Department of Microbiology and Parasitology, School of Molecular and Microbial Sciences, University of Queensland, St Lucia, QLD 4072, Australia1
Agency for Food and Fibre Sciences, Animal Research Institute, Department of Primary Industries, Yeerongpilly, Australia2
National Veterinary Assay Laboratory, Dobutsu Iyakuhin Kensajo, 1-15-1 Tokura, Kokubunji-shi, Tokyo, Japan3
Author for correspondence: Michael P. Jennings. Tel: +61 7 3365 4879. Fax: +61 7 3365 4620. e-mail: jennings{at}mailbox.uq.edu.au
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ABSTRACT |
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Keywords: serotyping antigen, infectious coryza, outer-membrane protein
Abbreviations: AP, alkaline phosphatase; HA, haemagglutination activity; HI, haemagglutination inhibition
a The GenBank accession numbers for the sequences determined in this work are AF491817AF491827.
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INTRODUCTION |
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The haemagglutinin antigen of H. paragallinarum plays a key role in serotyping, immunity and pathogenicity. The two serotyping schemes, the Page (Page, 1962 ) and Kume (Kume et al., 1983a
) schemes, are both performed using haemagglutination inhibition (HI) tests (Blackall & Yamamoto, 1990
). The most widely used serotyping scheme, that of Page, groups H. paragallinarum isolates into three serovars, A, B and C. Considerable attention has also been paid to the role of haemagglutinin antigens in pathogenicity (Blackall & Yamamoto, 1997
) and as protective antigens. For Page serovar A organisms, there is a close correlation between HI titre and both protection (Kume et al., 1980
; Otsuki & Iritani, 1974
) and clearance of the organism from the nostrils of vaccinated chickens (Kume et al., 1984
). Purified haemagglutinin antigen from a serovar A organism has been shown to induce protective immunity (Iritani et al., 1980
). For both serovar A and serovar C, the assessment of mutants lacking haemagglutination activity has shown that the haemagglutinin antigen plays a key role in colonization (Sawata & Kume, 1983
; Yamaguchi et al., 1993
). However, neither the protein sequence nor the gene encoding the haemagglutinin antigen has been identified. In this paper we report the isolation, identification and full-length sequence of a gene encoding a haemagglutinin antigen (HagA) of H. paragallinarum, as well as the overexpression and purification of recombinant haemagglutinin (rHagA) from E. coli.
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METHODS |
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H. paragallinarum whole-cell haemagglutinin purification.
H. paragallinarum strain 0083 was grown overnight (37 °C/static) in TMB, a broth medium prepared by omitting agar from Test medium supplemented with chicken serum and reduced nicotinamide adenine dinucleotide (TM/SN) (Reid & Blackall, 1987 ). Cells were centrifuged, washed twice in phosphate-buffered saline (PBS, pH 7·2), resuspended in 50 mM Tris/HCl, 10% (v/v) glycerol, pH 8·0, and lysed by sonication. The cell lysate was fractionated using ammonium sulfate to precipitate proteins at 020%, 2040% and 40+% ammonium sulfate saturation. Precipitated proteins were resuspended and analysed by immunoblotting using mAb4D. The 020%, 2040% and 40+% precipitated fractions were run on a 12% SDS-polyacrylamide gel, according to Laemmli (1970)
. The proteins were transferred to a nitrocellulose membrane (Protran, Schleicher and Schuell), using semi-dry transfer (Trans-blot semi-dry transfer cell, Bio-Rad) according to the manufacturers instructions. mAb4D was used at a dilution of 1/1000 and the secondary antibody at 1/7500 (goat anti-mouse IgGAP, Promega). Activity of the AP (alkaline phosphatase) conjugate secondary antibody was detected by incubation with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Amresco) with a development time of 1618 h at room temperature.
N-terminal sequencing.
