Characterization and comparative analysis of the genes encoding Haemophilus parasuis outer membrane proteins P2 and P5

Abstract

Haemophilus parasuis is a swine pathogen of significant industry concern, but little is known about how the organism causes disease. A related human pathogen, Haemophilus influenzae, has been better studied, and many of its virulence factors have been identified. Two of these, outer membrane proteins P2 and P5, are known to have important virulence properties. The goals of this study were to identify, analyze, and compare the genetic relatedness of orthologous genes encoding P2 and P5 proteins in a diverse group of 35 H. parasuis strains. Genes encoding P2 and P5 proteins were detected in all H. parasuis strains evaluated. The predicted amino acid sequences for both P2 and P5 proteins exhibit considerable heterogeneity, particularly in regions corresponding to predicted extracellular loops. Twenty-five variants of P2 and 17 variants of P5 were identified. The P2 proteins of seven strains were predicted to contain a highly conserved additional extracellular loop compared to the remaining strains and to H. influenzae P2. Antigenic-site predictions coincided with predicted extracellular loop regions of both P2 and P5. Neighbor-joining trees constructed using P2 and P5 sequences predicted divergent evolutionary histories distinct from those predicted by a multilocus sequence typing phylogeny based on partial sequencing of seven housekeeping genes. Real-time reverse transcription-PCR indicated that both genes are expressed in all of the strains.

FIG. 1.

Southern blots of NciI-digested H. parasuis genomic DNA from the strains indicated hybridized to an ompP2 (left) or ompA (right) probe. (Left) Lanes: 1, H425; 2, 831542; 3, 24054; 4, 15677; 5, Nagasaki. (Right) Lanes: 1, 831542; 2, 17321; 3, 15677. The relative positions of the DNA size markers are indicated to the left.

FIG. 2.

Alignment of H. parasuis P2 (A) and P5 (B) predicted protein sequences. Predicted extracellular-loop domains are indicated in boldface within the consensus sequence. Dots represent amino acid identity with the consensus sequence, and dashes represent gaps. Lowercase letters indicate that the corresponding substituted amino acid and the consensus amino acid have similar physical properties; uppercase letters indicate an amino acid substitution with dissimilar physical properties. An “x” in the consensus sequence indicates an amino acid present in some strains that is not in the consensus.

FIG. 2.

Alignment of H. parasuis P2 (A) and P5 (B) predicted protein sequences. Predicted extracellular-loop domains are indicated in boldface within the consensus sequence. Dots represent amino acid identity with the consensus sequence, and dashes represent gaps. Lowercase letters indicate that the corresponding substituted amino acid and the consensus amino acid have similar physical properties; uppercase letters indicate an amino acid substitution with dissimilar physical properties. An “x” in the consensus sequence indicates an amino acid present in some strains that is not in the consensus.

FIG. 2.

Alignment of H. parasuis P2 (A) and P5 (B) predicted protein sequences. Predicted extracellular-loop domains are indicated in boldface within the consensus sequence. Dots represent amino acid identity with the consensus sequence, and dashes represent gaps. Lowercase letters indicate that the corresponding substituted amino acid and the consensus amino acid have similar physical properties; uppercase letters indicate an amino acid substitution with dissimilar physical properties. An “x” in the consensus sequence indicates an amino acid present in some strains that is not in the consensus.

FIG. 2.

Alignment of H. parasuis P2 (A) and P5 (B) predicted protein sequences. Predicted extracellular-loop domains are indicated in boldface within the consensus sequence. Dots represent amino acid identity with the consensus sequence, and dashes represent gaps. Lowercase letters indicate that the corresponding substituted amino acid and the consensus amino acid have similar physical properties; uppercase letters indicate an amino acid substitution with dissimilar physical properties. An “x” in the consensus sequence indicates an amino acid present in some strains that is not in the consensus.

FIG. 2.

Alignment of H. parasuis P2 (A) and P5 (B) predicted protein sequences. Predicted extracellular-loop domains are indicated in boldface within the consensus sequence. Dots represent amino acid identity with the consensus sequence, and dashes represent gaps. Lowercase letters indicate that the corresponding substituted amino acid and the consensus amino acid have similar physical properties; uppercase letters indicate an amino acid substitution with dissimilar physical properties. An “x” in the consensus sequence indicates an amino acid present in some strains that is not in the consensus.

FIG. 2.

Alignment of H. parasuis P2 (A) and P5 (B) predicted protein sequences. Predicted extracellular-loop domains are indicated in boldface within the consensus sequence. Dots represent amino acid identity with the consensus sequence, and dashes represent gaps. Lowercase letters indicate that the corresponding substituted amino acid and the consensus amino acid have similar physical properties; uppercase letters indicate an amino acid substitution with dissimilar physical properties. An “x” in the consensus sequence indicates an amino acid present in some strains that is not in the consensus.

FIG. 3.

P2 protein structural representations for H. parasuis strain 15677, the strain with greatest similarity to the consensus sequence (left); H. parasuis strain H465, the strain with least similarity to the consensus sequence (middle); and, for comparison, H. influenzae strain Rd KW20 (right). Amino acid residues are colored according to hydrophobic potential with gradation from yellow (0.5) to blue (−2.0).

FIG. 4.

P5 protein structural representations for H. parasuis strain NADC1, the strain with greatest similarity to the consensus sequence (left); H. parasuis strain D74, the strain with least similarity to the consensus sequence (middle); and, for comparison, H. influenzae strain Rd KW20 (right). Amino acid residues are colored according to hydrophobic potential with gradation from yellow (0.5) to blue (−2.0).

FIG. 5.

Unrooted neighbor-joining trees derived from H. parasuis ompP2 (A), ompA (B), and MLST (C) sequences. Branches corresponding to partitions reproduced in less than 50% of bootstrap replicates were collapsed. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (10,000 replicates) are shown next to the branches.