Outer membrane proteins of Pasteurella multocida

Veterinary Microbiology 144 (2010) 1–17

Contents lists available at ScienceDirect

Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic

Review

Outer membrane proteins of Pasteurella multocida Tama´s Hatfaludi, Keith Al-Hasani, John D. Boyce, Ben Adler * Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Department of Microbiology, Monash University, Clayton, VIC 3800, Australia

A R T I C L E I N F O

A B S T R A C T

Article history: Received 18 September 2009 Received in revised form 23 January 2010 Accepted 28 January 2010

Pasteurella multocida is a ubiquitous pathogen which causes a range of diseases in diverse animal species. Components of the bacterial outer membrane, such as trans membrane proteins and lipoproteins, play key roles in the interaction of the pathogen with the host environment and in the host immmune response to infection. In this review, we evaluate the current knowledge of P. multocida outer membrane proteins and their role in pathogenesis and immunity. ß 2010 Elsevier B.V. All rights reserved.

Keywords: P. multocida OMPs Pathogenesis Adhesion Immunity

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . OM proteins: why are they of particular interest? . . . . . . . . . . . . . . . 2.1. Permeability of the OM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Functional groupings of OM proteins . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Structural proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. Small b-barrel membrane anchors . . . . . . . . . . . . . . . 3.1.2. Peptidoglycan-associated lipoproteins . . . . . . . . . . . . 3.2. Transporter proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. Nonspecific porins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. Energy-dependent transport-efflux. . . . . . . . . . . . . . . 3.3. Binding proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. Energy-dependent transporter-influx—iron transport 3.3.2. Siderophore receptors . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.3. Transferrin and lactoferrin receptors . . . . . . . . . . . . . 3.3.4. Haemoglobin receptors . . . . . . . . . . . . . . . . . . . . . . . . 3.3.5. Binding lipoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Adhesins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.1. PtfA – PM0084, PM0085 – type 4 fimbriae . . . . . . . . 3.4.2. ComE1—PM1665 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.3. FhaB—FhaB1 (PM0057), FhaB2 (PM0059). . . . . . . . . . 3.5. Putative adhesins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Membrane-associated enzymes . . . . . . . . . . . . . . . . . . . . . . . . .

* Corresponding author. Tel.: +61 3 9902 9177; fax: +61 3 9902 9222. E-mail address: [email protected] (B. Adler). 0378-1135/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2010.01.027

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2 2 2 3 3 3 9 9 9 10 10 10 10 11 11 12 12 13 13 13 13 14

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T. Hatfaludi et al. / Veterinary Microbiology 144 (2010) 1–17

4. 5.

3.6.1. PM1426 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6.2. NanH—PM0663, NanB—PM1000 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6.3. GlpQ—PM1444 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7. Protein assembly machinery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7.1. Oma87—PM1992 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8. Other proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8.1. PlpE—PM1517 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . OMPs as vaccines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Members of the genus Pasteurella are Gram-negative, non-motile, facultative coccobacilli measuring 0.3–1.0 mm by 1.0–2.0 mm (Boyce et al., 2010). The species Pasteurella multocida is subdivided into four subspecies; multocida, which includes the type species and three other gallicida, septica and tigris (Boyce et al., 2010). P. multocida subspecies multocida, the most important pathogenic member of the genus, has a broad disease spectrum and can infect many wild and domestic animal species. Diseases caused by P. multocida include fowl cholera in birds, atrophic rhinitis in pigs, haemorrhagic septicaemia in ungulates, enzootic pneumonia in cattle, sheep and goats, snuffles in rabbits and, more rarely, wound abscesses and meningitis in humans, predominantly following cat- or dog-inflicted injuries (Boyce et al., 2010). In this review, we focus on P. multocida subspecies multocida to provide a comprehensive coverage of its major outer membrane (OM) proteins. P. multocida strains can be differentiated by a number of methods. The capsule and lipopolysaccharide (LPS) form the basis for classification into serogroups (A, B, D, E, or F) and serotypes 1–16, respectively. Different serogroups and serotypes tend to be associated with particular diseases. Fowl cholera is generally caused by type A:1 or A:3 strains, haemorrhagic septicaemia by type B:2 or E:5 strains and atrophic rhinitis by toxigenic type D strains. P. multocida is a commensal organism in many mammalian hosts. However, it can potentially cause disease in almost all species if it gains access to the bloodstream. Clearly, pathogenesis is a result of complex interactions between specific host factors (e.g. species, age, immune status) and specific bacterial virulence factors (e.g. LPS, capsule, adhesins, OM proteins). Therefore disease pathogenesis is versatile and depends on the bacterial strain, the animal model used and the changing response of both the host and bacteria to the interaction (Boyce et al., 2010). The cell surface protects Gram-negative bacteria against a range of harsh environments and is critical for interaction of the bacterium with the host. Virulent P. multocida strains produce a polysaccharide capsule. The serogroup A capsule contains hyaluronic acid, while serogroup D and F capsules contain heparin and chondroitin, respectively. The OM functions as a selective barrier that prevents the entry of many toxic molecules into the cell, a property that is crucial for bacterial survival in many environments. At the same time, the proteins

14 14 14 14 14 15 15 15 15 15 15

embedded in the OM fulfill a number of roles that are critical for the bacterial cell, such as nutrient uptake, transport of molecules in and out of the cell, and interaction with the environment and host tissues (Fig. 1). 2. OM proteins: why are they of particular interest? Altogether, 20–30% of all genes in bacteria encode membrane proteins, and about 50% of the total OM mass consists of protein (Koebnik et al., 2000). However, of the many known OMPs, to date proteins belonging to only about 25 distinctive structural groups have been solved (http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html). As cytoplasmic proteins are usually soluble and easier to characterise then membrane proteins, they have often been the subject of proteomic studies. Indeed, despite progress in recent years, detailed knowledge of membrane proteins remains elusive. 2.1. Permeability of the OM Compared to the inner membrane, the OM is relatively permeable to small molecules. How then, do molecules move so readily across the OM? Porins form nonspecific water-filled channels in the OM that facilitate the entrance and exit of hydrophilic small molecules. These nonspecific channels allow the diffusion of ions and molecules up to a molecular mass of 600 Da. Porins are highly immunogenic and are conserved within bacterial families, making them attractive vaccine candidates. Structural studies have shown that most porins are transmembrane proteins containing three identical b-barrel subunits. The diffusion speed of solutes depends on the difference in concentration between the periplasm and the extracellular milieu, and on the molecular mass of the solute (Nikaido, 2003). Specific channels constitute another passive transport mechanism. In contrast to porins, specific channels possess a binding site for one or more substances (up to 5 kDa) (Koebnik et al., 2000). They mediate the spontaneous diffusion of specific classes of nutrients. In contrast to passive transport, larger molecules are transported across the OM by active, energy-dependent mechanisms. It would be difficult to ‘‘design’’ specific channels for these molecules without making them nonspecifically permeable to other solutes, thus compromising the bacterial resistance to environmental toxic compounds. Some compounds are transported into the cells by utilising TonB-dependent receptors or gated