The 020% ammonium sulfate fraction was separated by SDS-PAGE and semi-purified by electroelution (Bio-Rad). The eluted 39 kDa protein band was run on a 12% Tris-Tricine polyacrylamide gel. The proteins were blotted onto PVDF membrane (Polyscreen PVDF Transfer Membrane, NEN Life Science Products) using CAPS buffer [10 mM 3-(cyclohexylamino)-1-propanesulfonic acid, pH 11) with a Trans-blot semi-dry transfer cell (Bio-Rad) according to the manufacturers instructions. The PVDF membrane was soaked in Milli Q water (Millipore) for 10 min with shaking and stained with 0·1% (w/v) Coomassie blue R250, 50% (v/v) methanol, 10% (v/v) acetic acid for 5 min. The membrane was destained [50% (v/v) methanol, 10% (v/v) acetic acid] and rinsed in Milli Q water. The N-terminal sequence of the
39 kDa band was obtained using a PE Biosystems 492cLC protein sequencer.
PCR and inverse PCR.
Primers HA1 and HA2 (Table 2), based on N-terminal sequence and alignments of the P5/OMP regions of closely related organisms, were used to amplify the 900 bp core region of the putative haemagglutinin coding sequence from strains 0083, 0222 and Modesto. Chromosomal DNA from each strain was digested overnight with the restriction enzymes BfaI or HindIII (New England Biolabs). Restriction enzymes were heat-inactivated, according to the manufacturers specifications, and the DNA precipitated with 3 M sodium acetate and ethanol. The digested chromosomal DNA was self-ligated to form circular DNA using T4 DNA ligase (Promega). Internal primers were designed to amplify either the upstream (HA5/HA6) or downstream (HA3/HA7) sequences of the core region (according to the position of the restriction enzyme site within the core region). These inverse PCR amplification products were identified on a 1 % (w/v) agarose gel and were purified and sequenced to obtain the full-length sequence of the H. paragallinarum haemagglutinin gene (hagA).
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DNA sequencing.
ABI Prism Big Dye Primer Cycle Sequencing Ready Reaction with AmpliTaq DNA Polymerase, FS' (PE Applied Biosystems) was used for DNA sequencing. Following ethanol precipitation, samples were sent to the Australian Genomic Research Facility (AGRF) for automated sequencing by an ABI 373A automatic sequencer (Applied Biosystems International, Perkin Elmer).
Cloning, overexpression and purification of HagA protein
Cloning.
H. paragallinarum strain HP14 (Page serovar A) was streaked on TM/SN (Reid & Blackall, 1987 ) and incubated at 37 °C overnight in the presence of 5% CO2. A lysate was prepared by harvesting one plate of HP14 into 100 µl sterile PBS and boiling this suspension for 10 min. The hagA gene was amplified from strain HP14 using primers HA12 and HA13 (Table 2
). The
1·1 kb PCR product was extracted using QIAQuick Gel Extraction kit (Qiagen) and cloned into pGEM-T Easy (Promega). The hagA gene was subcloned from the resulting plasmid into a pQE30 His-tag fusion vector (Qiagen) by digestion with BamHI (from primer) and PstI (from vector), generating pQE30hagA. This plasmid was transferred into an expression strain, E. coli M15(pREP4), by electroporation followed by selection on LuriaBertani (LB) agar supplemented with 0·05% (w/v) glucose, 100 µg ampicillin ml-1 and 25 µg kanamycin ml-1. A representative clone containing the recombinant plasmid was selected for purification of rHagA. Other recombinant DNA methods used were essentially as described by Maniatis et al. (1989)
.
Expression and purification.
