INTRODUCTION

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Mannheimia haemolytica is the etiological agent of bovine and ovine pneumonic pasteurellosis, a disease that causes considerable economic losses to the cattle and sheep industries (17, 20). Capsular serotyping provides the primary basis for the classification of strains and epidemiological typing of M. haemolytica, which has traditionally been subdivided into 13 serotypes (1, 49). The association of different serotypes with infections of cattle and sheep (17, 20) suggests that serotype-related strain differences occur in host specificity and virulence. For example, serotype A1 and A6 strains account for almost all cases of bovine pneumonic pasteurellosis, whereas serotype A2 and A7 isolates are the major causes of disease in sheep. It has also been shown that bovine and ovine isolates of the same serotype, e.g., A1, A2, or A6, can be distinguished by differences in chromosomal genotype (13) or outer membrane protein (OMP) profiles (10). The inference is that natural populations of M. haemolytica consist of distinct evolutionary lineages that are differentially adapted to either cattle or sheep (13).

Leukotoxin is a key virulence factor in the pathogenesis of pneumonic pasteurellosis (6, 32, 35, 46, 47). It is a member of the RTX (repeats in toxin) family of gram-negative bacterial cytotoxins, which includes the alpha-hemolysin of Escherichia coli (30, 45). Most RTX toxins interact with different cell types from a variety of species, but the cytotoxins produced by M. haemolytica, as well as those from Actinobacillus actinomycetemcomitans and Actinobacillus pleuropneumoniae (ApxIIIA), have both cell type- and species-specific effects. The leukotoxin of M. haemolytica interacts only with the alveolar macrophages, neutrophils, and lymphocytes of ruminants and is believed to promote bacterial proliferation by killing or incapacitating these cells (4, 7, 23, 40). It has been postulated that leukotoxin target cell specificity underlies the host specificity of M. haemolytica infections, and it has recently been demonstrated that ₂ integrins are the putative leukotoxin receptors (2, 22, 28). Restriction endonuclease analysis (5), together with studies on the neutralizing activity of monoclonal antibodies (19), has demonstrated interserotypic variation of the M. haemolytica leukotoxin determinants, but the significance of allelic diversity for the pathogenesis of pneumonic pasteurellosis and for host specificity is not known.

The purpose of the present study was to investigate nucleotide sequence variation in the M. haemolytica leukotoxins and to determine how this variation relates to differences in virulence and host specialization. Horizontal DNA transfer and recombination are now recognized as important evolutionary mechanisms, complementing mutation, in the diversification of molecules involved in virulence, such as those encoding cell surface structures and other macromolecules for which there is an adaptive advantage in structural diversity (8, 15, 24, 29, 36, 44). Here we used an established framework of evolutionary relationships among strains of M. haemolytica (13) to study the molecular evolution of the leukotoxin (lktA) gene and to determine the role of horizontal DNA transfer and recombination in leukotoxin evolution.

	MATERIALS AND METHODS

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Bacterial strains and growth conditions. The complete lktA gene was sequenced in 31 M. haemolytica, 6 Mannheimia glucosida, and 4 Pasteurella trehalosi isolates. M. glucosida represents serotype A11 strains of P. haemolytica, which have recently been reclassified as a separate species (3), and P. trehalosi was recognized as the T biotype of P. haemolytica until its reclassification (43). Partial lktA sequences were obtained for a further 13 serotype A2 M. haemolytica isolates of bovine and ovine origin. The 54 isolates have been well characterized in previous studies (10-14) and were chosen to represent selected evolutionary lineages, serotypes, and hosts of origin. Properties of these isolates are presented in Table 1.

Preparation of DNA. Cells from 0.5 ml of overnight cultures were harvested by centrifugation for 1 min at 13,000 × g and washed once in sterile distilled H₂O. DNA was prepared with the InstaGene Matrix (Bio-Rad) according to the manufacturer's instructions and stored at 20°C.

