Improvement Of Haemophilus Parasuis Natural Transformation And Function Studies Of AT2, AT3

Haemophilus parasuis(HPS) is the etiologic agent of Glasser’s disease. As a commensal bacterium in the swine upper respiratory tract, HPS can invade host and cause systemic disease under certain conditions. In this study, we modified the existing methods for natural transformation by DNA methylation modification and adding USS squence. In our study, we hope to improve the transformation efficiency and expand the natural competence strains. At the same time, functional study of autotransporters in H. parasuis by construction Δat2pd, Δat3pd and Δat2pdΔat3pd mutant in CF7066 was carried out explore to how H. parasuis resists natural immunity. 1. Construction of recombinant plasmidPrimers cloning upstream and downstream homology arms of at2, at3, htr A were designed according to the SH0165 whole genome sequence. Connect the homology arms and corresponding resistance cassette by overlapping PCR, and connect the recombinant fragments with plasmid vector p K18 mobsac B or p SHK3 to obtain the recombinant plasmid.2. Improvement and evaluation of natural transformation methodWe transform of different H. parasuis strains by using recombinant plasmid p K3 UK and p S3 UK to prove whether the shuttle vector can be used in natural transformation of H. parasuis. p S3 UK was also transformed to SH0165 to get the vivo methylated p S3 UKm. The transformation efficiency of p S3 UKm was compared with p K3 UK and p S3 UK to evaluate whether DNA methylation modification can improve the natural transformation efficiency of H. parasuis. 3. Construction and identification of Δat2pd, Δat3pd and Δat2pdΔat3pd mutant in CF7066The recombinant plasmid(p K3 UK, p K2UE) was transformed to CF7066 to generate the mutants 7066Δat2pd::ermR, 7066Δat3pd::kanR and 7066Δat2pd::ermR Δat3pd::kanR. The mutants were identificated by PCR, Southern blot, RT-PCR and sequencing.4. Functional study of at2 and at3By plotting the OD600-time curve of CF7066 mutants, we found there is no significant difference between the wild-type strain and the mutants. Serum resistance test showed the deletion of at2 and at3 in CF7066 led to an increased sensibility to the complement in serum. Our quantification PCR showed that the cap D, gal U, gal E and omp P2 were upgulated in the mutants, which is consistant with the increased abilities of bacterial adhesion and serum resistance. Phagocytosis assays with porcine alveolar macrophages 3D4/21 suggest that both at2 and at3 contribute to phagocytosis resistance.In conclusion, we found in this study that the H. parasuis-E.coli shuttle vector p SHK3 can also be used for natural transformation. DNA methylation modification can improve the natural transformation efficiency of H. parasuis. By construction Δat2pd, Δat3pd and Δat2pdΔat3pd mutants in CF7066, we preliminary demonstrated the contribution of AT2 and AT3 in H. parasuis serum resistance and phagocytosis resistance.