Genetic organisation of the capsule transport gene region from Haemophilus paragallinarum

DE SMIDT, O., ALBERTYN, J., BRAGG, R.R. & VAN HEERDEN, E. 2004. Genetic organisation of the capsule transport gene region from Haemophilus paragallinarum Onderstepoort Journal of Veterinary Research, 71:139–152 The region involved in export of the capsule polysaccharides to the cell surface of Haemophilus paragallinarum was cloned and the genetic organisation determined. Degenerate primers designed from sequence alignment of the capsule transport genes of Haemophilus influenzae, Pasteurella multocida and Actinobacillus pleuropneumoniae were used to amplify a 2.6 kb fragment containing a segment of the H. paragallinarum capsule transport gene locus. This fragment was used as a digoxigenin labelled probe to isolate the complete H. paragallinarum capsule transport gene locus from genomic DNA. The sequence of the cloned DNA was determined and analysis revealed the presence of four genes, each showing high homology with known capsule transport genes. The four genes were designated hctA, B, C and D (for H. paragallinarum capsule transport genes) and the predicted products of these genes likely encode an ATP-dependent export system responsible for transport of the capsule polysaccharides to the cell surface, possibly a member of a super family designated ABC (ATP-binding cassette) transporters.


INTRODUCTION
Haemophilus paragallinarum is a gram-negative, polar staining, non-motile bacterium.In 24-h cultures it appears as short rods, or coccobacilli, 1-3 µm in length and 0.4-0.8µm in width, with a tendency for filament formation.This organism causes an acute respiratory disease of chickens known as infectious coryza (IC), a disease first recognised as a distinct entity in the late 1920s.Since the disease proved to be infectious and primarily affected the nasal passages, the name "infectious coryza" was adopted (Blackall 1989).The major economic effect of the disease is an increased culling rate in meat chickens and a reduction in egg production (10-40 %) in laying and breeding hens.The disease is limited primarily to chickens and has no public health significance (Yamamoto 1991).All the commercially available bacterins against IC consist of inactivated broth cultures of a combination of two or three different serotypes.Although vaccines against IC have been used in South Africa since 1975, it became apparent in the 1980s that the vaccines were becoming less effective in controlling the disease (Bragg, Coetzee & Verschoor 1996).This may be due to the emergence of previously unknown serovars, serogroups or changes in the population dynamics.Vaccine efficiency is therefore a problem and an alternative to available vaccines is needed.
Capsules are found on the surface of a wide range of bacteria and are often important for virulence.These polysaccharide structures have been the subject of intensive investigation because of their usefulness as vaccines for prevention of bacterial infections (Lee 1987;Boulnois & Roberts 1990).Many researchers sought to understand the role of the capsule in virulence by identifying the genes involved in capsular polysaccharide export and biosynthesis.The genetic organization of the group II capsule gene loci of Haemophilus influenzae type b (Kroll, Zamze, Loynd & Moxon 1989;Kroll 1992), Escherichia coli K1 and K5 (Boulnois, Roberts, Hodge, Hardy, Jann & Timmis 1987;Jann & Jann 1990), Pasteurella multocida M1404 (B:2) (Boyce, Chung & Adler 2000) and Actinobacillus pleuropneumoniae serotype 5a (Ward & Inzana 1997) have been determined and are very similar.In each of these species, a central DNA segment necessary for capsular polysaccharide biosynthesis is flanked by DNA encoding proteins for capsule export.Substantial homology exists in the genes required for capsular polysaccharide export among these species, suggesting a common evolutionary origin (Frosch, Edwards, Bousset, Kraube & Weisgerber 1991).
Genetically defined acapsular mutants have been shown to have reduced virulence in a number of organisms (Boyce et al. 2000).A mutant defective in the export of the P. multocida capsule was constructed by allelic exchange and virulence assays showed the acapsular P. multocida to be 10 6 fold less virulent than their encapsulated counterparts (Boyce & Adler 2000).Similar studies have been conducted on the bexA gene of H. influenzae (Kroll, Hopkins & Moxon 1988).A frame shift mutation engineered at a restriction site within the open reading frame resulted, when introduced into the cap locus in the chromosome, in the expression of a mutant phenotype.The noncapsulated mutants of A. pleuropneumoniae reported by Inzana, Todd & Veit (1993) showed extreme stability and induced a protective immune response without any symptoms of disease.This not only proves the capsule's involvement in virulence but also offers the opportunity to investigate the possibility of producing live vaccines.
In an attempt to understand the genetic organization of the capsular genes of H. paragallinarum degenerate PCR primers, based on the capsule loci of H. influenzae, A. pleuropneumoniae and P. multocida, were used to amplify a section of the capsule transport genes of H. paragallinarum.This section was employed as a probe to clone the full-length transport region.