3

Fig. 1. Outer membrane (OM) and OM associated proteins of P. multocida. The bacterium is depicted as a Gram-negative organism with inner (blue) and outer membranes (green) and periplasm. Structures which are known for proteins from any bacterial species are depicted as such, while models are shown when structures are not known. The following functional category groups are present. Structural proteins: Small b-barrel membrane anchors, OmpA (1BXW) and PmOmpA (courtesy of Carpenter et al., 2007) are shown on the left. Transport proteins: OmpF (2OMF) in blue is a representative of a nonspecific porin. TolC (1EK9) and AcrAB (2DRD) are the tripartite system of an efflux pump. Tp32 (1XS5) a PlpB homologue and MetNI (3DHW) represent the methionine ABC transporter system. Binding proteins: FepA (1FEP) and HasR (3CSL) are representative of a TonB dependent iron uptake system. Membrane-associated enzymes: These are represented with phospholipase OMPLA (1QD6) and the two sialidases NanH (2VW1), NanB (2KVK5). Adhesins: These are present with OmpX (1QJ8), the type 4 pili, the TAD locus and filamentous haemagglutinin protein FhaB. Protein assembly machine: That of Omp85 is represented only with the filamentous haemagglutinin secretion protein FhaC (2QDZ) that belongs to the same superfamily. The PDB code (Protein Data Bank accession code) is given in parentheses after the protein. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

channels. For the efflux of substances, export channels of the TolC family are used. Other means of transport across the OM include secretion through secretins, a chaperoneusher pathway used for the components of P pili, while autotransporters are proteins that contain all the information required for their own transport (Nikaido, 2003). 3. Functional groupings of OM proteins Previous groupings of OM proteins have separated them into OM integral and lipoproteins, and then further separated these two groups into functional categories. Indeed, bacterial OM lipoproteins have been grouped according to functional type (http://www.mrc-lmb.cam. ac.uk/genomes/dolop/), either as structural proteins, enzymes, receptors or transporters performing essential functions at the membrane–aqueous interface (Babu et al., 2006). Although unified by common structural features, they carry out diverse functions in diverse organisms. Similarly for integral membrane proteins, the functional categories include: small b-barrel membrane anchors; nonspecific porins; specific channels; energy-dependent transport-efflux; energy-dependent transporter-influx;

protein secretion pore; OM usher proteins; adhesins; membrane integral enzymes; and protein pore-forming toxins (Nikaido, 2003; Wimley, 2003). However, not all of these categories have been found among the reported OMPs of P. multocida. Here we have chosen to generate a common grouping for the OM lipoproteins and integral membrane proteins, where proteins are categorized solely by functional characteristics; the main functional categories are structural proteins, transport proteins, binding proteins, adhesins, protein assembly machines, and membrane-associated enzymes. The available information on 72 OM or OM associated proteins of P. multocida is summarised in Table 1. 3.1. Structural proteins 3.1.1. Small b-barrel membrane anchors 3.1.1.1. OmpA—PM0786. OmpA is a two-domain protein, which provides a physical linkage between the OM and peptidoglycan; it is also referred to as a slow porin as it mediates a slow diffusion rate. Its presumed primary function is to stabilize the cell envelope structure through

4

Table 1 Reported outer membrane proteins of P. multocida. Current gene name

Pm Number

Theor. Predicted function mass [kDa]

Atten. Gene Iron mutanta pre-valence receptors & in diff. regulatorsc sero-types [%]b

Gene expression In In Under Defined Under chicken chicken iron iron nutrient bloodd liverd limitatione sourcesf limitationg

Reaction Vaccination studiesi with Native Recombinant conval. protein proteink serumh or OM extractj

Key refo Passive immun.l

Chicken Mouse pfhR pfhB1

PM0040 PM0057

81.2 286.6

pfhB2

PM0059

431.5

PM0076

74.5

ptfA dppA

PM0084 PM0085 PM0236

14.9 51.9 59.2

hgbA

PM0300

109.6

ompW

PM0331

21.7

pm0336

PM0336

113.7

hgbB

PM0337

113.2

parC

PM0369

83.6

ompH_1

PM0388

37.3

ompH_2

PM0389

38.6

pm0442

PM0442

23.7

tolC1

PM0527

50.6

pcP

PM0554

15.5

hemR

PM0576

84.8

trpB

PM0578

43.5

OmpH2 – outer membrane porin Immunogenic membrane protein TolC1 – outer membrane efflux channel PCP – peptidoglycanassociated lipoprotein cross reacting protein HemR Haemoglobin binding receptor protein Tryptophan synthase beta chain

Hb, He, T yes

a1

yes yes

a1

37

Hok1 Hok2

o1 o26

1.83

+ Hok11

o2

Hok1

o1

a8

99

yes a1 no a2

95.2

1.83 Hb, He, T

5.67

Hb, He, T

0.88

2.12 2.33 2.25

14.41

2.67 a3

77.9

7.7

2.88

o3 o3 o1

k2

Ho

6 6.5 1.72 e1

no

1.37

2.86

Hok1 Hok2

o1, o4

Hok1

o4

04

1.76 Hok1 Hek3

Fr

o4, o5

o4 100

Fr

+

1.67 no

+

a4

j1

+ He + Hej2

k1

Ho + Ho + Hek4 + Hok10 Hok1

Hb, T

3.94

1.61

10.6

+

+ Ho

l1

o4, o6, o7 o4, o6

Hok1

o4, o8

k1

o4, o9

Ho 3.17

k5

Hok1 Hok6

Hok2

o4, o8, o10 o1, o11 o4, o12


est

PfhR – putative porin FhaB1 – filamentous haemagglutinin protein FhaB2 – filamentous haemagglutinin protein Outer membrane esterase Type 4 fimbriae subunit Type 4 fimbriae subunit TonB dependent outer membrane haemoglogin/ haemin binding receptor HgbA TonB-dependent haemoglobin receptor OmpW outer membrane protein TonB-dependent receptor, putative HgbB Haemoglobin/ haptoglobin binding protein DNA topoisomerase IV, subunit A OmpH1 – outer membrane porin

hbpA

PM0592

59.4

pm0612

PM0612

11.4

PM0659

214.3

nanH

PM0663

93.2

pm0741

PM0741

89.4

phyA

PM0773

80.7

ompA

PM0786

37.9

pm0803

PM0803

90.9

omp16

PM0966

16.1

pm0979 pm0998

PM0979 PM0998

14.5 30.6

nanB

PM1000

121.3

pm1021

PM1021

26.3

mglC

PM1040

35.8

pm1050

PM1050

37.2

pm1064

PM1064

30.0

Hb, He, T

8.76

Hok2

3.41

1.58 +

o1

Hok1

o4

k1

o8

Ho

87.2

o13

Hb, He, T

1.79

2.5

Hok1 Hok2

1.89

o1

o14

+

1.24

3.35

1.58f1

Hel1

o4, o15, o16, o17

Hok1

o4

+

Hok1 Hek7

o4, o8, o18

+

Hok1 Hok1

o4, o8 o1

4.8 6.63 e1

100

Hok1

100


pm0659

TonB dependent outer membrane haemoglogin/haemin binding receptor Hypothetical outer membrane protein Large extracellular alpha-helical protein, lipoprotein NanH – outer membrane integrated sialidase enzyme TonB dependent outer membrane haemoglogin/ haemin binding receptor PhyA Capsule polysaccharide export protein [Actinobacillus pleuropneumoniae serovar] or capsule polysaccharide modification protein LipA [Neisseria meningitidis MC58] OmpA - b-barrel membrane anchor protein Under iron limitation unregulated outer membrane protein Outer membrane protein P6 precursor; peptidoglycanassociated outer membrane lipoprotein Immunogenic lipoprotein Uncharacterised outer membrane protein NanB – outer membrane integrated sialidase enzyme Uncharacterised outer membrane protein MglC – betamethylgalactoside transporter inner membrane component Uncharacterised lipoprotein Uncharacterised lipoprotein

o13

o4 o4

1.57

Hok1

o11 o4 5

6

Table 1 (Continued ) Current gene name

Pm Number


pm1069

PM1069

47.7

pm1081

PM1081

90.8

tonBm

PM1188m

28.2

pm1282

PM1282

88.0

tufA

PM1357

43.2

phospho- PM1426 lipase A pm1428 PM1428

35.4 90.7

glpQ

PM1444

41.0

vacJ

PM1501

27.4

plpE

PM1517

37.3

pm1578

PM1578

35.7

pm1600

PM1600

91.0

pm1614

PM1614

49.6


Gene expression In In Under Defined Under chicken chicken iron iron nutrient bloodd liverd limitatione sourcesf limitationg

Reaction Vaccination studiesi with Native Recombinant conval. k serumh protein protein or OM extractj


