 1028.2 1062.8 1069.2 1110.2

1542.6 1666.2

-

3069.0 3087.5 3189.3 3210.5 3333

3064.8 3086.8 3188.0 3209.9 3333.0

3HexNAc, 3Hex, 3Hep, 2Kdo, Lipid A-OH 3HexNAc, 4Hex, 3Hep, Kdo-P, Lipid A-OH 3HexNAc, 3Hex, 3Hep, 2Kdo, PEtn, Lipid A-OH 3HexNAc, 4Hex, 3Hep, Kdo-P, PEtn, Lipid A-OH 3HexNAc, 4Hex, 3Hep, Kdo-P, 2PEtn, Lipid A-OH

Core OS

-

-

976.8 996.6 1036.8 1057.8 1068.6 1109.1 1130.1

-

1955.6 1995.2 2075.6 2117.6 2139.2 2220.2 2262.2

1952.7 1994.7 2075.8 2117.8 2138.9 2219.9 2261.9

OAc, 3HexNAc, 3Hex, 3Hep, Kdo 2OAc, 3HexNAc, 3Hex, 3Hep, Kdo OAc, 3HexNAc, 3Hex, 3Hep, PEtn, Kdo 2OAc, 3HexNAc, 3Hex, 3Hep, PEtn, Kdo 2OAc, 3HexNAc, 4Hex, 3Hep, aKdo OAc, 3HexNAc, 4Hex, 3Hep, PEtn, aKdo 2OAc, 3HexNAc, 4Hex, 3Hep, PEtn, aKdo


608.3 639.3

763.8 770.8 804.8 812.3 853.3

1145.8 1157.3 1207.8 1218.8 1280.3

-

2294.0 2316.0 2417.5 2438.7 2562.3

2293.2 2315.1 2416.2 2438.1 2561.2

2Hex, 3Hep, 2Kdo, Lipid A-OH 3Hex, 3Hep, Kdo-P, Lipid A-OH 2Hex, 3Hep, 2Kdo, PEtn, Lipid A-OH 3Hex, 3Hep, Kdo-P, PEtn, Lipid A-OH 3Hex, 3Hep, Kdo-P, 2PEtn, Lipid A-OH

Core OS

-

-

559.3 568.3 621.0 629.8 640.3 702.1

1119.3 1137.7 1281.8 1404.7

1120.6 1138.6 1244.0 1261.6 1282.7 1406.0

1121.0 1139.0 1244.0 1262.0 1283.2 1406.2

2Hex, 3Hep, aKdo 2Hex, 3Hep, Kdo 2Hex, 3Hep, PEtn, aKdo 2Hex, 3Hep, PEtn, Kdo 3Hex, 3Hep, aKdo 3Hex, 3Hep, PEtn, aKdo


760.8 791.6

1007.6 1014.8 1048.6 1055.8

-

-

3025.8 3047.3 3148.8 3170.4

3023.9 3045.8 3146.9 3168.8

4Hex, 2HexNAc, 3Hep, 2Kdo, Lipid A-OH 5Hex, 2HexNAc, 3Hep, Kdo-P, Lipid A-OH 4Hex, 2HexNAc, 3Hep, PEtn, 2Kdo, Lipid A-OH 5Hex, 2HexNAc, 3Hep, PEtn, Kdo-P, Lipid A-OH

(M-H) -

Molecular Mass (Da) Observed Calculated

Proposed Composition


21

Core OS

Observed Ions (m/z) (M-4H)4- (M-3H)3-

(M-2H)2-

822.4

1097.0

-

-

3293.8

3291.9

5Hex, 2HexNAc, 3Hep, 2PEtn, Kdo-P, Lipid A-OH

-

-

933.7 986.2 995.2 1005.8 1067.3 1076.4

-

1869.4 1974.4 1992.4 2011.6 2136.6 2154.8

1869.6 1974.7 1992.7 2013.8 2136.9 2154.9

4Hex, 2HexNAc, 3Hep, Kdo 4Hex, 2HexNAc, 3Hep, PEtn, aKdo 4Hex, 2HexNAc, 3Hep, PEtn, Kdo 5Hex, 2HexNAc, 3Hep, aKdo 5Hex, 2HexNAc, 3Hep, PEtn, aKdo 5Hex, 2HexNAc, 3Hep, PEtn, Kdo

(M-H) -

Molecular Mass (Da) Observed Calculated

Proposed Composition


Serovar/Strain (Description)


22


23


24


25

The Myriad Properties of Pasteurella multocida Lipopolysaccharide.

Immunogenic and toxic properties of a purified lipopolysaccharide-protein complex from Pasteurella multocida.

Production of mouse monoclonal antibodies to Pasteurella multocida type A and the immunological properties of a protective anti-lipopolysaccharide antibody.

Characterization of the common antigenic lipopolysaccharide O-chains produced by Bordetella bronchiseptica and Bordetella parapertussis.

Characterization of the immunogenicity of formaldehyde detoxified Pasteurella multocida toxin.

Assessment of Pasteurella multocida A Lipopolysaccharide, as an Adhesin in an In Vitro Model of Rabbit Respiratory Epithelium.

Antibodies to outer membrane proteins but not to lipopolysaccharide inhibit pulmonary proliferation of Pasteurella multocida in mice.

Letter: Joint infection by Pasteurella multocida.

Pasteurella multocida endocarditis.

Sequence of the dermonecrotic toxin of Pasteurella multocida ssp. multocida.

Letter: Joint infection by Pasteurella multocida.

Development of a rapid multiplex PCR assay to genotype Pasteurella multocida strains by use of the lipopolysaccharide outer core biosynthesis locus.

Comparative analysis of lipopolysaccharide and lipid antigens of Leptospira interrogans serovars.

Pasteurella multocida septicaemia in pigs.

[Pasteurella multocida bacteremia and prosthetic joint infection].

[The first report of human chronic sinusitis by Pasteurella multocida subsp. multocida in Japan].

Bacteremia due to Pasteurella multocida.

Hematological changes in calves exposed to a mixture of lipopolysaccharide and crude leukotoxin of Pasteurella haemolytica.

Conditions for transformation of Pasteurella multocida by electroporation.

Identification and characterization of lipopolysaccharide-induced TNF-alpha factor gene from Japanese flounder Paralichthys olivaceus.

Identification and characterization of lipopolysaccharide-binding proteins on human peripheral blood cell populations.

Isolation and characterization of a lipopolysaccharide mutant of Legionella pneumophila.

Letter: Treatment of Pasteurella multocida meningitis.

Persistence of the carrier status in haemorrhagic septicaemia (Pasteurella multocida serotype 6:B infection) in buffaloes.