Infectomics Analysis Of Actinobacillus Pleuropneumoniae And The Mechanism Of Its Intracellular Survival In Porcine Alveolar Macrophage

Actinobacillus pleuropneumoniae(APP)is a small Gram-negative bacterium with strong tissue tropism and the infected piglets are characterized by pulmonary cellulosic exudation and hemorrhagic necrosis,which is highly infectious and lethal.In recent years,there is mainly mixed infection and co-infection,especially the addition of antibiotics in feed is prohibited,the bacteria carrying rate in pigs may increase.Mining new disease prevention strategies from the perspective of host anti-infection is a new direction in the future.However,at present,the research on the immune interaction between APP and piglets is little,especially the regional(local)immune study on lung tissue is much less.The lung has its unique structure,physiological function and tissue micro-environment.Under the stimulation of pathogens,unique regional immunity may occur.Therefore,this subject established the model of APP-infected piglets,detected the differentially expressed genes profile of peripheral blood monocytes(PBMC)of diseased piglets by transcriptomics,detected and analyzed the differentially expressed proteins in alveolar lavage fluid and serum by proteomics technology,and comprehensively analyzed the obtained data.The immune response characteristics of peripheral blood and lung of piglets that were infected with app were obtained.Based on the data revealed that APP inhibited lung immunity in the early stage and promoted it in the later stage,we constructed APP transposon mutants library and screened their function on regulating immunity.Finally,the immunology mechanism of APP virulence factor Adh promoting the survival of bacteria in alveolar macrophages was discussed.The details are summarized as follows:1.Changbai piglets were used for intranasal challenge with Actinobacillus pleuropneumoniae 5b(APP L20 3.1*10¹⁰CFU/pig),and comprehensive evaluation was made from clinical score,blood routine,lung pathological changes and bacteriological examination.The body temperature of infected piglets increased from6 hours after infection.At 96 hours,the body temperature rose to more than 40?;After 48h,there were anorexia,depression,purulent nasal juice,cough and dog sitting breathing.The number of leukocytes and neutrophils increased significantly.X-ray examination showed that the lung gradually appeared cloud shadow,developing from point to sheet;The lung texture gradually thickened.From 24 hours,the lung showed massive congestion,swelling and blunt and round edges.At 120 hours,the degree of lung injury increased,and there was massive congestion and necrosis.In conclusion,a stable experimental piglet infection model was established.2.The pulmonary immune system of porcine pleuropneumonia was detected by infectious omics.The study confirmed that in the early stage of APP infection,the pulmonary immune response was inhibited and the systemic inflammatory response was strong;After 12 hours,lung inflammatory factors began to rise and lasted for a long time,and a large number of inflammatory cells infiltrated,causing excessive inflammation and tissue damage.It was found for the first time that APP inhibited the host immune response and inherent apoptosis signal pathway,which may be a new pathogenic mechanism.KEGG enrichment analysis found that a large number of differential genes related to T cell receptor signaling pathway were down regulated,such as CD28 molecule(CD28),mitogen activated protein kinase 1(MAPK1),p21activated kinase 2(PAK2),etc and the inhibition was more significant with the infection time.App may induce the host to secrete immuno-modulatory proteins in the early stage,thereby inhibiting complement activation and neutrophil chemotaxis.3.In order to find out the bacterial components of APP that inhibit immunity in the early stage and induce excessive inflammation in the later stage,a mariner transposon mutation library was constructed by using the mariner transposon system.The transposon clones that inhibit/promote the inflammation response were screened.After sequencing and analysis,a total of 23 genes were obtained.By measuring the transcriptional level differences of IL-6,IL-8 and GM-CSF after the interaction between transposon mutant and wild strain and PAM,it can be divided into"induced inflammatory response level"mutant and"inhibited inflammatory response level"mutant.The transposon clones with gene inactivation were selected for confirmation in mouse infection model.The pathogenicity of the transposon clones with down-regulated proinflammatory factors was significantly weakened and the lung lesions were alleviated;transposon clones with up-regulated proinflammatory factors had significantly increased pathogenicity and aggravated lung lesions.It provides a basis for the study of immunomodulatory pathogenesis and vaccine of APP.4.The virulence factor of APP transports adhesin head Adh effects the virulence of bacteria.Its effects on the bacteria killing and cytokine secretion of porcine lung macrophages(PAM)were studied from the perspective of immune regulation.By comparing the knockout strain 5b?Adh with the 5b WT strain,it was found that Adh could promote the synthesis of LPS,up regulate the cation transport regulator 2(CHAC2)expression in PAM,improve the intracellular level of GSH,reduce the intracellular ROS,and be conducive to the survival of APP in cells.At the same time,the up regulation of CHAC2 can inhibit IL-1?and IL-6 and TNF-?.Expression of.TLR4/NF-?B pathway regulates the expression of CHAC2.It was found that transfection of overexpression plasmid pc DNA-CHAC2 into PAM cells could significantly increase the survival number of 5b?Adh strain in cells,while sh RNA-CHAC2 could reduce the number of intracellular bacteria in 5b.Overexpression of CHAC2 could significantly reduce the level of ROS and significantly increase the number of intracellular bacteria of 5b?Adh strain.Based on the above studies,CHAC2 mainly affects the changes of ROS by regulating the level of GSH,thus changing the difference of intracellular bacteria between the two strains.And CHAC2 activated NOD1/NF-?B pathway and regulated the release of inflammatory factors related to PAM cells during APP infection and promotes the killiing ability of PAM cells.Similarly,PAM cells will also start the expression of anti-inflammatory factors and maintain the homeostasis in vivo under the induction of APP.At this time,it may be conducive to the immune escape of APP.The LPS level of 5b was significantly higher than that of 5b?Adh.It can activate TLR4/NF-?B pathway or induce the expression of inflammatory factors by regulating CHAC 2.In a conclusion:This study established a piglet APP infection model,and revealed the response characteristics of PBMC,serum and BALF in piglets at different stages of APP infection from gene and protein levels by infectious omics.At the same time,a transposon library was established to screen some inflammatory regulatory genes of APP and confirm the infection in mice.Finally,it was confirmed that Adh,the main functional region of adhesin,the virulence factor of APP,not only participated in the pathogenesis by adhesion,but also prolonged the intracellular survival time and amount of APP and enhanced the inflammatory response.These provide a basis for the pathogenesis of porcine pleuropneumonia and vaccine research and development.