Genomic Approach To Study Virulence Associated Factors Of Haemophilus Parasuis

Haemophilus parasuis is one of the colonizers as the normal microflora of porcine upper respiratory tract, but it is also the etiological agent of Glasser's disease, which is characterized as fibrinous polyerositis, arthritis and meningitis in veterinary clinics.Currently, nearly all the countries and regions in the world involved in swine industry has been reported for prevalence and outbreaks of such disease. Swine production has dramatically changed in recent years. New trends in production, which include the early weaning of piglets and the management of specific pathogen free herds, have contributed to an increase in the prevalence and severity of bacteria diseases, including those caused by H. parasuis. Clinical veterinary significant challenges due to the existence of strains with different phenotypes particularly lack of knowledge of those virulent and avirulent strains. In addition, still little is known about virulent associated factors of H. parasuis, factors contribute to the H. parasuis pathogenesis and molecular basis of host-pathogen interaction during infection. Thus, there is the urgent need to decipher the complete nucleotide sequence of H. parasuis, which could provide much vaulable information for molecular biology of H. parasuis in further. Collected the identified H. parasuis strains in our lab, we chose the SH0165 strains, which belonged to the dominat epidemic serotype 5 in China, for further complete genome sequencing. After finishing the complete genome sequence, we systemically analyzed the composition of virulent related factors, induced genes during in vivo infection and possible mechanism of H. parasuis virulence evolution. And summaried the specific tasks as the following:1. H. parasuis pupulation genotyping and selection the strain for genome sequencingTo obtain the representative strain,17 strains chosen from different regions as the representative strain in China, with another 17 strains considered as the international serotyping strains together, these strains were used to analysis the phylogenetic tree basing on the concatenated 18 conserved genes. The result indicated that there were at least two genotypes in H. parasuis population, and most of prevalent strains in China belonged to genotype A. In addition, ompP2 and sodA genes could be the potiential targets to differentiation the virulent and avirulent strains. And SH0165 as the domaint serotype 5 belonged to the genotype A within Chinese putative lineage was chosen for further genome sequencing analysis.2. Complete genome sequence of H. parasuis SH0165With the shotgun as DNA sequencing strategy, draft assemblies were compiled using Consed, based on 20,102 reads, to give sixfold coverage of the genome. Gaps among contigs were closed either by primer walking with selected clones, which were identified by analysis of the forward and reverse links between contigs with the Perl/Tk algorithm, or by sequencing of the DNA amplicons generated by PCR. And the architecture of SH0165 genome was confirmed by restriction enzyme digestion and long PCR covered the genome. The genome of H. parasuis strain SH0165 is composed of 2,269,156 bp in a single circular chromosome with an average G+C content of 39.99%,2,292 predicted coding sequences (CDS),202 pseudogenes, and 76 structural RNAs. The categories of gene function, protein features, metabolite pathways, phage-related genes, transport associated genes, pesuogenes and signal tranduction and regulation associated genes in SH1065 genome were also summarized in detail.3. Functional analysis of homologous virulent associated genes within SH0165 genomeWith available eight complete genomes in Haemophilus genus, by comparative analysis of virulent genes in different strains and species, we also collected the research literature associated virulent genes within Haemophilus genus. The comprehensive map of 96 potential virulent genes in SH0165 was classficated into seven different functional cateogries. Transcription analysis of these genes in vivo and in vitro by real-time PCR concluded possible 18 major virulent genes or operon genes. The following PCR scanning for above 18 genes or operon genes indicated these genes were conserved in Chinese epidemic strains. The available data implied these 18 genes or operon genes may play an important part in H. parasuis infection which contributed to the diease outcome.4. Transcriptome analysis of genes within H. parasuis SH0165 induced during in vivo infectionNext generation sequencing technology, Illumina Solexa Technology, was applied to analysis of transcriptome of H. parasuis during brain and lung infection, the result showed two prophages in SH0165 genome were significant induced up-expression, indicated phage related genes may play a role in H. parasuis brain in infection. There were 305 genes both induced up-expression in brain and lung infection, these genes were manly involved in nucleotide biosynthesis, energy production and conversion, antioxidant fuction, which implied H. parasuis adapted the mechanism to proliferation during infection by improvement the efficieny to achieve biosynthesis and energy metabolism. These also reveal the corresponding enzymes involved in nucleotide biosynthesis, energy production and conversion as putative antibiotic targets for the treatment of H. parasuis infection. 5. Phylogenomic analysis of virulent phenotype evolution of H. parasuisWith available 20 genomes in Pasteurellaceae,16S rDNA,35 conserved genes, and phylogneomic mapping of coverved genes were used to construct the phylogenetic tree in family. The result showed that there were two lineages in Pasteurellaceae, H. parasuis were genetic similar to Actinobacillus pleuropneumoniae (Ap) with in the same lineage, but different from other Haemophilus species in genus, which indicated the taxnomy of H. parasuis were different from Haemophilus genus, forming into an unique genus in Ap lineage.Comparative analysis of 20 collected virulent and comensal species in Pasteurellaceae, the result showed the mechanism of reduction evolution in members of Pasteurellaceae. By comparative analysis of draft 29755 and complete SH0165 genome, the constitution and number of phage genes were the major difference in two H. parsuis genomes. Combined the feature of SH0165 genome suggested that phage mediated virulent genes transfer and metabolic genes inactivation lead to SH0165 strain in a phase of rapid changes in genome, and this process may be the possible mechanism through which avirulent strain rapid evolve to an emergent virulent strain.