Chicken Mouse yes

a5

Hb, He, T

99.7

Hok1

o4

Hok1 Hok2

o1

2.5

o19

Hok2

He, T

o1

o1

Hok1 Hb, He, T

Ho

1.8

k1

o4 k2

Ho

o1

Hok6

o4, o10

Hok1

no

a6

1.57

+ Hek8

1.8

Hok1

o1

+ Hek8

o20

o4 o4

+

Hok1

o8


Outer membrane protein P1 precursor TonB dependent outer membrane haemoglogin/haemin binding receptor TonB – supplies energy for iron transport TonB dependent outer membrane haemin binding receptor TufA – translation elongation factor Tu-A phospholipase A, OMPLA TonB dependent outer membrane haemoglogin/haemin binding receptor GlpQ – glycerophosphodiester phosphodiesterase VacJ lipoprotein homolog, Surface lipoprotein H. influenzae Protective outer membrane lipoprotein Uncharacterised lipoprotein LPS-assembly protein; organic solvent tolerance protein Outer membrane antigenic lipoprotein B

PM1622

95.8

comE1

PM1665

12.6

nanP

PM1709

36.1

pm1720 pepP

PM1720 PM1724

29.2 50.2

metQ

PM1730

30.1

torD pm1809

PM1794 PM1809

22.7 67.0

hemY

PM1815

47.6

pm1819

PM1819

117.3

rfaF

PM1844

38.9

lctP pm1979

PM1852 PM1979

56.8 69.3

tolC2

PM1980

51.8

rps2 oma87

PM1984 PM1992

26.5 87.6

skp

PM1993

21.3

tbpA

82.0 Acc Nr. AY007725n

Putative tadD tadF flp1 rcpA rcpB

PM846 PM844 PM855 PM852 PM853

28.6 21.5 7.6 51.0 30.0

HasR – Ton B dependent haeme acquisition system receptor ComE1 – fibronectin and DNA binding protein NanP – periplamic sialate uptake component DNA uptake lipoprotein PepP – proline aminopeptidase P II MetQ – periplasmic methionine binding protein of ABC transporter TorD chaperone protein Uncharacterised outer membrane protein HemY Uncharacterised enzyme of haem biosynthesis [Haemophilus somnus 129PT] SrfB putative virulence factor RfaF ADP-heptose: LPS heptosyltransferase LctP L-lactate permease Uncharacterised outer membrane protein TolC2 – outer membrane efflux channel Ribosomal protein S2 Oma87 – Omp85 like protein assembly protein Skp export factor homolog Transferin binding protein in bovine strains TadD TadF Flp1 RcpA RcpB

Hoj3

Fr, T

Hok1

o21

o22

yes

a7

o13

1.89e1

yes

a6

2.2

+

Hok1

o4 o4

Hok1

o4, o8

Hok1

o4 o4 o14

yes

a1

o14 o11 3.3

o11 o8

1.6 +

no

a4

Hok1

2.58

1.62 99.7

2.68 31.5

yes

a5

2

2.6

Tr, T

o9

1.9 +

Hok1 Hek9

+

Hok1

+ Hol2

o4 o4, o8, o23


hasR

o8 o24

2.27 1.92

o25

7

8

Table 1 (Continued ) Current gene name

Pm Number

fhaC

PM0056 PM0058



Gene expression In In Under Defined Under chicken chicken iron iron nutrient d d e blood liver limitation sourcesf limitationg

Reaction Vaccination studiesi with Native Recombinant conval. k serumh protein protein or OM extractj


Chicken Mouse T. Hatfaludi et al. / Veterinary Microbiology 144 (2010) 1–17

FhaC – Filamentous haemagglutinin two partner secretion protein a Mutants tested for attenuation. Yes or no reports if the mutant is attenuated. a1 in mice (Fuller et al., 2000), a2 in mice (Bosch et al., 2002b), a3 in mice (Cox et al., 2003), a4 in mice and chickens (Hatfaludi et al., 2008), a5 in chickens (Harper et al., 2003), a6 in chickens (Hatfaludi et al. unpublished data), a7 in mice (Steenbergen et al., 2005), a8 in turkey internasaly (Tatum et al., 2005). b Prevalence of genes among different serotypes in P. multocida wild type (n = 245) and reference strains (n = 44), [% of positive strains in total] (Ewers et al., 2006). c Different iron sources bound by the protein and the presence of iron regulatory sequences. Hg – haemoglobin, He – haem, Fr – Fur regulated, T – TonB-box. d Genes differentially expressed during growth in chicken blood or liver as compared with growth in vitro in BHI. Average fold difference values are shown (Boyce et al., 2002, 2004). e Genes differentially expressed during growth under iron limited conditions as compared with growth in vitro in BHI. Average fold difference values are shown (Paustian et al., 2001). e1 (Boyce et al., 2006) f Genes differentially expressed during growth with defined iron sources. Genes corresponding to the listed values were grown with haemoglobin as the sole iron source, except f1 where ferric citrate was used. Average fold difference values are shown (Paustian et al., 2002a). g Genes differentially expressed during growth under nutrient limited conditions (Paustian et al., 2002b). h Antigens recognized by convalescent chicken sera (Al-Hasani et al., 2007). i Vaccination studies. Plus (+) or minus () represents if immunization showed protection and He or Ho indicate heterologous or homologous challenge. j Immunisation with native protein or OM extracts. j1 Native protein in chicken (Garrido et al., 2008), j2 mutant OM extract in mice (Sthitmatee et al., 2008), j3 OM extract in cattle (Prado et al., 2005). k Immunisation with recombinant protein. k1 (Hatfaludi et al. unpublished data), k2 (Bosch et al., 2004), k3 (Cox et al., 2003), k4 (Sthitmatee et al., 2008), k5 (Lee et al., 2007), k6 (Lo et al., 2004), k7 in turkey (Kasten et al., 1997), k8 (Wu et al., 2007), k9 sub-fragment (Mitchison et al., 2000), k10 (Luo et al., 1997), k11 in turkey (Tatum et al., 2009b). l Passive immunization, l1 with monoclonal antibody (Vasfi Marandi and Mittal, 1997), l2 with serum (Mitchison et al., 2000). m Not OMP. See Section 3.3.4.5. n TbpA is not present in PM70 strain, therefore accession number is shown. o Key references. o1 (Bosch et al., 2004), o2 (Tatum et al., 2005), o3 (Ruffolo et al., 1997), o4 (Boyce et al., 2006), o5 (Cox et al., 2003), o6 (Garrido et al., 2008), o7 (Luo et al., 1999), o8 (Al-Hasani et al., 2007), o9 (Hatfaludi et al., 2008), o10 (Lo et al., 2004), o11 (Tabatabai, 2008), o12 (Jablonski et al., 1996), o13 (Steenbergen et al., 2005), o14 (Tabatabai and Zehr, 2004), o15 (Carpenter et al., 2007), o16 (Dabo et al., 2003), o17 (Vasfi Marandi and Mittal, 1997) , o18 (Kasten et al., 1997), o19 (Bosch et al., 2002a), o20 (Wu et al., 2007), o21 (Prado et al., 2005), o22 (Mullen et al., 2007), o23 (Mitchison et al., 2000), o24 (Ogunnariwo and Schryvers, 2001), o25 (Harper et al., 2003), o26 (Fuller et al., 2000).