A 10 ml culture of E. coli M15(pREP4) containing pQE30hagA was grown at 37 °C with shaking overnight in LB broth supplemented with 0·05% (w/v) glucose, 100 µg ampicillin ml-1 and 25 µg kanamycin ml-1. The overnight culture was subcultured into 500 ml LB broth supplemented with 0·05% (w/v) glucose, 100 µg ampicillin ml-1 and 25 µg kanamycin ml-1 and grown at 37 °C, with shaking, to an OD600 of 0·30·5. Expression of rHagA was induced at 37 °C with 0·5 mM IPTG for 4 h. Cell lysis and purification of the polyhistidineHagA fusion were as recommended in the manufacturers instructions (Qiagen QIA-Expressionizt). Briefly, bacteria harbouring pQE30hagA were collected and centrifuged at 1000 g for 10 min at 4 °C. The pellet was washed with PBS and resuspended in 50 ml denaturing lysis buffer (100 mM NaH2PO4,, 10 mM Tris, 8 M urea, pH 8·0) followed by incubation at room temperature for 1 h with agitation. The cell debris was pelleted by centrifugation at 1000 g for 10 min at 4 °C and the supernatant incubated with pre-equilibrated Ni-NTA resin (Qiagen) for 30 min at room temperature with agitation. The Ni-NTA resin was equilibrated by incubation with 15 ml denaturing lysis buffer containing 20 mM imidazole for 30 min at room temperature with agitation, then washed twice with five bed volumes of wash buffer (100 mM NaH2PO4, 10 mM Tris, 8 M urea, pH 8·0, 20 mM imidazole, 500 mM NaCl). It was resuspended in wash buffer and packed into a 10 ml column and washed with a further five bed volumes of wash buffer. The His-tagged protein was eluted in three bed volumes of elution buffer (100 mM NaH2PO4,, 10 mM Tris, 8 M urea, pH 8·0, 250 mM imidazole) in 2 ml fractions. All eluted fractions were analysed by SDS-PAGE for presence of rHagA. The pooled fractions containing rHagA were dialysed against PBS containing 0·05% (w/v) SDS overnight at 4 °C.
Analysis of purified recombinant HagA protein.
The recombinant His-tagged HagA protein was analysed by immunoblotting using mAb4D. Purified rHagA (1 µg) was run on a 12% SDS-polyacrylamide gel, along with whole cells of HP14 as a positive control. The proteins were transferred to nitrocellulose membrane (Protran, Schleicher and Schuell) using semi-dry transfer (Trans-blot semi-dry transfer cell, Bio-Rad) according to the manufacturers instructions. mAb4D was used at a dilution of 1/50 (due to deterioration of mAb4D) and secondary antibody at 1/2000 (goat anti-mouse IgGAP conjugate, Promega). Activity of the AP conjugate secondary antibody was detected by incubation with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Amresco) with a development time of 2 h at room temperature.
Haemagglutination assay.
The assay for haemagglutination activity (HA) was performed as previously described (Blackall et al., 1990 ). Briefly, 50 µl diluent was added to the appropriate wells of a U-bottomed microtitre plate. Purified rHagA protein (50 µl) was added to the first well of the row. Doubling dilutions of the purified protein were made across the plate followed by the addition of 50 µl 0·5% (v/v) glutaraldehyde-fixed chicken red blood cells to each well. The plate was incubated at room temperature for 3060 min. The haemagglutination titre was read as the highest antigen dilution giving at least 50% haemagglutination or one HA unit. One HA unit is that dilution of the antigen that results in a 50% mix of shield and button and is read as the reciprocal of the dilution immediately preceding the first button. Appropriate positive and negative controls were included in the haemagglutination assay. The positive control was a whole-cell suspension of strain 0083 (Page serovar A), prepared as described previously (Blackall et al., 1990
). The negative controls consisted of rHagA dialysis buffer (PBS, 0·05% SDS) and a non-related His-tagged purified protein from H. paragallinarum. mAb4D and high-titre hyper-immune rabbit reference serotyping antisera to strains 0083 and Modesto (serovars A and C, respectively) were used in a HI assay as described previously (Blackall et al., 1990
).
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RESULTS |
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The rHagA protein purified from the Ni-NTA column was free of contaminant proteins as determined by SDS-PAGE analysis (Fig. 6a, lane 3). From a 500 ml culture, approximately 23 mg rHagA protein was purified at a concentration of 0·58 mg ml-1 as determined using a bicinchoninic acid protein estimation kit (Pierce).
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The purified rHagA was tested for activity in a HA assay (Blackall et al., 1990 ). A titre of 2200 HA units per mg rHagA protein was obtained. The negative controls (buffer and unrelated His-tagged protein; see Methods) did not display haemagglutination activity, confirming that neither the His-tag motif nor the buffer formulation was responsible for the haemagglutination activity.
