Identification And Immunogenicity Analysis Of Conserved Surface Proteins Of Actinobacillus Pleuropneumoniae

Gram-negative bacterium Actinobacillus pleuropneumoniae is an encapsulated coccobacillus and is the etiologic agent of a severe and contagious pleuropneumonia in swine, leading to great economic losses to the global pig industry. Based on differences in capsular polysaccharides,15serotypes of A. pleuropneumoniae have been recognized to date, with great diversity in virulence and interlocal distributions. While reliable characterization of its serotypic diversity is critical for both mechanistic research and clinical/epidemiological application. The diversity of serotypes has caused great difficulties for the prevention and control work of A. pleuropneumoniae. Currently, vaccination of A. pleuropneumoniae are usually carried out by inoculating inactivated whole-cell bacterins, and/or several available subunit vaccine targets （e.g. exotoxins ApxⅠ, ApxⅡ and ApxⅢ, and outer membrane proteins）. But these vaccination strategies play partial protection against challenges with homologous or heterologous serotypes of A. pleuropneumoniae. Live attenuated vaccines are promising but have not been used in pig farms due to safety issue. Thus, novel vaccination antigens that are able to be expressed by various A. pleuropneumoniae serotypes need to be exploited to provide effective protection against this porcine pathogen. In this study,39conservative outer membrane protein and outer membrane lipoprotein of stable expressed were identified by comparative genomic hybridization and transcriptional profiling of A. pleuropneumoniae. Among the39surface proteins,12of them were successfully expressed in E. coli. and their immunogenicity was estimated by homologous challenge in the mouse model.1. Comparative genomic hybridization of A. pleuropneumoniaeThe ORF datasets of three fully sequenced genomes of A. pleuropneumoniae serotype3strain JL03, serotype5b strain L20and serotype7strain AP76were collected to predict conserved surface proteins. In2097ORFs of A. pleuropneumoniae JL03,501proteins were predicted to reside at the cell surface,375proteins had signal peptides, and52were classified into outer membrane proteins. We found1851orthologs among the three serotypes of A. pleuropneumoniae. Finally,318orthologs encoding putative surface-exposed antigens （271membrane proteins and47lipoproteins） were extracted for the further analysis.A. pleuropneumoniae probe set consisted of60-mer high density oligonucleotides representing the JL03genome. Each probe was designed end to end along the JL03 chromosomal backbone. Test DNA extracted from fourteen serotype standard strains was co-hybridized with reference genomic DNA extracted from strain JL03, respectively. For each microarray hybridization assay, test DNA and reference DNA were fluorescently labelled with Cy5-dUTP and Cy3-dUTP respectively. Compared to JLO3, the percentage of divergent genes in the test strains varied from2.9%to4.2%, and201chromosomal genes were found absent or highly divergent in at least one strain tested according to the analysis of CGH data. Some known genomic loci （CPS biosynthesis gene cluster, the LPS O-antigen biosynthesis gene cluster, the operons of Apx toxins） associated with serotype diversity were confirmed again, and several novel candidates were found for the first time. Five potential genomic islands （GI-1,-2,-3,-4,-5） were found in the JL03genome, which would be associated with serotype diversity. The lineage relationships could be indirectly represented by comparison between standard strains of various serotypes and strain JL03, dividing into four group,1.1（serotypes2,3,4,6）,2.1（serotypes5b and9）,2.2（serotypes1,7,11,12）,2.3（serotypes5a,8,10,13）. These findings laid a foundation for future research about the genetic diversity of A. pleuropneumoniae.2. Transcriptome analysis of A. pleuropneumoniaeThe microarrays used for studying transcription profiles totally featured2,866nonredundant genes identified in A. pleuropneumoniae. Total RNA was extracted from cells of fourteen serotype standard strains and reverse transcribed into cDNA using MMLV, then transcribed into cRNA. The synthesized cRNA was labelled with Cy3NHS ester and were hybridized to the microarray slides. Data analysis were based on hybridization signals. Fourteen standard strains of A. pleuropneumoniae serotypes were systematically compared with the serotype3strain JL03with respect to genomic composition and gene transcription profile. Transcriptional profiling found656genes steadily transcribed in all tested strains at whole-genome level. Combined with the results of the comparative genomic hybridization and transcriptional profiling, three predominant genomic loci in A. pleuropneumoniae, involved in biosynthesis of capsular polysaccharides, lipopolysaccharide O-antigen chain and Apx toxins, were revealed belonging to different serogroups. These genetic traces should directly lead to serotype diversity of A. pleuropneumoniae. In addition,39conserved genes encoding putative surface-exposed proteins, which could served as potential vaccine candidates, were found to be steadily transcribed in the fourteen serotype standard strains. These findings provided useful clues for exploiting new-style universal vaccines for this important pathogen. 3. Immunogenicity of the conserved surface antigenic proteins in A. pleuropneumoniaeCombined with the results of the comparative genomic hybridization and transcriptional profiling, twenty of these conserved genes above were successfully cloned to expression vector pET-28a, and twelve of them were expressed in Escherichia coli and their immunogenicity was estimated by homologous challenge with the serotype3strain JL03in the mouse model, and then3（APJL₀126, HbpA and OmpW） among these vaccine candidates were further tested in the natural host swine by homologous and heterologous challenges with the serotype3strain JL03and the serotype1strain4074. The results showed that these proteins could induce high titers of antibodies and lighten the clinical symptoms and pathological changes of the challenged pigs. Although none of the surface proteins characterized in this study could individually induce effective protective immunity against A. pleuropneumoniae, they are potential candidates for subunit vaccines in combination with Apx toxins.