the interaction of its C-terminus with peptidoglycan (Nikaido, 2003). In Escherichia coli this major multifunctional protein is expressed at high levels of around 100,000 copies per cell (Khalid et al., 2008), and ompA expression is tightly regulated at the post-transcriptional level. Besides acting as a structural stabilizer, OmpA has been reported to function as an adhesin and invasin and to participate in biofilm formation. It can be an immune target as it is may be involved in immune evasion, and it acts as a receptor for several bacteriophages. Many of these properties are related to its monomeric structure, particularly the four short protein loops that emanate from the protein to the outside (Smith et al., 2007) (Fig. 1). P. multocida OmpA was first identified as a 36 kDa, twodomain, heat-modifiable protein using monoclonal antibodies against OM vesicles. Subsequent work from the same group showed that OmpA-specific monoclonal antibodies do not protect mice against lethal challenge with P. multocida (Vasfi Marandi and Mittal, 1997). OmpA can function as an adhesin, mediating the adherence of P. multocida to host cells and extracellular matrix molecules (Dabo et al., 2003). The N-terminal domain of the protein displays structural similarity to the transmembrane bbarrel section of the E. coli OmpA, whereas the C-terminal domain is homologous to the Neisseria meningitidis RmpM C-terminal domain. The P. multocida OmpA N-terminal domain is predicted to span the lipid bilayer, while the Cterminus sits in the periplasmic region (Fig. 1) and is thought to interact with peptidoglycan (Carpenter et al., 2007). A simulation model of the molecular dynamics and environmental interactions of OMPs with P. multocida OmpA suggested a role for electrostatic forces in mediating interactions between the C-terminal domain and the periplasmic surface of the membrane under low salt conditions (Carpenter et al., 2007; Khalid et al., 2008). Immunoblot analysis using recombinant P. multocida OmpA expressed in E. coli revealed that the protein was both immunogenic and expressed in vivo (Al-Hasani et al., 2007; Dabo et al., 2003). OmpA was also identified by mass spectrometry in the sarcosine-insoluble membrane fraction of in vitro grown bacteria. The in vitro expression ratio of OmpA from cells cultured under iron-depleted growth conditions was similar to the control. Interestingly, the expression of ompA (pm0786) was similar within the natural chicken host and in vitro (Boyce et al., 2006). 3.1.2. Peptidoglycan-associated lipoproteins 3.1.2.1. PCP-Lpp—PM0554. P. multocida PCP (peptidoglycan-associated lipoprotein cross-reacting protein), is a 15.6 kDa surface exposed lipoprotein with 80% similarity to Haemophilus influenzae PCP, where it is the target of serum bactericidal activity (Deich et al., 1990). The P. multocida PCP also displays similarity to the OM lipoprotein SlyB from E. coli and Burkholderia multivorans. SlyB in the latter organism contributes to the integrity of the cell envelope (Plesa et al., 2006). The gene encoding PCP (PM0554) was identified in P. multocida (Table 1) by in vivo expression technology (IVET) screening, indicating that the protein is expressed during infection (Lo et al., 2004). However, DNA microarray analysis indicated that pm0554

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was down regulated in vivo compared to in vitro growth (Boyce et al., 2006). Vaccination of mice or chickens with either the lipidated or non-lipidated form of recombinant PCP, expressed and purified in E. coli, induced no protection against homologous P. multocida challenge (Lo et al., 2004). 3.1.2.2. P6-like protein-Omp16—PM0966. A 16 kDa protein was isolated from a rabbit strain of P. multocida using a method for purification of pili and identified as PM0966. The protein showed high similarity to P6 protein of H. influenzae which has been shown to elicit a protective immune response in the chinchilla otitis media model (Green et al., 1993). Bioinformatics analysis indicated that PM0966 belongs to the OmpA-like peptidoglycan-associated lipoprotein superfamily (Babu et al., 2006). Kasten et al. (1997) showed that immune sera from turkeys reacted against the recombinant P6-like protein in all 16 P. multocida serotypes. However, immunisation of turkeys with the recombinant P6-like protein purified from baculovirus, elicited high antibody titres in the birds, but failed to protect them from infection. 3.2. Transporter proteins 3.2.1. Nonspecific porins Porins are water-filled open channels that span the OM and allow the passive penetration of hydrophilic molecules. Nonspecific porins can be distinguished from the specific and ligand-gated porins by their poor substrate selectivity and the high probability of these presenting an open conformation in the absence of any specific substrate (Nikaido, 2003). Representative major trimeric porins in E. coli include OmpF, an osmotically regulated cationselective porin (Fig. 1), and PhoE, a phosphate limitation-induced, anion-selective porin. 3.2.1.1. OmpH—PM0388 (OmpH1); PM0389 (OmpH2). Protein H, or porin H, is the a major OM protein present in the P. multocida envelope (Lugtenberg et al., 1986) and is a 37 kDa channel-forming, transmembrane porin with 38% amino acid similarity to the H. influenzae P2 porin (Vachon et al., 1986). Luo et al. (1997) purified native OmpH from a P. multocida A:3 strain and showed that it could elicit homologous protection in chickens. In contrast, the recombinant protein stimulated little protection. Alignment of OmpH sequences from 15 P. multocida serotypes indicated that the proteins were highly conserved (72– 100% identity), with major variation confined to two hypervariable regions predicted as large exposed loops. These loops were present in the predicted secondary structure. Vaccination studies showed that a cyclic synthetic peptide (Cyclic-L2), mimicking the predicted loop 2, induced 70% protection in chickens (Luo et al., 1999). We believe this to be the first report where a synthetic peptide mimicking a conformational epitope stimulated protection against a bacterial infection. Vasfi Marandi and Mittal (1997) also purified and characterised a 32 kDa major OMP from a P. multocida capsular serotype D strain. N-terminal sequencing of the protein showed that it had very high similarity to OmpH. Variations in the molecular mass of OmpH among different

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P. multocida strains (32–39 kDa) have also been reported. In a subsequent study a murine backpack tumour mouse model was used to deliver high levels of IgG monoclonal antibody specific for OmpH; the antibody protected mice against homologous challenge with P. multocida. OmpH expression is regulated by the Fur (ferric uptake regulator) protein and responds to glucose and iron concentrations (Bosch et al., 2001). Garrido et al. (2008) identified two copies of ompH (ompH1 and ompH2) (Table 1) in serogroup A strain PM108 and while contiguous on the genome, these genes were shown to be independently transcribed. Sequence analysis revealed significant differences compared to the corresponding sequences in the genome of strain Pm70. OM protein extracts from a fur, ompH1 and ompH2 triple mutant were used to immunise mice prior to challenge with a virulent heterologous P. multocida strain. The OM protein extract from the triple mutant conferred total protection. In the same study the heat killed triple mutant strain, was used as a vaccine, and induced 60% heterologous protection in mice. Lee et al. (2007) showed that in mice, full length recombinant OmpH, when expressed with an N-terminal thioredoxin (Trx) fusion, was able to elicit 70% protection against homologous challenge. In the same study, it was shown that three sub-fragments of the same recombinant OmpH protein stimulated lower protection in mice (30– 50%) without any preference for any of the sub-fragments. However, statistical significance was marginal. A 39 kDa protein (Cp39) found in a crude capsular extract of strain P-1059 (serotype A:3) was shown to be a capsule-associated adhesin, and a cross-protective antigen among P. multocida capsular serotype A strains. Based on properties of Cp39 and OmpH, the two proteins are identical, as the authors also suggested. Vaccination studies with OmpH, in both native and recombinant form, showed that it stimulated significant protection against heterologous challenge in chickens (Sthitmatee et al., 2008). Interestingly, protective immunity could be stimulated either with OmpH or in its absence. OM extracts from a triple fur, ompH1, ompH2 mutant were protective (Garrido et al., 2008), although the numbers of animals were small thus making statistical analysis difficult. Recombinant OmpH protein can also confer homologous protection (Lee et al., 2007; Sthitmatee et al., 2008). These observations by two independent groups may be related to absence of fur gene, which results in the over expression of Fur-regulated OM proteins (IROMPS). Garrido et al. (2008) showed that a single fur mutant evoked no protection. However, it is possible that the disruption of both ompH genes, causes other proteins to be expressed at elevated levels on the bacterial surface in amounts sufficient to trigger a protective response. Therefore, OmpH may well be one of several protective proteins. 3.2.2. Energy-dependent transport-efflux 3.2.2.1. TolC—PM0527 and PM1980 (IbeB). TolC proteins are key components of both the type I secretion system and efflux pumps (Fig. 1). The crystal structure of E. coli TolC

