Functional Studies Of NanH And At1 Genes In Haemophilus Parasuis

Haemophilus parasuis(H. parasuis)is a gram-negative bacterium, which is a species of opportunistic pathogen in swine upper respiratory tract.This paper mainly talked about the neuraminidase Nan H and the autotransporter protein AT1,and explore their main function in Haemophilus parasuis. The main results are as follows: 1. Verification of Neuraminidase in H. parasuisThe neuraminidase activity of wild strain JS0135, the mutant JS0135Δnan H::kan(△nan H) and the complementary strain JS0135-Cnan H(Cnan H)were tested. The results showed that the neuraminidase activity of Cnan H kept the same as the strain △nan H, also significantly lower than the wild strain. The adhesion ability of Cnan H was the same as the strain △nan H, also significantly higher than the wild strain.These data suggested that the complementation strains did not work well, but the deletion mutant was constructed successfully. The growth curve of wild strain JS0135 and mutant stain △nan H showed that the nan H deletion did not affect the growth of H. parasuis. Also,we observed that the in vitro growth of the strains did not need sialic acid as nutrition. 2.The adhesion ability、serum resistance and oxidation resistance of the △nan HWild type JS0135 and △nan H mutant were cultured with porcine kidney epithelial cells(PK-15) and pig iliac endothelial cell(PIEC)to test their adhersion ability. The results showed that the adhesion ability of △nan H was stronger than the wild type strain.Later, wild type strain JS0135 and △nan H mutant stain were incubated with normal pig serum, and the results showed that the adhesion ability of wild type strain was stronger than △nan H mutant. We also foud the complement classical pathway may play a role in the killing of the △nan H strain. The complement deposition on the JS0135 and the △nan H mutant were measured and compared. The results showed certain proteins associated with the mutant cells, but not associated with the wild type JS0135. Ig G binding was also detected in the △nan H strain,which was more than that in the wild type strain when incubated with NS, by western blot analysis. Mass spectrometry analysis showed that the complement C3 deposited on the surface of the mutant strain, and verified by western blot. Moreover, through flow cytometry assay we found in the mutant surface deposition of complement C3 on the surface of the mutant strain was significantly higher than that of wild type.In addition, the wild type strain JS0135 and △nan H mutant stain were incubated with H2O2. The results showed that the oxidative resistance of wild type strain was much stronger than the △nan H strain. 3. Construction and functional study of the at1 gene in H. parasuisBy natural transformation, we got at1 deletion mutant containing kan resistance in Haemophilus parasuis JS0135(Δat1). Through the incubation of the △at1 with PK-15 cells, we find that at1 gene may not be involved in the cellular toxicity. The results of adhesion assay indicated that at1 deletion resulted in an increased adhesion compared with the wild type strain.We also foud that the deletion of the at1 gene did not affect the biofilm formation and agglutination of the strain. Meanwhile, the oxidative resistance of wild type strain was stronger than the △at1 mutant, suggesting that the at1 gene may be involved in bacterial oxidation tolerance.In conclusion, based on the above results, we demonstrated that sialidase may play an important role in the pathogenesis of Haemophilus parasuis.The sialidase can reduce the bacterial surface deposition of the complement C3 to enhance bacterial resistance to serum killing. At the same time, the sialidase can increase the oxidation tolerance of strains to facilitate their infection and invasion in the host. And moremver the autotranspoter AT1 is involved in the oxidation tolerance of strains.