The Mechanisms Of Trimeric Autotranspotrer Adhesion Adh In The Interaction Between Actinobacillus Pleuropneumoniae And Host PAMs

Actinobacillus pleuropneumonia (APP) is a kind of strong tissue tropismGram-negative bacteria. Infection of piglets leads to pleural pneumonia andhemorrhagic necrotizing pneumonia and result in highly contagious and fatalrespiratory diseases. Adhesion to host tissue is an vital factor to bacterial colonizationand pathopoiesis. The newly discovered trimeric autotransporter adhesin (TAA) is animportant pathogenic virulence factor that can cause bacterial infection. Alveolarmacrophages(PAMs) is one kind of immune defense cells in the lungs that firstcontact with the pathogenic microorganisms and participate the host innate andadaptive immunity. The study on the interaction between PAMs and bacteria has beenbecome a new hotspot in bacterial pathogenesis studying. Our group firstly confirmedthe APP5b L20stain, a virulent serotype of APP, possess a TAA and named as Apa1,we further identified the Adh head domain is the key functional domain of Apa1. Thisstudy carried out the following work on Adh.1. The pathogenic mechanism of Adh mediated infection of APP piglet’ PAMcells by taking advantage of the Adh deletion strain. PAM difference were observedafter infected with APP wild-type (5b WT) and Adh mutant (5b ΔAdh) stain, and10cytokines released by these infected cells were measured by ELISA and cytokineschip. The results showed only IL-8level is much higher in the5b WT infected pigletscompared to that of the5b ΔAdh infected. Pharmaceutical inhibitors experimentsindicated both the IL-8mRNA and protein group were significant decreased afterphosphorylation of P38was inhibited in the5b WT（p<0.01）.However, inhibition ofthe phosphorylation of P38showed no effect on that of the5b ΔAdh infected PAMs.Westenblot results confirmed this higher release of IL-8is mediated by thephosphorylation of P38MAP kinase pathway. The Neutrophil chemotactic experimentin vitro showed the deletion of Adh decreased the recruitment of neutrophil by20%, The apoptotic study of PAMs indicted Adh can activate Fas and Bax, etc. followed byactivation of caspase-8,9,3to mediate apoptosis of PAMs.2. The gene expression profiles of5b WT and5b ΔAdh stains during theinfection process to PAMs were measured by using the APP specific genome-wideexpression profile chip technology. This study found185key genes,92upregulatedgenes and93downregulated genes, of the current reported APP genome-wide2886genes were significantly regulated. The upregulated genes mainly involved in energymetabolism, gene transcription and translation, virulence and trimer transshipmentadhesin, etc. The downregulated genes mainly involved in amino acid metabolism,ABC transportation and metabolism of coenzyme, etc. In addition, we reported manynewly virulent factors that involved in the infection process of APP to PAMs. Weobtained87genes,46upregulated genes and41down-regulated genes, that regulatedby Adh via comparing the gene expression profiles of5b WT and5b ΔAdh stains. Theupregulated genes mainly involved in hypothesis proteins, lipid metabolism, energymetabolism, gene transcription and procession. The down-regulated genes mainlyinvolved in hypothesis proteins, amino acid metabolism, carbohydrate metabolism,secondary metabolites synthesis, etc.3. Screening and identification of interacting proteins of Adh on PAM. Theinteracting proteins of trimer autotransport adhesin on PAMs have not been reported.In order to confirm the receptors of Adh on the PAMs, the prokaryotic expressed Adhprotein was used as bait to bind the RIPA lysis of PAM for immunoprecipitation, thenthe pulled down proteins were performed for mass spectra. Referring to protein onlinesoftware, Uniprot database,24suspected proteins were obtained. One of the highestcontent of24suspected proteins is keratin, the second highest is the serum albuminand heat shock protein70(HSP70). The membrane surface distributed proteins mainlyare the olfactory receptor protein receptor and fibroblast growth factor receptor andintracellular protein is NOD receptor. Then these suspected proteins were displayedon the293T cell through transfection of the reconstruction pDisplay vectors. In thefollowing adhesion study suggested the protein OR5M11has the best adhesion abilityto Adh, and can promote the adhesion of APP to293T cells. In addition, the pre-treatof OR5M11antibody to PAMs can significantly reduce the Adh adhesion and the release of IL-8and, more importantiy, can reduce the apoptosis of PAMs that causedby APP5b WT. Flow cytometry detection indicated the human lung epithelial cellsA549cell line own OR5M11protein as well as PAMs, indicating Adh may mediatethe adhesion of APP to A549cell line.4. Study the pathogenic mechanisms of Adh in mice model. In order topathogenicity differences of these two bacteria to mice, we firstly established APPrespiratory tract infection mouse model by intranasal infection of BALB/c with APP5b WT and5b ΔAdh. Our results showed the lethality of5b ΔAdh to mice is muchlower than that of5bWT to mice when infected with2×108CFU，1×108CFU withslighter lung pathogenic lesion, smaller increased lung weight, lower total protein inBALF and lower levels of proinflammatory cytokines IL-1β and chemokine CXCL1in lungs. In contrast, more inflammatory cells were infiltrated in the alveolar,interstitial lung and bronchus of the5b WT infected mice. In order to identify whetherAdh mediated lung damage was due to the over recruited neutrophils, the chemokinereceptor antagonist G31P treatment can significantly decrease the amount of therecruited neutrophils and, more importantly, significantly alleviate lung injury causedby5b WT infection. The immunohistochemistry of lung tissues indicated Adhcontributes to the activation of MAPK signaling pathway mediated by APP whichconsistent with the results from PAMs. This study firstly conformed TAA plays animportant role in the bacterial infection process by activation of p38, inducing therelease of inflammatory cytokines and chemokine of neutrophils to initiate hoststrength immune responses to APP, thus enhanced the pathogenicity of APP.5. Return APP to susceptible animals, piglets, to study the Adh mediatedpathogenicity of APP. In this study, piglets of45-day were infected with5b WTstrains and5b ΔAdh, then their clinical symptoms and pathological changes wererecorded. The results indicated that Adh mainly participated the early phase ofpathogenesis of APP to piglets.5b ΔAdh infection significant lagged the clinicalsymptoms, reduced bacterial lung burden and decreased the release of inflammatoryfactors, particularly the IL-8. Piglets routine blood tests showed the neutrophilstypical peak appeared in the peripheral blood neutrophils of the5b WT infected miceat24h post-infection, but not in the5b ΔAdh infected mice. Immunohistochemistry experiments further confirmed the Adh involved in the secretion of IL-8, MAPKsignal transduction pathway activation and apoptosis in lung cells. Lung specificexpression microarray technology detected495significant differentially expressedgenes, including274up-regulated genes,221down-regulated, between the5b ΔAdhinfected pigs and5b WT infected pigs. The upregulated genes were mainlyconcentrated in the antigen processing and presentation, NOD-like receptor signalingpathways, Toll-like receptor signaling pathways, DNA replication, and thedown-regulated genes were mainly in regulation of internalization, the intercellularadhesion and formation of endosomes. Therefore, the microarray analysis suggestedAdh enhanced bacterial host pathogenicity mainly by decreasing the repression ofantigen processing involved genes. This study confirms the Adh of TAA is involved inthe pathogenesis of APP in bacterial susceptible animals models and lays foundationto the study of mechanism of tropism pneumonia.