Bacterial strains
Haemophilus paragallinarum strain 1742, obtained from the Department of Poultry Health, University of Pretoria, South Africa, was grown in TM/SN medium (1 % biosate peptone, 1 % NaCl, 0.5 % glucose, 0.1 % starch and 0.0005 % thiamine solution, oleic acid-albumin complex and chicken serum as supplements) as described by Blackall & Yamamoto (1990), in which 1.5 % agar was used to solidify the medium if required.In liquid culture the organisms were grown without aeration and on solid media in a candle jar at 37 °C.Escherichia coli strain Sure2 (Stratagene) was grown with aeration in Luria-Bertani (LB) broth (Sambrook, Fritsch & Maniatis 1989) under selective pressure with 60 µg/ml ampicillin in liquid and solid media when required.

Preparation and analysis of genomic and plasmid DNA
Genomic DNA was prepared from 20, 5 ml liquid cultures of H. paragallinarum grown for 16 h (Towner 1991).The cells were harvested by centrifugation at 3 000 g for 10 min at 4 °C and the mass of the pellet was determined.The pellet was washed in TEbuffer (10 mM Tris-HCl, 1 mM EDTA) pH 8 and centrifuged again at 3 000 g for 5 min at 4 °C.The pellet was re-suspended in 40 ml/0.5 g cells buffer (50 mM Tris-HCl, pH 8, 0.7 mM sucrose) and lysozyme (20 mg/ml) was added before the suspension was incubated on ice for 5 min.Six hundred microlitres EDTA (0.5 M, pH 8) and 500 µl 10 % SDS were added for each 0.5 g cells, gently mixed and placed on ice for 5 min.After the addition of 10 ml/0.5 g cells digestion buffer (1 % SDS, 50 mM Tris-HCl pH 8, 0.1 M EDTA, 0.2 M NaCl, 0.5 mg/ml proteinase K), the suspension was incubated at 55 °C for 3-16 h with mild shaking.One time the volume of pH calibrated phenol (pH 7.8) was added to the lysate and incubated a further 3 h at 25 °C with constant inversion.Cell debris was removed by centrifugation at 4 000 g for 10 min and the supernatant mixed with 0.1x the volume 5 M NaCl and placed on ice for 5 min.Genomic DNA was precipitated with 10 ml 100 % ethanol, spooled and washed in 1 ml 70 % ethanol.After drying, the pellet was suspended in 500 µl/ 0.5 g cells TE-buffer and incubated at 50 °C for 1 h or kept at 4 °C overnight before use.
Plasmid DNA was isolated by a rapid alkaline lysis method described by Sambrook et al. (1989) and suspended in TE-buffer containing 10 µg/ml RNase.Genomic and plasmid DNA were analysed by restriction enzyme digestion.Plasmid DNA was digested with EcoRI or HindIII for 1 h, while genomic DNA was digested using BamHI, EcoRI, HindIII, PstI or XbaI for 3-16 h.All the enzymes used in these digestions were obtained from Roche Molecular Biochemicals.

PCR analysis and cloning techniques
PCR analysis was performed in a Perkin-Elmer Geneamp 2400 thermocycler.Haemophilus paragallinarum genomic DNA (60 ng) was used as template and PCR reactions were carried out in 50 µl volumes.The reaction mixtures consisted of a 10x dilution of reaction buffer (100 mM Tris-HCl, 15 mM MgCl 2 , 500 mM KCl, pH 8.3), 2 pmol of each degenerate primer (Table 1) in different combinations, 0.2 mM dNTP mixture and 5 U of Taq polymerase (Roche).The reaction conditions consisted of an initial denaturation cycle of 94 °C for 5 min followed by 25 cycles of 94 °C for 30 s, 45 °C for 30 s, 72 °C for 2 min and a final elongation cycle of 72 °C for 5 min.The same reaction constituents and conditions were used for amplification of the partial H. paragallinarum capsule transport gene locus and for production of a DNA probe for screening.
PCR products were purified and DNA fragments were recovered from agarose gels with the GFXä-PCR DNA and gel band purification kit (Amersham Pharmacia Biotech).Purified fragments were cloned into either pGEM-T Easy or pGEM-3Z (Promega).
Escherichia coli strain Sure2 was grown to early log phase at 18 °C in SOB-media as described by Hanahan (1983).Competent E. coli cells were prepared according to the method of Inoue, Nojima & Okayama (1990).