revealed a channel-tunnel that spans the bacterial OM and periplasm. This structure provides a large exit duct for protein export and multidrug efflux after its recruitment by the substrate-engaged inner membrane complexes (e.g. AcrAB), building a tripartite system (Fig. 1). There is also accumulating evidence that these systems are involved in multidrug resistance in bacteria (Piddock, 2006). In our laboratory, we identified two P. multocida TolC proteins, PM0527 and PM1980 (IbeB) (Table 1) (Hatfaludi et al., 2008). PM0527 was identified as an OM protein through mass spectrometry of the membrane fraction of in vitro grown bacteria (Boyce et al., 2006). Mutation of either pm0527 or pm1980 resulted in increased susceptibility to numerous chemical compounds. Both proteins clustered with other multidrug efflux proteins in a TolC family phylogenic tree. These proteins contain amino acids predicted to form a circular network of salt bridges at the periplasmic tunnel entrance similar to H. influenzae, where these oppositely charged residues are responsible for the anion selectivity in ion transport by the TolC homologue. 3.3. Binding proteins 3.3.1. Energy-dependent transporter-influx—iron transport In vivo conditions are characterised by low free iron concentration due to the presence of host iron-binding glycoproteins such as transferrin, lactoferrin and haptoglobin. Bacteria have evolved a range of iron sequestering systems to survive in vivo; each of these iron systems utilises an OM receptor, a periplasmic binding protein, and an inner membrane ABC transporter. In all cases the energy required to drive the transport is supplied by a TonB system (Fig. 1). Three types of iron sequestering systems have been identified, which utilise siderophore, transferrin or haemoglobin receptors. There are major differences in the uptake mechanisms. Haem and siderophores can be taken up by the bacterial cell as intact molecules, whereas iron must be extracted from transferrin prior to transport (Krewulak and Vogel, 2008). 3.3.2. Siderophore receptors Siderophores are secreted from the bacterial cell and function to remove iron from the host iron-binding glycoproteins. The iron–siderophore complex is initially bound by specific protein receptors on the surface of the bacterial cell and the iron is then transported into the cell. Two siderophore binding proteins from E. coli are FepA, a TonB-dependent Fe-siderophore (Fig. 1), and FhuA, a specific ferrichrome iron transporter. These proteins generally contain an OM-spanning b-barrel C-terminal domain, and a N-terminal ‘hatch’, which interacts with the inner membrane proteins (Koebnik et al., 2000; Nikaido, 2003). 3.3.2.1. 76 kDa, 84 kDa and 96 kDa proteins. When grown under conditions of iron deprivation, some P. multocida strains express a siderophore called multocidin. In an attempt to identify siderophore binding receptor proteins, Choi-Kim et al. (1991) reported three iron-regulated OM proteins with molecular masses of 76 kDa, 84 kDa and


96 kDa, which were expressed only when the bacteria were grown in iron-restricted medium, or in vivo. Convalescent turkey sera reacted with all three proteins. To demonstrate receptor function, radioactively labelled iron–multocidin (59Fe–multocidin) complex was used in binding assays and bound only bacteria, or OM protein extract from bacteria, grown in iron-restricted medium. However, specificity of binding could not be determined using inhibition assays. Antisera raised against OM protein extracts did not significantly alter the binding of the 59Fe–multocidin complex. Ikeda and Hirsh (1988) expressed a prominent 84 kDa OM protein from P. multocida A:3 grown under irondepleted conditions. This protein was immunogenic in turkeys and present in 15 different serotypes of P. multocida grown under iron-depleted conditions, although with varying molecular masses (84–92 kDa). It is possible that Ikeda’s 84 kDa protein and the 59Fe–multocidin complex binding protein are the same. 3.3.3. Transferrin and lactoferrin receptors The second mechanism of iron uptake involves surfaceexposed iron-binding proteins that interact directly with host iron-binding glycoproteins. Transferrin and lactoferrin each have a molecular mass of 80 kDa and are therefore too large to pass through the bacterial OM. Thus, additional steps are required to remove iron from these proteins at the external bacterial surface. Extraction of iron from transferrin and lactoferrin is usually facilitated by TbpB/ TbpA and LbpA/LbpB proteins, respectively. TbpB is a lipoprotein that is attached to the OM with an N-terminal lipid anchor and acts as an initial binding site for ironsaturated transferrin. In Nesisseria spp., TbpA is an integral, TonB-dependent OM protein that is predicted to have large surface exposed loops for binding and forcing the separation of iron from the transferrin (Krewulak and Vogel, 2008). 3.3.3.1. TbpA (Acc Nr. AY007725). Bovine P. multocida strains are able to utilise iron from bovine transferrin, but not from transferrin of other species. However, avian strains are unable to capture iron from any transferrin molecules including those from avian source. An 82 kDa protein was purified from OM extracts of bovine P. multocida strains grown under iron limiting conditions by affinity purification using biotin labelled transferrin (Ogunnariwo and Schryvers, 2001). This protein was identified as TbpA (Table 1). Unlike other species, P. multocida has a single, novel TbpA receptor capable of efficiently mediating iron acquisition from bovine transferrin without the involvement of a second receptor protein TbpB. The tbpA gene is flanked by a leucyl-tRNA synthase gene and an IS1060 element in contrast to other species where TbpB and TbpA are found in an operon. This genetic arrangement supports the idea that transposition may have been the mechanism by which this trait was introduced into P. multocida. Analysis of the TbpA amino acid sequence indicated that, like all transferrin receptors, it has a TonB box; however, it differs substantially from other members of the Tbp family. It is considerably smaller (ca. 20 kDa), as the predicted external loop is absent. Since

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there is no TbpB present in P. multocida, this suggests that portions of this loop in other TbpA proteins could be required for the interaction with TbpB. Veken et al. (1994) examined P. multocida B:2,5 haemorrhagic septicaemia strains for TbpA homologues. An 82 kDa protein was present in B:2,5 strains and was found to bind bovine transferrin. However, no proteins that bound transferrin could be isolated from a B:3,4 serotype strains. Shivachandra et al. (2005) using primers based on the P. multocida A:1 strain tbpA sequence (Acc Nr. AY007725), amplified the tbpA gene from chicken (A:1), buffalo (B:2), pig (D:1) and duck (F:3) P. multocida strains. Sequence analysis of the tbpA gene from serotype B:2 showed 98% identity to the A:1 strain. By contrast, Ewers et al. (2006) analysed 289 strains for distribution of virulence genes and no tbpA-specific PCR fragment was amplified from avian or pig strains. The tbpA gene was present in only 31.5% of the strains. Indeed, in this study tbpA was found exclusively in isolates from bovine (70.2% of all tested bovine strains), sheep (80%), or buffalo (57.1%) strains. 3.3.4. Haemoglobin receptors The iron component of haemoglobin, haem, can be utilised as a source of essential iron by many microorganisms (Krewulak and Vogel, 2008). Pathogenic bacteria commonly secrete exotoxins such as haemolysin that can lyse cells and release haem. P. multocida has been shown to lyse erythrocytes under anaerobic conditions. Known haem receptors in other species include HgbA of H. influenzae, HemR of Yersinia enterocolitica and HmbR of N. meningitidis. 3.3.4.1. HgbA—PM0300. A 109.7 kDa haemoglobin-binding protein was identified from a P. multocida serogroup D strain and designated as HgbA (Table 1) (Bosch et al., 2002b). In vitro and in vivo assays demonstrated that a P. multocida hgbA mutant bound haemoglobin to the same extent as wild type, although with slower kinetics. In agreement with this finding, the virulence of a P. multocida hgbA mutant was unaffected, suggesting the presence of other predicted haemoglobin receptors (PM0741, PM0745, and PM1282) (Table 1). However, a hgbA mutant from a library of signature tagged transposon mutants of a P. multocida bovine pneumonia strain that was fully attenuated in a mouse septicaemia model (Fuller et al., 2000). The hgbA gene (pm0300) is part of a single transcriptional unit together with pm0298 and pm0299. Mutation in either of the other two genes was lethal. This operon is iron regulated, and expression is triggered in the first 2 h following infection in mice (Bosch et al., 2002b). Expression of the P. multocida hgbA gene increased 6.0-fold under iron limitation (Paustian et al., 2001) and also during growth in blood of infected chickens (Boyce et al., 2002). 3.3.4.2. HgbB—Pm0337. From a P. multocida A:1 strain, Cox et al. (2003) identified another haemoglobin-binding protein of 113.5 kDa, designated HgbB (Table 1), that displayed 68 and 69% similarity to the H. influenzae HI0712 haemoglobin and H. influenzae HgpC haemoglobin–haptoglobin complex binding proteins. Bioinformatics analysis

