| Haemophilus parasuis is the agent of Gl?sser’s disease, as a commensal bacterium in the swine upper respiratory tract, it can invade host and cause systemic diseases under certain stress conditions. The Htr A(high temperature requirement A) proteins are very well conserved in evolution and present in both prokaryotic and eukaryotic organisms, they play an important role in quality control of proteins. In many pathogenic bacteria species, the Htr A cloesely related to their survival in host and virulence. This study focused on the effect of Htr A on adherence and survival under stress response in the H. parasuis, our main results are as follows: 1. Construction of Δhtr A mutants by natural transformation in H. parasuisA recombinant plasmid was constructed which is named p K18 mobsac B-htr A-UKD. The recombinant plasmid was transformed into standard serotype 5 strain Nagasaki by natural transformation, and the htr A mutants were screened on kan resistant plates. Then the mutants were further identified through PCR, RT-PCR and Southern blot. 2. Comparison the biological characteristics of wild strain Nagasaki and Nagasaki htr A mutant strain in environmental adaptationThe growth curves of wild strain Nagasaki and mutant stain Nagasaki-Δhtr A have been drawn under 37°C and 40°C, results showed that htr A-deletion has affected the growth of both strains. Moreover, the growth of mutant slowed significantly under 40°C and the viable count also reduced dramatically.We also have drawn the growth curves of both strains under the conditions of p H6.5 or p H8.5, the results showed that the two strains were affected remarkably under p H6.5, yet the OD600 of the mutant was lower than the wild strain under p H8.5 at each time point. So the enviroment adaption ability of the mutant was weaker than the wild strain. Observation on the of Nagasaki and Nagasaki-Δhtr A through SEM at two time points indicated that the bacterial surface of mutant was smoother than the wild strain. 3. Htr A is involved in the resist of phagocytosis, adherence and serum resistance in H. parasuisWild-type strain Nagsaki and Nagasaki-Δhtr A mutant were cultured with pig macrophages 3D4/2, the result showed that the resistant phagocytic capacity of Δhtr A mutant was stronger than the wild strain. Both strains were cultured with porcine kidney epithelial cells(PK-15) and pig iliac endothelium cells(PIEC), our findings demonstrated that the adherence index of Nagasaki-Δhtr A was significantly lower than wild strain at different time points(0.5h, 1.5h, 2.5h). When added recombinant Htr A, the adherence ability of wild-tye strain was enhanced remarkably compared with mutant. Serum resistance test showed the deletion of htr A in Nagasaki led to an increased sensibility to the complement in serum. 4. Identification of differentially expressed genes involved in survival and adherence after htr A deletion by q RT-PCRA few genes were selected which are relevant to survival and adherence to analyze their expression after htr A-deletion. It turned out that rfa E, thy A, cdt B, nan H, rfa F related to the adherence or virulence were down regulated significantly under 37°C after htr A-deletion, which was consistent with former phenotypic test. Moreover, clp P and ops X that upregulated were respectively related to heat shock and serum resistance. The majority of genes down regulated were related to adherence under 40°C, and two heat shock genes deg S, clp P were down regulated simultaneously. We could conclude that the expression of many functional gens had been affected by loss of htr A.In summary, we have proved that Htr A might involve in stress response, adhesive ability, resist of phagocytosis and serum resistance in H. parasuis. |
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