The Influence Of Hcp Protein Of T6ss On APEC Biological Characteristics And NLRP3 Inflammasome In The Chicken

As a Gram-negative bacterium,APEC（Avian Pathogenic Escherichia Coli）,can cause the diseases of poultry,such as air sacculitis,sepsis,etc.The extensive spread and virulence of APEC have caused threats in the aquaculture industry.Therefore,it is significant to study APEC’s virulence system and the pathogenic mechanism in the public health and the disease prevention.The virulence factor of APEC is numerous.And Type VI Secretion Systems（T6SS）involves in APEC’s pathogenic process through the secretion of effectors.Among them,Hcp（hemolysin coregulated protein）is involved in the assembly and function of T6SS.Previous studies have confirmed that hcp gene involved the regulation of APEC’s invasion and bacteria competition.However,the specific regulation mechanism of the protein encoded by hcp genes on APEC characteristics and pathogenic processes is unclear.This study constructed an overexpression strain of hcp gene.By which,we studied the effect of overexpression of hcp gene on APEC biological characteristics;The role of Hcp protein in the colonization of APEC was studied by tissue bacterial load test.In order to explore the antibacterial activity of Hcp in bacteria,Hcp protein of prokaryotic expression was used in vitro antibacterial experiment and the toxicity test of Escherichia Coli,and the tested strain was sequenced;To study the role of hcp gene in the process of APEC infecting the host,The effect of NLRP3 inflammasome is obtained by a total of culture with the constructed strain or the expressed protein.RT-q PCR and ELISA were used to measure the m RNA level of NLRP3 and Caspase-1 gene and the secretion of the cytokine（IL-1β,IL-18）.The specific research content and results are as follows:1.The effect of hcp gene on the biological characteristicsThe hcp overexpression strains were constructed to carry out the tests of biological characteristics.The results confirmed that the overexpression of hcp2a gene enhanced the biofilm formation capacity of the AE17（P<0.01）.In comparison to the wild strain AE17,overexpression of hcp2a、hcp2b gene reduced the viability of AE17 in the environment（H₂O₂,p H=5）（P<0.01）.In addition,when the hcp2a、hcp2b gene was expressed excessively in the environment（p H=10）,the survival rate of the AE17 had no significant change.After the hcp2a,hcp2b gene was overexpressed,the resistance to serum of AE17 had significant reduction（P<0.01）.The adhesion measurement results showed that the adhesion rate of the AE17-p STV28-hcp2a strain is 5.66 times than the control strain.Also,when the hcp2a gene was overexpressed,the adhesion of AE17 was significantly increased,and the hcp2b gene had no significant effect on the adhesion of AE17.By intramuscular injection of chicks and conducting tissue bacterial load tests,the results showed that the bacterial load increased significantly among the brain and lungs of Hcp active protein group.And this suggested that the hcp gene plays a role in the colonization process of APEC.2.Research on the antibacterial activity of Hcp proteinIn this study,we conducted an Escherichia coli toxicity experiment.The results showed that Hcp protein inhibited the growth of Escherichia coli（BL21）.The sequencing results of strains showed that the co-culture group of Hcp2a protein was screened to 1110 different genes,of which the expression of 418 genes was up,the expression of 692 genes was down.The GO note indicated that the different gene was mainly concentrated in the nucleoside monophosphate metabolic process,purine nucleoside triphosphate metabolism,riboside triphosphate metabolism,etc.KEGG analysis showed that the differential gene was mainly concentrated in microbial metabolism in different environments,biosynthesis of amino acids,biosynthesis of secondary metabolites,etc.The co-culture group of Hcp2b protein was screened to983 different genes,of which the expression of 447 genes was up,and the expression of 536 genes was down.The GO note indicated that the different gene was mainly concentrated in the nucleoside triphosphate metabolism,ATP biosynthesis,etc.KEGG analysis showed that the differential gene was mainly concentrated in pyruvate metabolism,metabolism of methyl butyrate,fatty acid degradation and other signaling pathways.Hcp protein was expressed and purified for micro-antibacterial test.The results showed that Hcp2a and Hcp2b protein had the inhibitory effect on indicators（CMC44102,ATCC25923,ATCC9150,S5,S10）.This suggested that Hcp was involved in regulating the survival of AE17 in bacteria.3.The influence of the constructed strain of hcp gene and Hcp protein on the NLRP3 inflammasome in the hostIn order to study the influence of the constructed strain and Hcp protein on the NLRP3 inflammasome in the host,RT-q PCR and ELISA were performed in this study.The results showed that the NLRP3、Caspase-1 gene m RNA of cells which were infected withΔhcp2a strain was significantly lower than the wild strain AE17 group,and the content of IL-1β,IL-18 was significantly lower;The same was true with the cells which were infected withΔhcp2b strain;After the cell was treated with the overexpressed strain of hcp gene,the level of NLRP3 gene m RNA was significantly increased in comparison to the control strain.After the treat of the Hcp2a protein of the prokaryotic expression on DF-1 cells,the expression of NLRP3 gene m RNA was significantly up in comparison to the control group.After 12h,the secretion of IL-1β,IL-18 increased significantly among the Hcp2a protein group;When cells were treated with the Hcp2b protein of the prokaryotic expression for 12h,the NLRP3 or Caspase-1 gene m RNA level was significantly raised.After 24h,the content of IL-18in the supernatant increased significantly.The above results indicate that the constructed strain of hcp gene and protein can affect the m RNA expression of NLRP3inflammasome and the secretion of cytokines（IL-1β,IL-18）.In summary,the T6SS-2 hcp gene overexpressed strain is successfully constructed.The overexpression of hcp gene has influenced the characteristic of AE17,including the biofilm formation,environmental tolerance,adhesion process and the ability to resist the sterilization of serum;The result of the toxicity test of Escherichia coli and transcriptome sequencing indicates that Hcp protein inhibits bacterial growth by affecting BL21 metabolism and amino acid synthesis.Antibacterial tests of Hcp protein show that Hcp protein can inhibit other bacterial growth and participate in bacterial competition of APEC.In addition,the results of experiments indicate that the hcp gene and its protein have an effect on the expression of NLRP3 or Caspase-1 gene m RNA,resulting in the change of secretion level of IL-1βor IL-18 downstream.