PCR amplification and DNA sequence analysis. M. haemolytica strains have been shown to possess only one lktA gene (5), and a direct PCR approach was adopted. The complete coding and flanking regions of the lktA gene were amplified with a Taq DNA polymerase kit (Boehringer Mannheim) according to the manufacturer's instructions. PCR error rates were shown to be insignificant by the complete sequence identity of duplicate amplifications of the lktA gene in isolate PH278. The lktA gene was amplified from the chromosomal DNA with the 5' primer lktA9 (5'-TCAAGAAGAGCTGGCAAC-3') and the 3' primer lktA7 (5'-AGTGAGGGCAACTAAACC-3'). The primers were designed from the published sequences for the lktA genes of serotype A1 (21, 30) and A11 (5) isolates. Primer lktA9 corresponds to residues 53 to 70 upstream of the lktA initiation codon; primer lktA7 corresponds to residues 105 to 122 downstream of the lktA termination codon. Primers were designed using the Primer Designer (version 2.0) computer program and synthesized with a Beckman Oligo 1000 DNA synthesizer. PCRs were carried out in a Perkin-Elmer 480 DNA thermal cycler using the following amplification parameters: denaturation at 94°C for 45 s, annealing at 62°C for 45 s, and extension at 72°C for 2 min. Thirty cycles were performed, and a final extension step of 72°C for 10 min was used. Production of a PCR amplicon of the expected size (~3 kbp) was confirmed by agarose gel electrophoresis, and the DNA was purified with a QIAquick PCR purification kit (Qiagen, Chatsworth, Calif.). The DNA was finally eluted in 30 µl of sterile distilled H₂O and stored at 20°C. Sequence reactions were performed with the ABI Prism Dye Terminator Cycle Sequencing Kit (Perkin-Elmer), and sequence analysis was performed with an Applied Biosystems 373A DNA Sequencer. Both strands of the lktA gene were sequenced with seven internal pairs of primers designed as sequence data became available.

Analysis of nucleotide and protein sequence data. Nucleotide sequence data were analyzed and edited with the SEQED (version 1.0.3) computer program (Perkin-Elmer Applied Biosystems). Statistical and phylogenetic analyses were carried out with MEGA (25) in conjunction with alignment programs written by T.S.W. Statistical analyses for clustering of polymorphic sites were carried out by the maximum chi-square method (42) with a program written by T.S.W. Predictions of hydrophilicity, hydrophobicity, antigenic index, and surface probability of protein sequences were performed with the PROTEAN program (DNASTAR Inc.).

Nucleotide sequence accession numbers. The GenBank accession numbers for the lktA gene sequences obtained in this study are given in Table 1.

	RESULTS

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Nucleotide and amino acid sequence variation. The complete nucleotide sequence of the lktA gene was determined for 31 isolates of M. haemolytica representing 12 capsular serotypes and the 22 electrophoretic types (ETs) previously defined by multilocus enzyme electrophoresis (MLEE) (13). The lktA gene was also sequenced for six isolates representing different ETs of M. glucosida (13) and for four isolates that each represent one of the capsular serotypes (T3, T4, T10, and T15) of P. trehalosi (14). Part of the lktA gene was also sequenced for an additional 13 serotype A2 isolates of M. haemolytica recovered from cattle and sheep in order to identify the allele type. In this case, the first 700 nucleotides at both the 5' and 3' ends of the gene were analyzed. Properties of these isolates are shown in Table 1.