Blotting techniques
Southern hybridisation was used as a method to identify fragments in digested genomic DNA that encode the capsule transport gene locus and colony hybridisation to identify positive clones containing the recombinant plasmids.
Genomic DNA was digested with BamHI, EcoRI, HindIII, PstI or XbaI and the fragments separated by agarose gel electrophoresis.The DNA was transferred to a Magnacharge nylon membrane (Micron separations, Inc.) by 1 h downward capillary transfer as described by Chomczynski (1992).DNA was linked to the membrane with the GS gene linker™ (BIO-RAD) prior to hybridisation.
Colony blotting to screen for the presence of clones containing the transport gene locus was performed on transformants grown for 16 h on LB plates containing ampicilin.Blotting proceeded as described by the DIG system users' guide for filter hybridisation (Roche Molecular Biochemicals).Colonies were lifted from the growth media and fixed on a magnacharge nylon membrane.The membrane was subjected to lysis in 10 % SDS and denaturation solution (0.5 M NaOH, 1.5 M NaCl) followed by neutralisation (1 M Tris-HCl pH 7.5, 1.5 M NaCl) and washing twice in SSC until all the cell debris was removed.
Hybridisation and colorimetric detection were performed as described in the DIG Nucleic Acid Detection Kit (Roche Molecular Biochemicals).

Probe labelling and screening methods
The 2.6 kb fragment used as a hybridisation probe was amplified from H. paragallinarum genomic DNA with primers HctD-1F and HctA-1R (Table 1).This fragment was prepared as the Hct-probe by random prime labelling with digoxigenin using the DIG labelling and detection kit.The amount of labelled DNA was determined by comparison of the intensity of the spots of a serial dilution of the Hct-probe to that of a labelled control (supplied by the manufacturer).

Sequencing and analysis
Plasmid construct pHctA-D was used as a template for sequencing.Sequencing reactions were performed with the ABI Prism Big Dye terminator V3.0 cycle sequencing ready reaction kit and data collected on an ABI Prism 377 DNA sequencer (Perkin-Elmer biosystems).Data was analysed using Sequencing analysis V3.3.Sequences were reverse complemented and compared by using Sequence Navigator V 1.0.1 and assembled using Auto-assembler V1.4.0 and DNAssist V2.0.

Sequence submission
Sequence of the transport gene locus was submitted to GenBank, accession number AY116594.

Partial amplification of the H. p paragallinarum capsule transport gene region
Genomic DNA was isolated from H. paragallinarum and used as a template for PCR amplification of the H. paragallinarum capsule transport genes.The capsule transport gene sequences of H. influenzae (bexA-D genes), A. pleuropneumoniae (cpxA-D genes) and P. multocida (hexA-D genes) were obtained from GenBank (accession no.X54987, U36397 & AF067175) and submitted to a multiple sequence alignment using DNAssist V 1.0.2.Six degenerate primers were designed (Table 1) from areas in these aligned gene sequences where the sequence was highly conserved.
The PCR performed with different oligonucleotide combinations (Fig. 1A), showed amplification of fragments of expected sizes in lanes 1 (~2.6 kb), 2 (~2.3 kb), 3 (~1.9kb), 4 (~1.6 kb) and 6 (~1.1 kb).The relative position of each of these fragments in the proposed H. paragallinarum transport gene region is indicated in Fig. 1B.Lanes 5 and 7 showed either non-specific or no amplification.More than one band was visible in some lanes due to non-specific priming and a low annealing temperature of 45 °C.The estimated ~2.6 kb fragment amplified by the oligonucleotides HctD-1F and HctA-1R (Fig. 1A, lane 1), was cloned into pGEM-T Easy and designated pHct.The nucleotide sequence of this frag-ment was determined and analysis revealed considerable homology with the capsule transport genes of related organisms (H.influenzae, A. pleuropneumoniae and P. multocida).This high degree of homology among the four species indicated that the sequenced 2638 bp insert in pHct represented part of the capsule transport gene region of H. paragallinarum.By comparison with the capsular transport genes of P. multocida, this fragment contained homologues of hexC and hexB as well as small regions of the 3' end of hexD and the 5' region of hexA.

