The genetic organization of the capsular polysaccharide biosynthesis region of Actinobacillus pleuropneumoniae serotype 15.

Nucleotide sequence determination and analysis of the cps gene involved in the capsular polysaccharide biosynthesis of Actinobacillus pleuropneumoniae serotype 15 revealed the presence of three open reading frames, designated as cps15ABC genes. At the protein level, Cps15A and Cps15B showed considerably high homology to CpsA (67.0 to 68.7%) and CpsB (31.7 to 36.8%), respectively, of A. pleuropneumoniae serotypes 1, 4 and 12, revealing the common genetic organization of the cps among serotypes 1, 4, 12 and 15. However, Cps15C showed no homology to any proteins of A. pleuropneumoniae serotypes, indicating that cps15C may be specific to serotype 15. This study will provide the basic molecular knowledge necessary for the development of diagnostics and a vaccine for A. pleuropneumoniae serotype 15.

Actinobacillus pleuropneumoniae is a Gram-negative bacterium and the etiologic agent of porcine pleuropneumonia, which causes serious economic losses to the pig-rearing industry [6]. To date, 15 serotypes are recognized, mainly on the basis of the antigenic diversity of capsular polysaccharides in the organisms [3, 19, 21, 22]. Since virulence differs among serotypes [6] and vaccines for A. pleuropneumoniae are serotype-specific [20, 25], serotyping is important and should be widely performed in veterinary diagnostic laboratories. However, only a few laboratories can prepare a full set of serotype-specific antisera for serotyping. Furthermore, cross-reactions are often observed among different serotypes, such as between serotypes 1, 9 and 11; serotypes 4 and 7; serotypes 3, 6, 8 and 15; this prevents the accurate and rapid typing of field strains [5,6,7].

Prevalent serotypes differ from country to country [5]. For example, the predominant serotypes are serotypes 1 and 5 in North America [5, 6], serotype 2 in most of Europe [5, 6], serotype 15 in Australia [3, 5, 6] and serotypes 2, 1 and 5 in Japan [5, 13]. Serotypes 1, 2, 5, 9 and 11 have been generally found to be more virulent than other serotypes [6]. However, approximately 15% of fattening pigs in a herd died due to acute pleuropneumonia caused by serotype 15 [14], indicating that serotype 15, unlike serotypes 3 and 12, should not be considered as low pathogenic [6]. The development of reliable serotyping tests and vaccines for serotype 15 would be important, because isolation cases of serotype 15 have recently increased in Japan [13] and North America [7], because cross-reactions are often observed among serotypes 3, 6, 8 and 15 [5, 6] and because no commercial vaccine is fully effective against serotype 15 challenge [25].

In this study, we determined the nucleotide sequence of the gene involved in the capsular polysaccharide synthesis (CPS) of A. pleuropneumoniae serotype 15 (cps15). The first aim of this study was to obtain the basic molecular knowledge necessary for the development of A. pleuropneumoniae serotype 15 diagnostics, such as PCR serotyping tests, which have been developed in other serotypes [1, 4, 12, 15, 17, 24, 27]. The second aim of this study was to obtain a basic molecular knowledge necessary for the development of vaccine, such as a genetically modified capsule-deficient mutant vaccine [9].

A. pleuropneumoniae serotype 15 strain HS143 was used to determine the nucleotide sequence of cps15. The organisms were cultivated with TSA agar (Difco, Sparks, MD, U.S.A.) supplemented with 5% defibrinated horse blood and 2% fresh yeast extracts at 37°C. In order to determine the nucleotide sequence of the cps15, internal region of cpxD of A. pleuropneumoniae serotype 15 (cpxD15) was PCR-amplified from the genomic DNA of serotype 15 strain H143, which was prepared as described previously [10]. PCR primers were designed from data previously deposited in databases (5′-ACY TCA GGC CCT AGC CAT AST GC-3′ and 5′-CAC ACG ATA AAC CGT YGG TAC ATC-3′) [26]. The amplified PCR products were purified and sequenced as described previously [11]. Since cps is usually flanked by cpxD in A. pleuropneumoniae[16, 26], an inverse touchdown PCR was then performed to obtain DNAs flanked by cpxD with primers invF (5′-GCA GTA GGC GGA ACA ACG GAA AAC ATT-3′) and invR (5′-ATA TCC CGC ACC GCC TAC AGT ACC TAA AAA-3′), which were designed on the basis of the nucleotide sequence of cpxD determined in this study. Purified genomic DNA from serotype 15 strain HS143 was digested with restriction enzymes EcoRI and HindIII, religated with T4 DNA ligase in order to generate circular template DNAs and used for the following inverse touchdown PCR, respectively. The inverse touchdown PCR was performed in a total volume of 50µl containing 1 X buffer (Toyobo, Otsu, Japan); 0.2 mM of each dNTP; 0.3 µM of each primer (invF and invR) and the template DNAs described above. The following amplification steps were used: 1 cycle at 94°C for 2 min (preheating); 5 cycles at 98°C for 10 sec and 74°C for 20 min (first step); 5 cycles at 98°C for 10 sec and 72°C for 20 min (second step); 5 cycles at 98°C for 10 sec and 70°C for 20 min (third step); 20 cycles at 98°C for 10 sec and 68°C for 20 min (forth step); 1 cycle at 68°C for 10 min (final step). Amplified DNAs (approximately 8 and 5 kilobase pairs) were purified by the QIA quick PCR amplification kit (Qiagen, Hilden, Germany) and submitted to nucleotide sequence determination with fluorescent dye terminators as described previously [11]. The nucleotide sequence determined has been deposited under accession number AB701753 in DDBJ/EMBL/GenBank.