Association of lktA alleles with evolutionary lineages and serotypes of M. haemolytica and the host species of origin. The association of lktA alleles with evolutionary lineages (represented by ETs) and serotypes of M. haemolytica, together with the host species of origin, is shown in Fig. 1. There were three principal findings, which are summarized below. First, lktA1 type alleles were associated exclusively with strains representing ETs of lineage A. However, allele lktA1.1 occurred only in bovine serotype A1 and A6 strains of ETs 1 and 2, whereas alleles lktA1.2 to lktA1.5 were present in ovine isolates of seven serotypes representing eight ETs. Second, lktA2 type alleles occurred in serotype A2 strains of ETs 16, 17, and 21. With the exception of two strains of ET 16, all isolates possessing this allele type were of bovine origin. The uncommon allele lktA3 was similarly associated only with bovine strains of ET 18. Third, the recombinant alleles lktA6 to lktA10 (see below) were associated with ovine strains having a wide range of genetic diversity. Alleles lktA6, lktA7, and lktA9 occurred in serotype A13, A16, and A14 strains representing ETs 15, 11, and 10, respectively; lktA8 type alleles were more widely distributed among serotype A2 isolates of ETs 19, 20, and 22 and among serotype A7 isolates of ETs 12 to 14; and allele lktA10 was associated with serotype A2 isolates representing ET 21. 

View larger version (25K): [in this window] [in a new window]   FIG. 1.   Association of lktA alleles with evolutionary lineage, capsular serotype, and host species of origin. The dendrogram shows the genetic relationships of ETs of M. haemolytica and was generated by the UPGMA method of clustering from a matrix of coefficients of pairwise genetic distances based on 18 enzyme loci (11). For MLEE data, genetic distance is defined as the number of detectable codon changes per locus (39). Three lineages, identified at a genetic distance of 0.28, are indicated by the letters A, B, and C. Bov, bovine; Ov, ovine.

Intragenic recombination. Comparison of the distribution of polymorphic nucleotide sites among lktA alleles representing each of the 10 allelic groups revealed the presence of mosaic structures of varying degrees of complexity. Thus, for pairs of alleles, certain regions of the gene were identical, or nearly so, in sequence whereas adjacent regions of the same alleles were very different. The maximum chi-square method (42) was used to make pairwise comparisons of sequences representing each allelic group, identify statistically significant clusters of polymorphic nucleotide sites, and determine the endpoints of recombinant segments (Fig. 2). On the basis of these analyses, the mosaic structures of single alleles representative of each group are illustrated schematically in Fig. 3.

View larger version (21K): [in this window] [in a new window]   FIG. 2.   Maximum chi-square comparisons for the following pairs of alleles: (a) lktA1.1 versus lktA2.1, (b) lktA4.1 versus lktA5.1, (c) lktA5.1 versus lktA6, (d) lktA6 versus lktA7, (e) lktA7 versus lktA8.1, (f) lktA8.1 versus lktA9, and (g) lktA1.3 versus lktA10. Vertical lines represent polymorphic nucleotide sites, and numbered nucleotides indicate positions where the chi-square value was a maximum (i.e., kmax) with respect to the partitions on the left and right. These positions mark the endpoints of partitions that represent recombinant segments. The probability that the expected maximum chi-square value in 1,000 randomly generated data sets was greater than the observed value for the real data was <0.05.

View larger version (40K): [in this window] [in a new window]   FIG. 3.   Schematic representation of the mosaic structures of alleles representative of the major allelic groups lktA1 to lktA10. The different colors indicate sequence identity and the likely origins of recombinant segments. The number of sites different from those in the corresponding region of the likely donor allele(s) (and the degree of divergence) are indicated below certain recombinant segments (see the text). All other segments exhibited 100% sequence identity to the corresponding regions of the donor alleles. Numbers above the proposed recombination sites indicate the position of the last nucleotide at the downstream end of the recombinant segment.