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of hgbB revealed the presence of putative Fur and TonB binding sites. However, inactivation of the hgbB did not result in reduced bacterial virulence in mice, nor did it decrease the ability of P. multocida to bind haemoglobin. Furthermore, when used as a vaccine antigen recombinant insoluble HgbB did not confer any protection to mice against wild-type infection. A truncated version of HgbB was unable to bind haemoglobin, suggesting that the Cterminus of HgbB is important for binding. The expression of hgbB did not change under iron limitation (Paustian et al., 2002b) or in the blood of infected chickens (Boyce et al., 2002). It has been proposed that other haemoglobinbinding proteins are present in P. multocida that are indeed constitutively expressed (Cox et al., 2003). 3.3.4.3. Multiple haemoglobin-binding proteins. Eight proteins have been identified as predicted haemoglobin receptors: PM0040, PM0236, HgbA (PM0300), HemR (PM0576), PM0592, PM0741, PM1081, PM1282 and PM0142 (Table 1) (Bosch et al., 2004). Each of these sequences contains putative TonB boxes. When the eight genes were cloned and expressed in E. coli, all proteins bound haemoglobin or haem or both. However, when used as vaccine antigens, none of the proteins protected mice from P. multocida infection. 3.3.4.4. HasR—PM1622. HasR (Fig. 1) is the OM receptor of a specific haem uptake system that binds excreted haembinding proteins (haemophores) (Krieg et al., 2009). Prado et al. (2005) identified HasR as a 96 kDa surface expressed, and highly immunogenic OM protein of a P. multocida A:3 strain (Table 1). Sequence analysis identified the presence of TonB boxes and a Fur box, suggesting that its expression may be regulated in the presence of iron. The HasR protein was identified together with 4 other proteins grown under iron deficient conditions. However the other proteins, with molecular masses of 107 kDa, 78 kDa, 69 kDa, 48 kDa, respectively were not identified. 3.3.4.5. TonB complex—PM1188. In all cases of iron transport (siderophore, transferrin and haemoglobin), transfer across the OM is an active process that requires energy. The proton motive force of the cytoplasmic membrane is coupled to the OM via three proteins: TonB, ExbB and ExbD (Fig. 1). TonB acts as an energy transducer, while ExbB and ExbD stabilize and recycle TonB once the iron molecule passes through the OM. TonB plays a role in positioning the periplasmic binding proteins near the OM transporter, thereby facilitating the binding of the iron or, iron containing molecules (e.g. ferric sidephore) to its respective periplasmic binding protein (Krewulak and Vogel, 2008). The proteins of the TonB complex are not classical OM proteins and they are embedded in the inner membrane. However, they warrant attention as their function is strongly interconnected with the iron transport receptors. Bosch et al. (2002a) showed that the exbB, exbD and tonB genes are physically linked on the chromosome, as they are in other members of the Pasteurellaceae. Despite this, the genes are transcribed independently and their expression is regulated in response to iron. Each of these

genes is necessary for P. multocida virulence in mice, as inactivation of any one of them increased the LD50 by more than 1000-fold. These data show that each of the components of the ExbB (PM1186), ExbD (PM1187), TonB (PM1188) complex support the infection process in P. multocida. 3.3.5. Binding lipoproteins 3.3.5.1. PlpB-MetQ—PM1730. In our laboratory, we recently characterised the 30.1 kDa PlpB protein (Table 1) as a MetQ homologue that is involved in methionine binding (unpublished data). It represents the periplasmic binding component of a methionine uptake ABC transporter (Fig. 1). PlpB was initially identified by mass spectrometry from OM preparations (Rimler, 2001) and proposed to be a 39 kDa cross-protective antigen (Tabatabai and Zehr, 2004). It was identified independently by mass spectrometry in the OM fraction of in vitro grown P. multocida but at a mass of 30.1 kDa (Boyce et al., 2006). Although PlpB was initially identified as a crossprotective antigen, recombinant PlpB failed to protect either mice or chickens against P. multocida challenge, despite stimulating an antibody response (Wu et al., 2007). Indeed it is likely that the cross-protective antigen is in fact PlpE. We have shown (Hatfaludi et al., unpublished data) that a P. multocida plpB mutant was avirulent in chickens and mice. Virulence was restored by complementation with the intact plpB gene. In vitro biochemical analysis using radiolabelled methionine demonstrated that soluble recombinant PlpB can bind both L- and D-methionine and that non-labelled L- and D-methionine could competitively inhibit binding of their labelled counterparts. Uptake assays using radiolabelled methionine and whole intact cells indicated that bacterial cells utilise mainly Lmethionine. These results show that PlpB is a novel, methionine transporter that is required for the in vivo survival of P. multocida both in chickens and mice. 3.3.5.2. Putative binding lipoproteins. Bioinformatics analysis of the P. multocida Pm70 genome sequence indicated two putative lipoproteins (HemR and PM1578) (Table 1) belonging to the periplasmic binding protein superfamily (Babu et al., 2006). HemR (PM0576) is the haemoglobinbinding protein identified by Bosch et al. (2004). Expression of hemR increased 10.0-fold during growth under iron limitation (Paustian et al., 2001) and 3.9-fold during growth in the liver of infected chickens (Boyce et al., 2002). The second lipoprotein, PM1578, is a predicted periplasmic protein of unknown function. Expression of the pm1578 gene was reduced 1.8-fold under nutrient limitation (Paustian et al., 2002b). 3.4. Adhesins Attachment to host cells and/or extracellular matrix proteins is a primary prerequisite for bacterial infections; adhesins that mediate such adherence are potential virulence factors. Many bacteria produce multiple adhesins; e.g. H. influenzae expresses the adhesins Hap, Hia/Hsf,


HMW1/2, and haemagglutinating pili (Erwin and Smith, 2007) and E. coli the b-barrel membrane protein OmpX (Koebnik et al., 2000) (Fig. 1). 3.4.1. PtfA – PM0084, PM0085 – type 4 fimbriae Type 4 fimbriae (pili) are long, filamentous appendages that have been identified in many species of Gramnegative bacteria. They are often key structures involved in the attachment of bacteria to host cell surfaces (Fig. 1). Fimbriae consist of repeated fimbrial subunits which range in molecular mass from 15 kDa to 20 kDa and whose Nterminal sequences are highly conserved. They can be classified into two groups, the classical type 4 pili and type 4-like pili (or type 4B). Type 4 fimbriae have been used as vaccines against ovine footrot and bovine keratoconjunctivitis caused by Dichelobacter nodosus and Moraxella bovis, respectively. The P. multocida A:1 fimbrial subunit protein, PtfA, was purified by reverse phase HPLC and the first 21 amino acids of the mature protein determined by N-terminal amino acid sequencing (Ruffolo et al., 1997). The sequence was highly similar to the type 4 fimbrial subunit proteins from a number of pathogenic bacteria. The ptfA gene, which encodes a protein of 144 amino acids with a deduced molecular mass of 15.2 kDa, was subsequently cloned. Interestingly, the P. multocida PtfA signal sequence of 12 amino acids was uncharacteristically longer than those of most other classical type 4 fimbrial subunit proteins, a feature seen only in H. influenzae. The biological significance of this feature is not yet clear. Siju et al. (2007) cloned, sequenced and expressed in E. coli the ptfA gene from P. multocida serogroup B:2. Sequence alignment showed that the P. multocida B:2 and A:1 genes were 78.4% identical at the nucleotide level, with the first 200 bp being 100% identical. Rabbit antiserum raised against the recombinant PtfA (serotype B:2) protein detected an 18 kDa protein in P. multocida B:2, A:1 and Pseudomonas aeruginosa whole cell lysates. 3.4.2. ComE1—PM1665 A novel, putative fibronectin-binding protein ComE1 with a calculated molecular mass of 12.7 kDa was identified by panning a phage library of P. multocida DNA (Mullen et al., 2008b). Recombinant ComE1 (PM1665) bound to immobilised fibronectin and to collagen (type I), but not to a range of other matrix macromolecules, including fibrinogen, laminin, hyaluronic acid, and plasminogen. The ComE1 protein (Table 1) was predicted to contain two helix–hairpin–helix (HhH) domains in its Cterminus. The recombinant ComE1 expressed as a GST fusion protein bound to both soluble and immobilised fibronectin. In contrast to other fibronectin adhesins, ComE1 interacts with the integrin-binding fibronectin type III (FnII) repeats FnIII9–10, rather then the N-terminal type I repeats. The ComE1 binding domain included the predicted helix–hairpin–helix motif as well as the conserved amino acid motif, VNINTA. ComE1 was shown to be localised on the bacterial surface by transmission electron microscopy, and binding of P. multocida to fibronectin was almost completely inhibited by anti-ComE1 antiserum (Mullen et al., 2008b).