Construction of a mini-library to isolate the entire capsule transport gene region
To facilitate the cloning of the full-length capsular transport region, the pHct insert was used as a probe (designated Hct) in southern blotting followed by colony hybridisation.Genomic DNA of H. paragallinarum was digested with five different restriction enzymes, transferred to a nylon membrane and hybridised with a digoxigenin labelled Hct-probe under stringent conditions.Southern blotting and hybridisation indicated that a HindIII fragment of ~6.15 kb (Fig. 2, lane 3) hybridised to the Hctprobe.Hybridisation products visible in lanes 1, 2, 4 and 5 at a position of ~21 kb correspond to the relative position of undigested genomic DNA or unresolved large restriction fragments when using restriction enzymes BamHI, EcoRI, PstI and XbaI.
The HindIII fragments resolved between 6 kb and 6.5 kb were excised from the gel, purified and cloned into vector pGEM-3Z to construct a minilibrary.Colony hybridisation was used as a screening method to identify positive clones containing the transport genes.A total of 93 colonies were visible within 1 day of transformation and two colonies showed hybridisation with the Hct-probe after screening under stringent conditions.
Plasmid DNA, extracted from the above-mentioned colonies and digested with HindIII, revealed the presence of a ~6.15 kb insert.To confirm that these plasmid constructs did contain the capsule transport region, the 5' and 3' terminal regions were sequenced.Sequencing confirmed that both clones were identical and also gave an indication of the orientation in which the ~6.15 kb fragment was ligated into the vector.Sequence alignment to known capsular genes, using the above-mentioned sequences, indicated that the ~6.15 kb fragment did in fact contain the relevant capsule region.
PCR reactions were performed to determine which part or parts of the Hct-probe features in the The PCR fragments expected were as follows: 2 600 bp (lane 1, Fig. 1A), 2 300 bp (lane 2, Fig. 1A), 1 900 bp (lane 3, Fig. 1A), 1 600 bp (lane 4, Fig. 1A), 1 100 bp (lane 5, Fig. 1A), 1 100 bp (lane 6, Fig. 1A), 860 bp (lane 7, Fig. 1A) ~6.15 kb fragment.These PCR reactions were performed using sequence specific oligonucleotides designed according to the sequence obtained from the 2.6 kb pHct fragment.Sequence specific oligonucleotide HctD-1R was used in combination with T7 (Fig. 3, lane 1) and HctA-1F in combination with SP6 (Fig. 3, lane 2) (SP6 and T7 have binding sites on opposite sides of the multiple cloning site of pGEM-3Z).Amplification of two bands were visible, a ~1.5 kb band in lane 1 representing the segment upstream and a ~2.1 kb band in lane 2 indicating the segment downstream from the Hct-probe sequence.These results and the high degree of sequence homology with the transport genes of related organisms, verified that the ~6.15 kb fragment represents the entire H. paragallinarum capsule transport region and was designated pHctA-D.
The nucleotide sequence of the full-length capsular transport region was determined through primer walking using the ~6.15 kb HindIII restriction fragment of pHctA-D.Analysis of the complete sequence revealed that the H. paragallinarum capsule transport gene region is 3 792 bp in length with a GC content of 37 %, comprising four open reading frames representing the four capsule transport genes designated hctDCBA (Fig. 4 and 5).

