Comparative genomic characterization of Actinobacillus pleuropneumoniae

Abstract

The Gram-negative bacterium Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumoniae, a lethal respiratory infectious disease causing great economic losses in the swine industry worldwide. In order to better interpret the genetic background of serotypic diversity, nine genomes of A. pleuropneumoniae reference strains of serovars 1, 2, 4, 6, 9, 10, 11, 12, and 13 were sequenced by using rapid high-throughput approach. Based on 12 genomes of corresponding serovar reference strains including three publicly available complete genomes (serovars 3, 5b, and 7) of this bacterium, we performed a comprehensive analysis of comparative genomics and first reported a global genomic characterization for this pathogen. Clustering of 26,012 predicted protein-coding genes showed that the pan genome of A. pleuropneumoniae consists of 3,303 gene clusters, which contain 1,709 core genome genes, 822 distributed genes, and 772 strain-specific genes. The genome components involved in the biogenesis of capsular polysaccharide and lipopolysaccharide O antigen relative to serovar diversity were compared, and their genetic diversity was depicted. Our findings shed more light on genomic features associated with serovar diversity of A. pleuropneumoniae and provide broader insight into both pathogenesis research and clinical/epidemiological application against the severe disease caused by this swine pathogen.

FIG. 1.

Circular representation of sequence conservation between A. pleuropneumoniae serovar 5b strain L20 and 11 strains belonging to different serovars. Circles are numbered from 1 (outermost circle) to 16 (innermost circle). The outermost two circles show CDSs, rRNAs and tRNAs in the L20 genome of A. pleuropneumoniae serovar 5b. The next 11 circles show the coordinates of BLAST hits detected through blastn comparisons (minimum sequence identity of 95% and expected threshold of 10⁻⁵) of the L20 reference genome against 11 A. pleuropneumoniae genomes, including two public complete genomes and nine contig sets of new genomes, and each circle is colored according to serovar reference strains: maroon for serovar 7 strain AP76, silver for serovar 3 strain JL03, teal for serovar 1 strain 4074, cyan for serovar 2 strain S1536, light purple for serovar 4 strain M62, red for serovar 6 strain Femϕ, blue for serovar 9 strain CVJ13261, olive for serovar 10 strain D13039, fuchsia for serovar 11 strain 56153, yellow for serovar 12 strain 1096, and orange for serovar 13 strain N273. Overlapping hits appear as darker arcs. The innermost two circles show GC content and GC skew plot of the L20 genome. Several known serovar-specific genomic regions with low sequence identity were numbered as follows: I, the ∼38-kb prophage region; II and III, the coding gene cluster involved in type I restriction-modification system; IV, the LPS O-antigen biosynthesis region; V, the CPS biosynthesis region.

FIG. 2.

Distribution of cellular function categories of core orthologous protein clusters.

FIG. 3.

A. pleuropneumoniae whole-genome phylogeny. (A) Dendrogram showing the phylogenetic relationship based on differences in genetic gain or loss of noncore genes among the 12 strains of diverse A. pleuropneumoniae serovars. The numbers on the branch represent the number of genic differences that occurs from the previous bifurcation node. (B) Maximum-likelihood tree estimated from a data set of 1,287 concatenated, conserved genic sequences in 12 A. pleuropneumoniae genomes. The numbers on the branch stand for the number of nucleotide substitutions per kilobase that occur prior to the next level of separation.

FIG. 4.

Schematic comparison of the genetic organizations of the CPS biosynthesis and export gene clusters in the reference strains of 12 A. pleuropneumoniae serovars. Three types of CPS have been defined in A. pleuropneumoniae as follows: type 1, serovars 2, 3, 6, 7, 9, 11, and 13 are composed of teichoic acid polymers linked by phosphate diester bonds; type II, serovars 1, 4, and 12 are composed of oligosaccharide polymers linked by phosphate bonds; and type III, serovars 5b and 10 are composed of repeating oligosaccharide units (26, 34).

FIG. 5.

Schematic comparison of the genetic organizations of the LPS O-antigen biosynthesis gene clusters in the reference strains of 12 A. pleuropneumoniae serovars. A. pleuropneumoniae serovars can be divided into two groups based on two different mechanisms for the assembly and translocation of O antigen: group I, the Wzy/Wzx-dependent pathway is present in serovars 2, 3, 4, 6, 7, and 13; and group II, the ABC-2 transporter-dependent pathway is present in serovars 1, 5b, 9, 10, 11, and 12.