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In addition to functioning as an adhesin, ComE1 has been shown to be involved in bacterial competence and DNA uptake. ComE1 has significant amino acid similarity to related DNA binding and uptake proteins (ComEA and ComE) from both Gram-positive and Gram-negative bacteria (Mullen et al., 2008a). Furthermore, recombinant ComE1 bound DNA via the same helix–hairpin–helix motif required for fibronectin binding. The DNA binding was not sequence specific and was confined to double stranded DNA. ComE1 was cloned and expressed from five members of the Pasteurellaceae family, H. influenzae; Actinobacillus pleuropneumoniae; Aggregatibacter actinomycetemcomitans; Mannheimia haemolytica; and M. succiniproducens. All of the homologues bound to both fibronectin and double stranded DNA, with a DNA binding constant (Kd) of 7.3 mM for P. multocida. Mutation of the gene in A. pleuropneumoniae showed it to be critical for both natural competence and fibronectin binding, as the mutant had a 104-fold reduced transformation frequency and was unable to bind to fibronectin. Based on the homology, it is assumed that it is similar in P. multocida (Mullen et al., 2008a). 3.4.3. FhaB—FhaB1 (PM0057), FhaB2 (PM0059) The filamentous haemagglutinin protein of Bordetella pertussis plays a critical role in adhesion to host cells. It is both transported to the bacterial surface and also exported to the extracellular milieu and is predicted to increase bacterial dispersion and colonisation (Locht et al., 2001) (Fig. 1). Analysis of the P. multocida Pm70 genome sequence identified two predicted filamentous haemagglutinin genes (fhaB1 and fhaB2) (Table 1); the encoded proteins shared 45% identity (Tatum et al., 2005). Fuller et al. (2000) in a signature-tagged mutagenesis screen of a P. multocida bovine pneumoniae strain identified FhaB1 and FhaB2 as critical for virulence. The fhaB2 mutant was fully attenuated in a mouse septicaemia model. Furthermore, Tatum et al. (2005) showed that a fhaB2 mutant in an avian P. multocida A:3 strain was attenuated for virulence in turkeys by intranasal route, and to a lesser extent by intravenous route. The mutant and parent were equally resistant to killing by serum complement. Recently, the same group reported that a combination of three fragments from the N terminus of FhaB2 elicited protection in turkeys (Tatum et al., 2009a). 3.5. Putative adhesins 3.5.1.1. The Tad locus The Tad (tight adherence) macromolecular transport system, present in many bacterial and archaeal species, represents an ancient and major subtype of type II secretion. The tad genes encode the machinery that is required for the assembly of adhesive Flp (fimbrial lowmolecular-weight protein) pili (Fig. 1), which can be essential for biofilm formation and colonisation (Tomich et al., 2007). In P. multocida the tad locus encodes the following predicted OMPs: TadD (PM0846); TadF (PM0844); RcpA (PM0852); RcpB (PM0853); Flp1 (PM0855) (Table 1) (May et al., 2001). The expression of the P. multocida tadF and flp1 genes was reduced 2.0-fold in

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response to haemoglobin (Paustian et al., 2002b). A P. multocida A: strain flp1 mutant, generated by signaturetagged mutagenesis, was fully attenuated in chickens, but the mechanism of attenuation was not examined (Harper et al., 2003). 3.6. Membrane-associated enzymes A representative member of this group is OMPLA—an OM phospholipase of E. coli that is the first OM enzyme whose three-dimensional structure was solved (Fig. 1). The predicted role of OMPLA is the hydrolysis of phospholipids on the outer surface of the OM, where they are normally not present (Koebnik et al., 2000). 3.6.1. PM1426 In P. multocida, a putative member of this group is PM1426 (Table 1) that is predicted to encode a phospholipase A. This protein was identified experimentally as an OM protein by resolving the membrane fraction of in vitro grown bacteria (Boyce et al., 2006). The pm1426 gene was down regulated under nutrient limited conditions (Paustian et al., 2002b). A pm1426 mutant showed no attenuation in competitive growth assay in chickens, and recombinant PM1426 conferred no protection in chickens against homologous P. multocida challenge (Hatfaludi et al., unpublished data). 3.6.2. NanH—PM0663, NanB—PM1000 Many pathogenic bacteria have evolved mechanisms to evade recognition by the immune system based on the surface exposure of sialic acid. A common theme in this host-mimicry is the incorporation of sialic acid as a component of either the capsular polysaccharide or LPS. Sialidases (neuraminidases) are enzymes that liberate sialic acid from sialyl-conjugated glycoproteins, glycolipids, or colominic acids by cleaving alpha-ketosidic linkages. It has been hypothesised that sialidases contribute to the virulence of some pathogenic organisms, especially those that inhabit and invade mucosal surfaces (Schauer, 2009). P. multocida is a pathogen that can colonise the respiratory system of susceptible hosts. Most isolates show sialidase activity. Mizan et al. (2000) identified two sialidase genes, one encoding NanH (80 kDa) and the other NanB (120 kDa) (Table 1); both proteins were shown to be outer membrane associated in P. multocida A:3 (Fig. 1). The Cterminus of NanB shows amino acid similarity to a family of autotransporters, while NanH does not. Insertional inactivation of nanH did not reduce sialidase production, but the enzyme activity was reduced and bacteria showed reduced growth rate on 2-30 sialyl lactose. NanB demonstrated sialidase activity on both 2-30 and 2-60 sialyl lactose, while NanH cleaved only 2-30 sialyl lactose. Both enzymes had high ranges of pH tolerance, thereby enabling activity in different host cell compartments. It has been proposed that these sialidases have a nutritional function in P. multocida and scavenging of sialic acid may contribute to the ability of the organism to colonise and persist on mucosal surfaces. Steenbergen et al. (2005) suggested that P. multocida takes up sialidic acid through the activity of NanH and

NanB and through the tripartite ATP-independent periplasmic (TRAP) sialate transporter. Indeed sialic acid metabolism is essential for systemic pasteurellosis; the nanP (pm1709) (Table 1) mutant was attenuated in a mouse infection model. The attenuation is consistent with the hypothesis that LPS sialylation is dependent on external sources of sialic acid, since failure to transport exogenous sialic acid would result in the expression of unsialated LPS. However, there is no direct evidence for sialylation of LPS. Recently, Tatum et al. (2009b) investigated the importance of sialic acid uptake in pathogenesis in an avian strain of P. mutocida. Non-polar deletion mutants of nanP and nanU did not incorporate any radiolabelled sialic acid into cellular components. The mutants were also highly attenuated in turkeys challenged either intranasally or intravenously. 3.6.3. GlpQ—PM1444 The P. multocida GlpQ is a lipoprotein with glycerophosphodiester phosphodiesterase activity, that shows 90% similarity to H. influenzae protein D and 81% similarity to GlpQ of E. coli (Lo et al., 2004). H. influenzae protein D has been shown to degrade glycerophosphorylcholine from the surface of epithelial cells to choline. The choline is then used to decorate the H. influenzae LPS, thereby providing a camouflage mechanism to avoid the host immune system (Weiser, 2005). The H. influenzae protein D was shown to stimulate heterologous immunity against infection with H. influenzae clinical isolates in rats (Akkoyunlu et al., 1996). GlpQ in P. multocida A:1 was identified using the IVET system, indicating that the protein is expressed during infection. However, P. multocida GlpQ is not surface exposed. Vaccination of mice and chickens with recombinant lipidated or non-lipidated GlpQ, expressed and purified in E. coli, did not stimulate protection against homologous challenge. Serum raised against the OM preparations of P. multocida recognized GlpQ, indicating that the protein is present in the OM. As GlpQ is a lipoprotein, but not surface exposed, it is likely that the protein is attached to the OM but faces the periplasm (Lo et al., 2004). 3.7. Protein assembly machinery