Synonymous and nonsynonymous substitutions. Visual inspection of the aligned amino acid sequences representing alleles lktA1, lktA2, lktA4, and lktA5 indicated that the distribution of polymorphic amino acid sites is nonrandom, because well-defined regions of amino acid conservation and heterogeneity occur throughout the leukotoxin molecule (Table 3). The numbers of synonymous substitutions per synonymous site (d_S) and nonsynonymous substitutions per nonsynonymous site (d_N) were estimated for the combined variable and conserved regions, and the d_S/d_N ratios were calculated. The d_S values for the variable (0.8269 ± 0.0800) and conserved (0.5724 ± 0.0322) regions were not significantly different, but the d_N value for the variable regions (0.1887 ± 0.0150) was an order of magnitude larger than that for the conserved regions (0.0214 ± 0.0026). The d_S/d_N ratios for the variable and conserved regions were 4.4 and 26.7, respectively, and provide evidence of strong selective constraint against amino acid replacement in the conserved regions of the gene and relaxed constraint in the variable regions.

View larger version (20K): [in this window] [in a new window]   FIG. 4.   Variation in frequency of synonymous and nonsynonymous nucleotide substitutions along the length of the lktA gene among alleles lktA1, lktA2, lktA4, and lktA5. pS and pN were calculated for subsets of 30 codons in a sliding window for the length of the gene.

Relationship between degree of amino acid variation and hydrophilicity-hydrophobicity. Kyte-Doolittle hydrophilicity-hydrophobicity profiles for leukotoxins encoded by lktA1, lktA2, lktA4, and lktA5 type alleles were remarkably similar (data not shown). The N-terminal half of the molecule (amino acids 1 to 400) consists of a series of hydrophobic and hydrophilic domains, and comparison of the plots with the data given in Table 3 revealed that hydrophobic domains representing codons 41 to 48, 65 to 72, 141 to 158, 172 to 190, 227 to 251, 262 to 318, and 355 to 401 correspond to regions of conserved amino acid sequence, whereas hydrophilic domains representing codons 1 to 40, 49 to 64, 73 to 86, 113 to 140, 159 to 171, and 191 to 226 correspond to regions of variable amino acid sequence. The hydrophobic domains also have low predicted antigenic indices and surface probabilities (data not shown).

	DISCUSSION

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The lktA gene of M. haemolytica is highly diverse and is represented by at least eight major allelic variants with a complex evolutionary history. These allelic variants have mosaic structures of varying degrees of complexity that reflect a history of extensive intragenic recombination. The frequency and sites of the recombinational exchanges are possibly related to the presence of chi sequences represented by "hot spots" of recombination at nucleotide positions 96 to 123 (alleles lktA6, lktA8, and lktA10), 1446 (lktA8 and lktA9), and 1881 (lktA4 and lktA7). The complete, or nearly complete, identity of homologous segments in donor and recipient alleles suggests that most of these recombination events occurred relatively recently and probably postdate the domestication of cattle and sheep; this applies to alleles lktA6 to lktA10 in particular. Additional evidence for a recent origin is provided by the fact that each allelic variant is represented by only a small number of alleles which exhibit very little interallelic diversity.

In addition to intragenic recombination, assortative (entire-gene) recombination has also contributed to genetic diversity in M. haemolytica. For example, the presence of lktA8 alleles in genetically diverse strains representing six ETs (Fig. 1) is clearly due to horizontal gene transfer, because the formation of identical recombinant alleles by convergent evolution in different lineages is highly unlikely. The pattern of sequence diversity described here clearly accounts for the seven variants of the lktA gene previously identified by restriction endonuclease analysis (5).

