Comparative genome analyses to understand the population biology and virulence of the pig pathogen Actinobacillus pleuropneumoniae

Abstract

The bacterium Actinobacillus pleuropneumoniae is an important respiratory tract pathogen of pigs leading to major swine health problems and huge economic losses to farmers worldwide. It is divided into at least 16 distinct capsular serovars and has highly diverse pathogenicity. A safe vaccine that offers complete protection against all serovars has yet not reached the market. In this study, a multi-strain genomic approach was used to screen universal vaccine candidates against A. pleuropneumoniae. Roary was used to identify core genes between 221 A. pleuropneumoniae strains from serovars 1-5, 5b, 6-16, and K2:O7 and nontypeables strains. By cross-referencing with previous literature, it was identified that 34 of the A. pleuropneumoniae conserved genes are predicted to code for virulence factors. These included genes involved in cell wall biogenesis, anaerobic respiration, metabolism, secretion and ATP synthesis. Of the 34 genes, nine genes (ompW, prc, rbsB, tatB, atpG, dmsA, nrfAB, napB and ccmH) were identified to encode for surface proteins or secretory proteins. These surface or secretory proteins are potential candidates for subunit vaccine. Another 19 genes (murD, glpR, rfaC, malP, secB, dnaK, aspA, lpdA, purF, guaB, atpGH, moaA, moaE, lldD, frdA, hemA, hemB, ureABC and ureEG) were identified to encode for cytoplasmic proteins. These cytoplasmic proteins are potential candidates for live-attenuated or differentiating infected from vaccinated animals (DIVA) vaccines. Diversity between different A. pleuropneumoniae serovars was also examined by reconstructing phylogenetic trees based on core genes and accessory genes separately. The core gene tree clearly divided the isolates into two main groups. The genomic relationships between A. pleuropneumoniae strains belonging to the same serovar were also studied. Mostly, the isolates of the same serovar were found to be closely related to each other. The exceptions were serovars 6, 12, K2:O7 and nontypeables, which showed greater genetic variation. Due to the presence of genetic variation between and within serovars, the identification of universal vaccine candidates is the best way forward to develop subunit or DIVA based vaccines against A. pleuropneumoniae. The isolates of A. pleuropneumoniae were also investigated by formulat- ing a multilocus sequence typing (MLST) scheme based on partial sequences of the genes gdhA, infB, recA, mdh, frdB, atpG and gph and 32 sequence types (STs) were observed. The analysis of these STs using eBURST revealed six clonal complexes and suggested that A. pleuropneumoniae is an intermediately clonal bacterium. Both the MLST and the core-gene phylogeny results also revealed a possibility of capsule switching in A. pleuropneumoniae which has implications for whole-cell bacterin vaccines. Additionally, an evolutionary genomics approach was used to identify core genes that show evidence of recombination and positive selection in A. pleuropneumoniae. Approximately, 61% of the core genes showed strong signals for homologous recombination (q-value < 0.05). Furthermore, the selection analysis indicated that 25 genes are under significant selection pressure. Extensive functional analysis of the positively selected genes demonstrated that genes coding for products relevant to bacterial cell membrane, metabolism, transcription, secretion and transportation are prone to positive selection pressure. This information will be useful for researchers for novel drug development against A. pleuropneumoniae.