b-Barrel proteins are present in the OMs of Gramnegative bacteria, mitochondria and chloroplasts. A central component required for the assembly of the proteins in the bacterial membrane is Omp85 (BamA). In N. meningitidis, Omp85 has been proposed to play either a direct role in the assembly of proteins in the OM, or in lipid assembly into the OM (Knowles et al., 2009). A member of the Omp85 superfamily, FhaC is shown in Fig. 1. 3.7.1. Oma87—PM1992 Oma87 of P. multocida belongs to the Omp85 family (Ruffolo and Adler, 1996) and shows 75% similarity to the D15 protective surface antigen of H. influenzae (Mitchison et al., 2000). Sequence analysis and proteinase K treatment indicated that the protein was surface exposed. Both native and recombinant Oma87 were strongly immunostained by


convalescent-phase serum. Antiserum produced against recombinant Oma87 in rabbits was used to passively immunise mice; the animals were protected against a low dose homologous A:1 strain challenge, but not against challenge with a heterologous A:3 strain (Table 1). Four sub-fragments of Oma87 from a P. multocida D:1 strain, were cloned and expressed in E. coli, as GST fusions. One of the sub-fragments, containing amino acids 18–130, reacted with convalescent chicken sera. However, vaccination with this sub-fragment failed to protect chickens against challenge with virulent P. multocida serotype A. 3.8. Other proteins 3.8.1. PlpE—PM1517 A lipoprotein, designated Pasteurella lipoprotein E (PlpE) from Mannheimia (formerly Pasteurella) haemolytica was found to be surface exposed and highly immunogenic in cattle (Confer et al., 2006) (Fig. 1), PlpE is a target for complement-mediated killing of M. haemolytica (Pandher et al., 1998). Addition of recombinant PlpE to the commercial M. haemolytica vaccine markedly enhanced the vaccine-induced immunity against experimental challenge with some Mannheimia serotypes (Confer et al., 2006). In P. multocida, Rimler (2001) reported a crossprotective antigen with a mass of 39 kDa identified in bacteria isolated from infected turkey tissue. This protein was initially incorrectly named as Pasteurella lipoprotein B (PlpB) (Tabatabai and Zehr, 2004). Indeed, proteomics analysis indicated that native PlpB has a molecular mass of 30.1 kDa (Boyce et al., 2006; Wu et al., 2007). The crossprotective antigen is likely to actually be the 39 kDa Pastuerella lipoprotein E (PlpE). Bioinformatics analysis of PlpE amino acid sequences from different strains of P. multocida showed that they shared 90–100% identity, but were only 24% identical to M. haemolytica PlpE. Immunisation with recombinant P. multocida PlpE conferred protective immunity, with 80–100% of mice, and 63– 100% of chickens protected against heterologous challenge (Wu et al., 2007). PlpE is the first P. multocida recombinant protein to stimulate high level cross-serotype protective immunity. 4. OMPs as vaccines At present there are commercial vaccines available for a range of diseases caused by P. multocida, including bacterins, toxoids and live attenuated vaccines. However, identity of protective antigens in P. multocida infections has remained surprisingly elusive. It is clear that LPS plays some role in immunity, but immunisation of mice with LPS elicits only partial protection, which is by definition restricted to the homologous serotype. To date, from many OM proteins tested in immunisation experiments (Table 1), only three have provided protection against P. multocida challenge. Soluble and insoluble PlpE both in mice and chickens protected against heterologous challenges (Wu et al., 2007). Peptides of OmpH protected mice against homologous, and chickens against heterologous challenges (Lee et al., 2007; Luo et al.,

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1999). Lastly, fragments of recombinant filamentous haemagglutinin protein FhaB2, elicited protection in turkeys (Tatum et al., 2009a). Yet, no vaccines based on individual protein antigens have been developed commercially. 5. Concluding remarks Knowledge of P. multocida OMPs has increased in the recent years. Many now have ascribed function as transporters, receptors involved in iron uptake or adhesins. However, bioinformatics prediction shows that the composition of P. multocida OM subproteome is far from comprehensive. Accordingly, further experimental efforts are required to define the full complement of OMPs and to ascribe a function to each protein. Acknowledgements We thank Priyangi Alwis for her ideas and assistance with the figure. Original research in the authors’ laboratory was supported by the Australian Research Council through the Centres of Excellence programme. References Akkoyunlu, M., Janson, H., Ruan, M., Forsgren, A., 1996. Biological activity of serum antibodies to a nonacylated form of lipoprotein D of Haemophilus influenzae. Infect. Immun. 64, 4586–4592. Al-Hasani, K., Boyce, J., McCarl, V.P., Bottomley, S., Wilkie, I., Adler, B., 2007. Identification of novel immunogens in Pasteurella multocida. Microb. Cell Fact. 6, 3. Babu, M.M., Priya, M.L., Selvan, A.T., Madera, M., Gough, J., Aravind, L., Sankaran, K., 2006. A database of bacterial lipoproteins (DOLOP) with functional assignments to predicted lipoproteins. J. Bacteriol. 188, 2761–2773. Bosch, M., Garrido, E., Llagostera, M., Perez de Rozas, A.M., Badiola, I., Barbe, J., 2002a. Pasteurella multocida exbB, exbD and tonB genes are physically linked but independently transcribed. FEMS Microbiol. Lett. 210, 201–208. Bosch, M., Garrido, M.E., Llagostera, M., Perez De Rozas, A.M., Badiola, I., Barbe, J., 2002b. Characterization of the Pasteurella multocida hgbA gene encoding a hemoglobin-binding protein. Infect. Immun. 70, 5955–5964. Bosch, M., Garrido, M.E., Perez de Rozas, A.M., Badiola, I., Barbe, J., Llagostera, M., 2004. Pasteurella multocida contains multiple immunogenic haemin- and haemoglobin-binding proteins. Vet. Microbiol. 99, 103–112. Bosch, M., Tarrago, R., Garrido, M.E., Campoy, S., Fernandez de Henestrosa, A.R., Perez de Rozas, A.M., Badiola, I., Barbe, J., 2001. Expression of the Pasteurella multocida ompH gene is negatively regulated by the Fur protein. FEMS Microbiol. Lett. 203, 35–40. Boyce, J., Harper, M., Wilkie, I., Adler, B., 2010. Pasteurella. In: Prescott, J.F. (Ed.), Pathogenesis of Bacterial Infections in Animals. Blackwell Publishing, IA, USA. Boyce, J.D., Cullen, P.A., Nguyen, V., Wilkie, I., Adler, B., 2006. Analysis of the Pasteurella multocida outer membrane sub-proteome and its response to the in vivo environment of the natural host. Proteomics 6, 870–880. Boyce, J.D., Wilkie, I., Harper, M., Paustian, M.L., Kapur, V., Adler, B., 2002. Genomic scale analysis of Pasteurella multocida gene expression during growth within the natural chicken host. Infect. Immun. 70, 6871– 6879. Boyce, J.D., Wilkie, I., Harper, M., Paustian, M.L., Kapur, V., Adler, B., 2004. Genomic-scale analysis of Pasteurella multocida gene expression during growth within liver tissue of chickens with fowl cholera. Microbes Infect. 6, 290–298. Carpenter, T., Khalid, S., Sansom, M.S., 2007. A multidomain outer membrane protein from Pasteurella multocida: modelling and simulation studies of PmOmpA. Biochim. Biophys. Acta 1768, 2831–2840. Choi-Kim, K., Maheswaran, S.K., Felice, L.J., Molitor, T.W., 1991. Relationship between the iron regulated outer membrane proteins and the

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