The haemagglutination activity of rHagA, in conjunction with the recognition of this protein by monoclonal antibody mAb4D in immunoblots, confirms the identity of the recombinant protein with the haemagglutinin of H. paragallinarum characterized by previous workers (Iritani et al., 1980 ; Takagi et al., 1991b
). HI assays did not demonstrate inhibition of haemagglutination of rHagA protein using mAb4D or reference polyclonal serotyping antisera.
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DISCUSSION |
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In the present study, a haemagglutinin antigen of serovar A H. paragallinarum, strain 0083, was identified and partially purified. A single protein band of the expected molecular mass corresponding to the haemagglutinin was identified by a haemagglutination-inhibiting monoclonal antibody, mAb4D (Takagi et al., 1991a ), in a Western blot. The band was isolated and the N-terminal sequence determined. The resultant N-terminal sequence identified this haemagglutinin of H. paragallinarum as a member of a family of outer-membrane proteins (the OmpA family) including P. (M.) haemolytica PomA and A. actinomycetemcomitans Omp29, as well as H. influenzae P5, which functions as an adhesin (Webb & Cripps, 1998
). The full-length sequence of the gene encoding this haemagglutinin (hagA) was obtained using inverse PCR technology. The hagA gene was sequenced and the deduced amino acid sequence contained a sequence identical to that originally obtained by N-terminal sequencing of the partially purified single band identified by mAb4D (Fig. 1
), confirming that the cloned gene encoded the same protein that was originally identified by mAb4D and N-terminal sequencing (Fig. 2
).
The hagA gene of H. paragallinarum strain HP14 was cloned and overexpressed in E. coli. The 37 kDa purified recombinant protein (rHagA) was recognized, as a single band, by the serovar A anti-haemagglutinin monoclonal antibody, mAb4D. The size of rHagA is consistent with the estimated molecular mass deduced by Iritani et al. (1980)
. A small size difference was observed between the strain 0083 haemagglutinin (this study; Fig. 1
, lane 2 and Iritani et al., 1980
) and the haemagglutinin protein of strain HP14 (Fig. 6a
, lane 2; 6b, lane 2). This molecular mass difference is presumably due to either the amino acid sequence differences in the hagA genes between the two strains or perhaps post-translational modifications (see below).
The sequencing of the 11 serotyping reference strains revealed a surprisingly small degree of sequence variation amongst the strains, given that the haemagglutinin is presumed to be the major H. paragallinarum serotyping antigen. We had expected to find amino acid sequence variations that correlated with the serological differences, but only limited variation was observed and, apart from a single conserved residue in serovar B sequences (Arg88), none of these sequence variations correlated with the serological groupings of the strains. The serotyping reactions have two components: the haemagglutinin that aggregates the chicken red blood cells, and the antisera which are added to the reaction to inhibit the haemagglutination activity. The sequence variation within the HagA protein did not explain the phenotypic differences observed among the strains in the HI assay. Thus, it is clear that alternative explanations are required to explain the antibody binding that differentiates the serovars in a HI assay; for example (a) post-translational modifications of the HagA protein may enable expression of particular phenotypes to allow serotypic variation amongst strains to develop and (b) another surface protein(s) may be involved in the serotypic differences observed rather than the haemagglutinin protein identified, i.e. steric hindrance of the haemagglutinin function may occur if another membrane protein interferes with the interaction between serotyping antibodies and the haemagglutinin protein. If this is the case, then the difference between the serovars may result from variations in the expression or sequence of these other proteins. Alternatively, (c) there are multiple haemagglutinins (Kume et al., 1983b ).
