Construction And Analysis Of The Pan-genome Of Glaesserella Parasuis

Gl?sser’s disease,caused by Glaesserella parasuis(G.parasuis)and characterised by fibrous polyposis and arthritis,is an important disease in the modern,day-old,isolated production systems of the world pig industry,causing huge economic losses incurred by the pig industry in various countries are enormous.Antibiotic treatment of the disease often fails to achieve the desired effect and the available commercial vaccines for G.parasuis are poorly cross-protective.Therefore,the prevention of G.parasuis needs to be based on an in-depth understanding of the genetic background and to investigate the causes of the development of G.parasuis resistance mechanisms and immune heterogeneity.In this study,we propose to use nanopore sequencing technology to sequence the whole genome of several clinical isolates of G.parasuis,to further construct the G.parasuis pangenome and analyze its main features,to identify core and non-essential genes and to investigate the functions and biological processes involved in both types of genes;to design and construct a multi-epitope vaccine using the core genome as the target,and to investigate the cross-protection potential of the multi-epitope vaccine.In addition,a pan-genomic association study was conducted for three biological traits: G.parasuis virulence,ecology and biofilm formation ability,and a forward genetic strategy was used to identify genes associated with trait variation under natural conditions.The main results are as follows.(1)Whole-genome sequencing of 16 G.parasuis clinical isolates was performed using Oxford Nanopore Technology(ONT),and a nanopore sequencing assembly process for G.parasuis was established by comparing the genome quality obtained from different assembly and error correction strategies.The results showed that the median sequence length of the assembled genomes was 2369871 bp(IQR:2341178～2392938 bp),with a median GC% of 40.02%;the genomes were compared with the direct homologous gene set of Amoeba phylum,showing a median of 97.28%of complete genes.system,with assembly numbers CNA0038229,CNA0038230,CNA0038231,CNA0038232,CNA0038233,CNA0038234,CNA0038235,CNA0038236,CNA0038237,CNA0038238 CNA0038239,CNA0038240,CNA0038241,CNA0038242,CNA0038243,CNA0038244.by comparing the G.parasuis m RNA transcripts with BLAT of the assembled genome and with BLAST of the coding region,it is clear that insertional deletions are the main systematic error of ONT and directly affect the The coding region was correctly predicted and had to be corrected to improve the quality of the genome.The results then compared the advantages and disadvantages of assembling bacterial genomes using different sequencing platforms(Illumina,Pac Bio,ONT)and showed that ONT data alone could directly assemble a contiguous and null-free genome,and with the Homopolish error correction algorithm could achieve the same accuracy as using Illumina’s highly accurate short read-length data correction,resulting in a complete and highly accurate genome in a short period of time.The genome can be assembled with the same accuracy as with Illumina’s highly accurate short read length data.(2)Using 121 G.parasuis natural population isolates to complete the construction of a pan-genome,it was found that the G.parasuis pan-genome consists of 8885 genes(of which the core genome consists of 1133 genes and the non-essential genome consists of 7752 genes)and is an open pan-genome with the ability to continuously acquire exogenous genes to adapt to the environment,and its rich genetic diversity is the main reason for the lack of cross-protection of existing vaccines.This rich genetic diversity is the main reason for the lack of cross-protection of existing vaccines.Based on pan-genomic and reverse vaccinology,five B-cell epitopes,six helper T-cell epitopes and three cytotoxic T-cell epitopes were identified and screened from the outer membrane proteins of the G.parasuis core genome,and the vaccine protein constructed by fusing multiple epitopes was physiochemically stable and reliable in three-dimensional model,and had multiple strong hydrogen bonding interactions with Toll-like receptor 2 molecules.The vaccine protein obtained by prokaryotic expression was approximately 33 k D,and immunization tests in mice showed that the polyclonal antibodies produced by immunization could bind to different serotypes and indistinguishable strains of G.parasuis,indicating that the vaccine construct is a potential universal G.parasuis vaccine.(3)On the basis of the G.parasuis pan-genome construct,the biological functions of the core and non-essential genomes were analyzed.The results show that the core genome is fully involved in the life processes of G.parasuis,including metabolism,transcription and translation,signal transduction,etc.It is the backbone of the rest of the genome.The non-essential genome is also biologically essential,involved in fundamental biological processes,and is mainly enriched in mobile elements,defense mechanisms,carbohydrate transport and metabolism,which confer greater environmental resilience to G.parasuis(e.g.in hypoxic environments under pathological conditions in the host).The core genome is distributed with various types of virulence factors,which are complemented by non-essential genomes,mainly enriched in biofilm formation,adhesion,immune regulation,invasion and other categories,providing a genetic basis for the complex pathogenesis of G.parasuis and improving the chances of survival of the organism.Drug resistance genes are abundantly distributed in the non-essential genome,suggesting that horizontal transfer of genes is the main way in which G.parasuis acquires drug resistance.Based on pan-genomic association studies,the main genes associated with three important traits of G.parasuis: virulence,ecology and biofilm formation,and their mechanisms of action were analyzed in depth: genes associated with strong virulence were involved in metabolic,genetic and environmental information processing signaling pathways,which facilitated G.parasuis colonization in the host and resistance to survival stress;genes associated with strong biofilm formation were mostly associated with denitrification.The genes associated with strong biofilm formation are mostly related to denitrification process,which is beneficial to the survival of the bacteria inside the biofilm under low dissolved oxygen environment.In this study,the whole genome sequences of 16 G.parasuis clinical isolates were successfully sequenced using nanopore sequencing technology,and the G.parasuis pangenome was constructed by combining the published genome sequences.On this basis,a universal G.parasuis multi-epitope vaccine was designed and constructed,and the molecular genetic basis of the major biological traits of G.parasuis was investigated and analysed.The molecular genetic basis of the major biological traits of G.parasuis was also analysed,providing experimental materials and scientific references for the development of novel vaccines and therapeutic drugs based on the G.parasuis pan-genome.