The nucleotide sequence of the DNA (8,551 bp) comprising cps15 was determined. Three open reading frames (ORFs) were located between the cpx15D and lysA genes (the CPS export gene and the diaminopimelate decarboxylase genes, respectively), which are conserved in A. pleuropneumoniae and flanked by the cps (Fig. 1). The ORFs were designated as cps15ABC genes (Fig.1) and encoded the Cps15A to Cps15C proteins, respectively. At the amino acid level, Cps15A showed considerably high homology to ORF1 of Actinobacillus suis[18] as well as did to Cps1A, Cps4A and Cps12A (CPS phosphotransferase) of A. pleuropneumoniae serotypes 1, 4 and 12, respectively [2, 16] (Table 1 ). Cps15B showed overall homology to a glycosyl tranferase of Mannheimia varigena and to ORF2 of A. suis[18] as well as did to Cps1B, Cps4B and Cps12B (glycosyl transferase family protein) of A. pleuropneumoniae[2, 16] (Table 1). Cps15C showed no homology to any proteins of A. pleuropneumoniae, whereas it showed homology to a hypothetical protein of Corynebacterium resistens[23] and to a protein involved in CPS biosynthesis of Neisseria meningitidis serogroup Z [8, 28] (Table 1). These findings suggested that a horizontal gene transfer of the cps gene across the taxonomically and phylogeneticaly unrelated bacterial classes, including Gram-positive bacteria C. resistens and N. meningitidis belonging to β-Proteobacteria, might have occurred during capsule evolution. The G+C contents of cps15A, cps15B and cps15C were 26.9, 26.8 and 34.2%, respectively (Table 1), which is lower than the 41% (overall G+C content of A. pleuropneumoniae) [26], indicating that the cps15ABC genes might have been acquired by horizontal gene transfer.

Fig. 1.

Schematic diagram of the DNA region involved in CPS of A. pleuropneumoniae serotype 1 (accession numbers (nos.) AF518558 [2] and ADOD00000000 [26]), 4 (accession nos. GU585380 [unpublished] and ADOF00000000 [26]) and 12 (accession. nos. AY496881 [16] and ADOL00000000 [26]). Arrows of the same color indicate open reading frames of encoding proteins exhibit amino acid sequence homology.

Table 1.

Identity of Cps protein of Actinobacillus pleuropneumoniae serotype 15 (Cps15) compared to those of A. pleuropneumoniae and other bacterial species

Serotype-specific enzymes that are involved in CPS biosynthesis are probably responsible for the dissimilarities among the CPS chemical structures [26]. However, it has been reported that the CPS structures produced by A. pleuropneumoniae serotypes 1 to 13 and 15 can be divided into three groups according to the basic differences in their chemical compositions and structures: Group I (serotypes 1, 4, 12 and 15), with CPS composed solely of repeating oligosaccharide units linked by phosphates; Group II (serotypes 5 and 10), with CPS composed of repeating oligosaccharide units; Group III (serotypes 2, 3, 6–9, 11 and 13), with CPS composed of teichoic acid polymers linked by phosphate diesters [16, 21, 26]. The genetic organization of the cps genes provided molecular evidence to support the CPS grouping of A. pleuropneumoniae serotypes [16, 26]. The present study revealed that A. pleuropneumoniae serotype 15 carries a gene for the CPS phosphotransferase (cps15A) which may be involved in the chemical linkage of phosphates in the linear CPS backbone [26]. This, in turn, indicates that A. pleuropneumoniae serotype 15 belongs to Group I. This study also revealed that the genetic organization of the cps genes of A. pleuropneumoniae serotype 15 corresponds to the CPS structural classification, as do serotypes 1–13 [16, 21, 26].

As shown in Fig. 1, the genetic organization of the cps was essentially common among A. pleuropneumoniae serotypes 1, 4, 12 [16, 26] and 15 [this study]. However, the orientation of the cps15ABC gene against cpxD and lysA genes was different from that of other A. pleuropneumoniae serotypes [16, 26] (Fig. 1). The different orientation between the cps15ABC genes and cps genes of other serotypes indicated that an inversion might have occurred only in A. pleuropneumoniae serotype 15.

In conclusion, the nucleotide sequence of the cps15 gene has been determined in this study. We believe that the present results will provide the basic molecular knowledge necessary to develop diagnostics and a vaccine for A. pleuropneumoniae serotype 15.