The idea that a post-translational modification may occur has some support in the literature. Recent reports of post-translational modifications of prokaryotic surface proteins have opened up a new aspect of microbial pathogenesis. There are a growing number of reports suggesting not only that many bacteria glycosylate their surface proteins but also that this process can be critical to pathogenicity (Tuomanen, 1996 ). Examples include significant pathogens such as Neisseria spp. (Power et al., 2000
; Stimson et al., 1995
), mycobacteria (Dobos et al., 1996
) and streptococci (Erickson & Herzberg, 1993
). A post-translational modification of Campylobacter coli flagellin, which involves a terminal sialic acid moiety, has also been identified (Doig et al., 1996
). Variation in the glycosylated structures between strains is implicated in the discrimination of serotype-specific epitopes of C. coli (Doig et al., 1996
). The haemagglutination activity of Myxococcus xanthus fimbriae has also been shown to be inhibited by the addition of specific sugars, which indicates a function for glycosylation in agglutination by this organism (Dobson et al., 1979
). Carbohydrate analysis has previously revealed the presence of sialic acid in the purified haemagglutinin from whole cells of H. paragallinarum (Iritani et al., 1980
). In addition, Iritani et al. (1980)
found that treatment of purified haemagglutinin with glycosidase inhibited haemagglutination activity. This suggests that the haemagglutinin protein may be a glycoprotein, and if the region of the protein involved in agglutination or haemagglutination inhibition carries the carbohydrate moiety, then the differences in glycosylation could give rise to serovar specificity.
Takagi et al. (1991b) have previously reported the cloning of a genomic fragment containing a serovar A haemagglutinin of H. paragallinarum strain 221 in the vector pBR322 and expression of recombinant haemagglutinin in E. coli strain C600. This E. coli strain was shown to possess haemagglutination activity and protected chickens against infectious coryza upon challenge (Takagi et al., 1991b
). However, neither the sequence of the gene nor the resultant protein was obtained. Takagi et al. (1991b)
also did not purify the recombinant protein, but instead used E. coli expressing the protein as the immunogen in vaccination trials and were able to induce HI antibody in the chickens. In the case of our clone, the rHagA was overexpressed by E. coli M15(pREP4) in an insoluble form, most probably as inclusion bodies (data not shown) due to the lack of the H. paragallinarum signal sequence, which prevents the protein from being secreted and presented on the outer membrane of E. coli. This is consistent with our observations that the E. coli M15(pREP4) cells expressing rHagA are unable to directly haemagglutinate chicken red blood cells (data not shown) unlike the clone of Takagi et al. (1991b
).
The deduced amino acid sequence of HagA is closely related to that of the H. influenzae P5 protein, as shown in Fig. 3. It is believed that the P5 outer-membrane protein of non-typable H. influenzae (NTHi) may play a role in NTHi pathogenesis by acting as an adhesin that binds to respiratory mucin (Webb & Cripps, 1998
). Due to the significant amino acid sequence similarity between the H. paragallinarum HagA and H. influenzae P5 proteins, it is possible that a similar mechanism may play a role in infection by H. paragallinarum. A role for H. influenzae P5-mediated attachment to host structures is suggested by the observation that sialic-acid-containing oligosaccharides of respiratory mucin bind P5 (Webb & Cripps, 1998
). Although the host ranges are quite different for these organisms, it is possible that such highly conserved proteins may share similar functions.
In conclusion, the hagA gene has been cloned and overexpressed in E. coli and the recombinant protein has been shown to be a functional haemagglutinin. Physicochemical and immunological analyses are consistent with this gene encoding the 39 kDa haemagglutinin previously described by other workers (Takagi et al., 1991b
; Iritani et al., 1980
). The full-length DNA sequence has been determined in 11 H. paragallinarum serotyping strains. There is no correlation between sequence variation in this gene and the serovar of the typing strain sequenced, suggesting that the immunological differences that underlie the Page and Kume serotyping schemes do not rely solely on antibodies directed at this amino acid sequence. Serovar differences may be due to other haemagglutinins in H. paragallinarum, three of which have been previously described in serovar A organisms (Kume et al., 1983b
), or blocking antibodies directed at alternative proteins or post-translational modifications. Investigations into the use of the rHagA protein as a vaccine against infectious coryza are under way.
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ACKNOWLEDGEMENTS |
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Received 5 July 2001;
revised 21 January 2002;
accepted 20 February 2002.
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