Origin of the mosaic alleles lktA6 to lktA10. The majority (5 of 8) of the M. haemolytica alleles, namely lktA6 to lktA10, consist of two to four distinct segments that are identical or nearly identical to segments from lktA1, lktA2, lktA4, and lktA5 type alleles (Fig. 3). To account for the complex mosaic structure of lktA alleles, we propose an evolutionary model that posits that new alleles have been created by a series of gene transfers and recombination events (Fig. 5). We hypothesize that these alleles have been formed by a sequential series of intragenic recombinational exchanges culminating in the formation of alleles lktA8 and lktA9. A model representing the proposed sequence of events leading to the formation of alleles lktA6 to lktA10 is shown in Fig. 5. High percentages of the DNA contents of alleles lktA6 to lktA9 (96.7, 94.1, 52.8, and 87.8%, respectively) originate from M. glucosida and P. trehalosi alleles of types lktA4 and lktA5 (Fig. 3). Strong support for a common origin of alleles lktA6 to lktA9 is provided by the fact that homologous segments have identical, or nearly identical, nucleotide sequences. The simplest explanation for this finding is that alleles lktA6 to lktA9 are derived from an ancestral allele that itself was formed by a recombination event involving lktA4 and lktA5 type alleles (Fig. 5). Such recombination events between these two species are not uncommon, because two examples of intragenic recombinational exchanges were discovered in the present study.

View larger version (20K): [in this window] [in a new window]   FIG. 5.   Proposed sequence of recombination events in the evolution of lktA leading to the formation of lktA8 and lktA10 type alleles in the ovine-specific lineages represented by ETs 12 to 14 and 19 to 22. The central role of the bovine lktA2 allele in the evolution of ovine alleles lktA6, lktA8, lktA9, and lktA10 is clearly seen. The mosaic structures of the alleles are as shown in Fig. 3.

Host switching of bovine serotype A2 strains to sheep has led to the evolution of new recombinant alleles in ovine strains. Recombinational exchanges involving lktA2 alleles have played a significant role in leukotoxin evolution, as evidenced by the fact that lktA2-derived segments are present in lktA6, lktA8, lktA9, and lktA10 alleles (Fig. 5). The following evidence suggests that the latter four alleles have evolved as a consequence of host switching of serotype A2 strains from cattle to sheep. First, lktA2 alleles are associated only with bovine or bovine-like serotype A2 strains (Fig. 1). Partial sequence analysis of the lktA gene from a wider range of serotype A2 strains confirmed that, with two exceptions, lktA2 type alleles occur only in bovine strains of ETs 17 and 21 (Table 1). Although two exceptional lktA2-possessing serotype A2 isolates (ET 16 [Fig. 1]) were isolated from sheep, these strains possess OMP profiles characteristic of bovine isolates (10). Furthermore, recent sequence analysis of the OMP pomA gene (50) from bovine and ovine isolates (R. L. Davies, unpublished data) has confirmed that these two isolates are related to bovine and not ovine serotype A2 strains. Therefore, we suspect that the two ET 16 isolates are not true ovine-adapted strains but instead represent a bovine-adapted clone that recently spread to sheep. Second, lktA4 and lktA5 type alleles are present in the species M. glucosida and P. trehalosi, respectively, bacteria that occur only in sheep (3, 13, 20). In support of this, recombinant segments derived from lktA4 and lktA5 type alleles were not identified in any of the alleles associated with bovine strains. Third, the recombinant alleles lktA6 to lktA10 are associated only with ovine strains of serotypes A2, A7, A13, A14, and A16; with the exception of A2, none of these serotypes are known to occur in cattle (17). Partial sequence analysis of the lktA gene from additional serotype A2 strains confirmed that lktA8 and lktA10 alleles occur only in ovine isolates of ETs 19 to 22 (Table 1).

Molecular evolution and leukotoxin structure. The leukotoxin molecule of M. haemolytica consists of well-defined regions of conservation and heterogeneity which correspond to different functional domains (9, 16, 26, 31, 33, 45, 48). In particular, there are three highly conserved regions that are common to all RTX toxins (45, 48). The N-terminal half of the molecule consists of a series of hydrophobic putative membrane-spanning domains, separated by hydrophilic regions, that are involved in pore formation; the hydrophobic domains are followed by a second region of approximately 200 amino acids, rich in -turns, that is involved in cell binding and LktC-mediated toxin activation; the third conserved region consists of amino acids 733 to 786 in M. haemolytica and forms six glycine-rich tandem repeat domains that are involved in Ca²⁺ binding.