DISCUSSION
Analysis of the H. paragallinarum hctDCBA gene cluster revealed a clear bias toward codons rich in nucleotides A and T (37 % GC content) consistent with the 39 % GC content of the H. influenzae capsule gene cluster (Kroll, Loynds, Brophy & Moxon 1990) and 37 % GC content of the H. influenzae genome overall (Roy & Smith 1973).It also correlates with the calculated GC contents of A. pleuropneumoniae (40 %) and P. multocida (37 %).The gene lengths and region size correlate well with those of related organisms, all belonging to the family Pasteurellaceae (Table 2).Blast searches of the combined, non-redundant nucleotide and protein databases at the National Centre for Biotechnology Information (NCBI) indicated that H. paragallinarum hctDCBA were highly homologous at both the nucleotide and amino acid levels to H. influenzae bexDCBA (Kroll et al. 1990), A. pleuropneumoniae cpxDCBA (Ward & Inzana 1997) 6 Alignment of the relatively hydrophilic portions of HctB, BexB and OppB.The number in brackets is the position of the first amino acid in each sequence.Identical amino acids in all three genes are boxed, and the matches of the OppB sequence to the Dassa/Hofnung consensus are underlined GenBank accession numbers of capsular transport sequences from related bacterial species seria meningitidis ctrABCD (Frosch, Muller, Bousset & 1992) (Table 2).
The predicted amino acid sequences of the hct genes showed significant identity with the capsule transport genes of related organisms.The predicted HctA protein showed on average 77 % identity and 85.4 % similarity with the A proteins of H. influenzae, A. pleuropneumoniae and P. multocida.HctA contains the ATP-binding domains A (GXLGRXGXGKS) and B (XXDNLRFI) (Walker, Sarste, Runswick & Gay 1982) at amino acids 1080-1092 and 1131-1138 respectively (Fig. 5, regions C1 and C2), which are conserved in the BexA and CpxA homologues (Kroll et al. 1990;Fath & Kolter 1993;Ward & Inzana 1997).The nucleotide homology as well as the high degree of similarity between homologous proteins, supports the speculation that hctA might encode an ATP-binding protein component of a polysaccharide export apparatus.
HctB protein showed an average of 58 % identity and 84.4 % similarity with its corresponding homologues and is predicted to be a hydrophobic protein over most of its length, containing at least six potential membrane-spanning a-helical domains (Kyte & Doolittle 1982;Kroll et al. 1990 ).A short relatively hydrophilic region starting at amino acid 990 (Fig. 5, region B) aligned with a similar region in OppB of Salmonella typhimurium (Hiles, Gallagher, Jamieson & Higgins 1987) and BexB from H. influenzae (Kroll et al. 1990).Furthermore, each showed a marginal sequence similarity to a consensus thought to be involved in intermolecular interactions in the oligopeptide transporter (Dassa & Hofnung 1985).Fig. 5 (region B) shows the position of this sequence on HctB and Fig. 6 shows an alignment of the relatively hydrophilic portions of HctB, BexB and OppB.HctB is therefore a candidate for an integral innermembrane component of the putative polysaccharide exporter.
The multiple protein sequence alignment of HctC with the respective C proteins of H. influenzae, A. pleuropneumoniae and P. multocida showed a lower homology (average of 45.8 % identity and 68.2 % similarity) in comparison to HctA and B with their corresponding homologues.Transposon mutagenesis of bexC (Kroll et al. 1990) suggested that this gene might be a periplasmic protein.Prediction of protein subcellular localisation of the HexC protein performed with PSORT (Nakai & Kahehisa 1991), suggested an inner membrane protein, possibly with a periplasmic domain, concurring with the transposon mutagenesis data on BexC (Chung et al. 1998).
The N-terminus of BexC containing phosphatase activity suggests that the protein is either excreted into the periplasm with cleavage of an N-terminal leader peptide or anchored in the bacterial inner membrane by an uncleaved N-terminal domain to protrude into the periplasm.It is therefore a candidate for a periplasmically orientated component of a capsular polysaccharide exporter.Ward & Inzana (1997) predicted the CpxC protein of A. pleuropneumoniae to be relatively hydrophilic with hydrophobic domains near the N and C-termini that may serve as membrane anchors.Three long hydrophobic stretches of amino acid sequence with membrane-spanning potential allowing the possibility of anchoring at more than one site have been identified in BexC (Kroll et al. 1990).Similar stretches of sequence are present in HctC at amino acids 421-442, 458-475 (Fig. 5, regions A1 and A2) at the proposed N-terminal and 753-777 at the C-terminal (Fig. 5, region A3).Considering this information and the facts known about the HctC homologues, it is proposed that this protein serves as the second component of a protein complex involved in polysaccharide export across the cytoplasmic membrane (Reizer, Reizer & Saver 1992).
HctD showed an average of 42.6 % identity and 65.3 % similarity with the predicted D proteins of H. influenzae, A. pleuropneumoniae and P. multocida.HctD showed similarity of 63 % with BexD and 62.5 % with CrtA from H. influenzae and N. meningitidis respectively.CtrA from Neisseria meningitidis is believed to be an outer membrane protein with porin properties (Frosch et al. 1992).In addition, BexD and its homologues is believed to be outer membrane associated (Kroll et al. 1990;Rosenow, Esumah, Roberts & Jann 1995), mutations in the bexD gene coding for this corresponding protein accumulated polysaccharides in the periplasmic space (Bronner, Clarke & Whitfield 1994).Based on these similarities with CtrA and BexD, HctD it is probably an outer membrane protein involved in capsular polysaccharide transport across the outer membrane, possibly with porin properties.
These data are therefore consistent with the hypothesis that the hctABCD gene cluster encodes proteins that form an export complex for capsule polysaccharides.The findings will greatly facilitate the investigation at molecular level of the role of the H. paragallinarum capsule in pathogenesis.However, confirmation of the importance of each gene product and elucidation of the function of each protein will require characterization of the phenotypic impact of in-frame deletions or other mutations in the respective genes.In-frame deletions might lead to reduced virulence with the possible as a live vaccine.

TABLE 2
Comparison of capsular transport gene and protein sizes and % identity and similarity between proteins from H. paragallinarum 1742 with those of related bacterial species a Open reading frame of each capsule transport gene and corresponding nucleotide size in base pairs b Predicted proteins for each capsule transport gene and protein size in amino acids c