Comparative Genomics Analysis And Study On Subunit Vaccine Of Porcine Extraintestinal Pathogenic Escherichia Coli

Escherichia coli is the most widely studied model bacterium, according to its pathogenic characteristics, it can be divided to commonsal E. coli, intraintestinal pathogenic E. coli and extraintestinal pathogenic E. coli. ExPEC strains possess certain specific virulence traits that enable them to invade and colonize extraintestinal sites and cause a wide range of infections, such as urinary tract infections, meningitis, sepsis, respiratory tract infection, pneumonia, osteomyelitis and so on.Recent years, multiply and highly frequent pig diseases become the main constraint on the development of our pig industry. Since2004, our lab has isolated872ExPEC strains from extraintestinal tissues of sick pigs in chinese pig farms and performed an epidemiologic study, the result showed that porcine ExPEC becomed one of the bacterial pathogens that threat the development of our pig industry. But nowadays, research reports about ExPEC have focused on isolates of human and avian origin, with little attention on porcine ExPEC infections, and reports about porcine ExPEC are few, with no porcine isolates yet to be sequenced. Additionally, the various serotypes owned by E. coli together with the ubiquitous presence of antibiotic resistance in the isolated porcine ExPEC strains both bring big challenges to the control of porcine ExPEC infections. Thus our lab obtained the first complete genomic sequences for high and low pathogenic porcine ExPEC strains by the second generation sequencing technology, analyzed their virulence factors, researched their biological characteristics and elaborated their pathogenesis. Besides, we choosed two conservative outer membrane protein and evaluated their immunogenicity as subunit vaccine candidates against porcine ExPEC infections. And summaried the tasks as the following:1. Complete genome sequencing and analysis of porcine ExPEC highly virulent strain PCN033and weakly virulent strain PCN061Among the isolated porcine ExPEC strains, we screened highly virulent strain PCN033and weakly virulent strain PCN061in a mouse model, got the first complete genomic sequences for porcine ExPEC. PCN033genome comprise a circular chromosome of4,987,958base pairs, and three plasmids of3,319bp,4,086bp and161,511bp, respectively. PCN061genome comprise a circular chromosome of4,603,777bp, and six plasmids of2,014bp,5,754bp,6,222bp,34,692bp,103,644bp and145,722bp, respectively. We did detailed gene annotation, classification of gene function, pseudogene prediction, analysis of basic genomic elements, including genomic islands, prophage regions and insertion sequences. All-against-all BLASTP comparison of all proteins from PCNO33, PCN061and commsensal E. coli HS strains showed that PCN033had1056special CDSs. Additionally, we constructed a phylogenetic tree basing on the concatenated conserved genes of Escherichia strains. The result showed that PCN033and PCN061were clustered with enteroaggregative E. coli042and commensal E. coli HS, respectively.2. Comparative genomic analysis of highly and weakly virulent strainsBy comparing the sequences of PCN033and PCN061, we found that the majority of PCN033-specific sequences were horizontally transferred DNAs. Almost half of these mobile genetic elements contain bacteriophage-associated sequences. Among those specific sequences, genes for virulence determinants including adhesins, lipopolysaccharides, capsular polysaccharides, iron-transport systems and secretion systems, and the genes involved in metabolism were identified. Additionally, a potential virulent plasmid, PCN033p3, harboring many typical ExPEC virulence factors was identified in PCN033. We detailedly analyzed the above different aspects of the two strains and summarized the genotypic basis of their pathogenic difference.3. Comparative analysis of the biological characteristics of highly and weakly virulent strainsWe used BALB/c mice as model to investigate the pathogenicity of ExPEC strains PCN033and PCN061. The50%lethal doses (LD50) of PCN033and PCN061were3.4×105and2.3x107colony forming units (CFU) g-1, respectively. PCN033could mutiply more effectively in mouse blood compared with PCN061. Additionally, histological examination showed that the meninges of mouse infected with PCN033presented obviously meningitic manifestation. Based on the genetic variation between PCN033and PCN061, we verified corresponding phenotypic differences including flagella, motility, biofilm and metabolic capacity. The result revealed that PCN033was motile, able to produce flagella and metabolize more carbon substrates, exhibited a lower degree of biofilm formation than PCN061. Comparative analysis of the biological characteristics of highly and weakly virulent strains explained their pathogenic difference in terms of phenotype.4. Immunogenic characterization of outer membrane porins OmpC and OmpF of porcine ExPECWe chosed two conservative outer membrane porins OmpC and OmpF of porcine ExPEC. These two proteins were cloned and expressed to investigate their immunogenicity. Intraperitoneal immunization of mice with the purified recombinant proteins OmpC and OmpF stimulated strong immunoglobulin G (IgG) antibody responses. Both IgG1and IgG2a subclasses were induced, with a predominance of IgGl production. After challenge with2.5×107CFU (5xLD50) of the highly virulent ExPEC strain PCN033,62.5%and87.5%protection was observed in mice immunized with OmpC and OmpF, respectively. In addition, both anti-OmpC and anti-OmpF sera can mediate complement-dependent opsonophagocytosis. Phylogenetic analysis showed that the ompC gene was ubiquitously present in all E. coli strains, whereas the ompF gene was mutated in certain strains. Furthermore, the selection analysis indicated that gene ompC may be subject to strong immune pressure. Our results demonstrated that OmpC is a promising vaccine target against ExPEC infections in swine.