The Mechanism Of Pagc Protein Affecting Intestinal Colonization Of Salmonella Pullorum

Salmonella pullorum is a gram-negative intestinal pathogen that matches poultry.It is a major threat to young poultry under the age of 3 weeks and has a high mortality rate after infection,which has seriously affected the development of poultry industry and public health safety,and has caused huge economic losses to China's chicken industry.PagC protein belongs to the bacterial outer membrane protein,which is widely present in various serotypes of salmonella,but its biological function has not been elucidation,especially in salmonella pullorum.In this study,the pagC deletion strain of S.pullorum was constructed,and the mechanism of pagC affecting intestinal colonization of S.pullorum by affecting bacterial extracellular vesicle production and biofilm formation was preliminarily clarified,which helps to further elucidate the pathogenic mechanism of the bacteria.Relevant researches are as follows:1.Construction of the pagC deletion strain of salmonella pullorum and biological characteristics analysisIn this study,the pagC of CVCC1800 of S.pullorum was successfully knockouted by lambda-Red homologous recombination method.The pagC of salmonella pullorum CVCC1800 was cloned into plasmid pBR322 and electron-transformed into the deletion strain to construct the pagC complement strain.Compared the wild strains of Salmonella pullorum(WT),the pagC deletion strains(?PagC)and the pagC complement strain(C?PagC)biological characteristics.Growth curve measurements showed no significant difference in ?PagC and WT growth rate.Animal experiments showed that the deletion of the pagC enhanced the colonization ability of Salmonella pullorum in the intestine,and the bacterial load increased at least 10 times compared with the wild strain.2.Effect of pagC deletion strain on intestinal colonization of Salmonella pullorumIn this experiment,extracellular vesicles in WT,?PagC and C?PagC culture supernatants were extracted by ultrafiltration and ultracentrifugation,and then purified by density gradient centrifugation.Transmission electron microscopy and SDS-PAGE showed that 30%of Optiprep had higher purity extracellular vesicles.Subsequently,the purified extracellular vesicles were subjected to mass spectrometry and found to contain a lot of enzymes related to glucose metabolism.The extracellular vesicles significantly inhibited the biofilm formation of Salmonella pullorum in vitro.The quantitative results of Broadford and SynaptoRed C2 showed that the extracellular vesicles secretion of the pagC deletion strain was significantly reduced.It is speculated that after the pagC was knockout,Salmonella pullorum is reduced in secretion extracellular vesicles,and the biofilm is not easily decomposed,which in turn enhances the colonization of bacteria in the host intestinal tract.3.Development of bacteria ghost based on pagC deletion of S.pullorumBecause there is no differential diagnosis target for Salmonella pullorum vaccine in the market,it is difficult to purify the disease.In this study,a cell penetrating peptide(CPP)was used to prepare ghost based on ?PagC.The results showed that CPP inactivation was the best when the concentration was 50 ?mol/L and ?PagC was OD600=0.4.The bacteria ghost has provided a target for differential diagnosis while retaining antigenicity,and laid a foundation for the subsequent development of a differential diagnosis ghost vaccine against Salmonella pullorum.