| Acinetobacter baumannii is an opportunistic pathogen that has received much attention and research because of its antibiotic resistance,survival,and revealed virulence mechanisms.Acinetobacter baumannii often triggers pulmonary and blood infections,causing pneumonia or bacteremia,mainly manifested by clinical symptoms such as high fever,dyspnea and abortion.The bacterium is widely distributed in almost all natural environments including water,soil,air and so on.In addition,this strain has a wide range of hosts and can cause higher mortality rate of livestock and is very harmful to the livestock industry,as well as infect humans and seriously damage people’s health.Therefore,to prevent the infection caused by A.baumannii,studies on the pathogenic mechanism of A.baumannii as well as the mechanism of host immune response are essential.In this paper,the mechanism of host immune response caused by A.baumannii infection of bovine origin was investigated by two models,in vitro infection and in vivo infection.The main results and innovation points of this paper are as follows:(1)Firstly,a virulence factor evolution tree of HNAB01 strain which is a bovine Acinetobacter baumannii isolate in our laboratory was constructed.A multiple sequence evolutionary tree constructed according to the eight major virulence factor genes(ace I,tss B,aba R,car O,omp A,tss G,blp2 and lpt D)of HNAB01 strain.The results showed that HNAB01 strain had the closest relative to ATCC 17978 strain and also a close relative to the highly virulent LAC-4 strain.The HNAB01 strain was proved to be highly virulent.Secondly,the HNAB01 strain was used to infect bovine fibroblasts in vitro.The results showed that HNAB01 strain exhibited obvious cytotoxicity,causing 80% of the cells to undergo morphological changes post 12 h of infection.However,all cellular morphological changed and the cells began to die post 14 h of infection.The strong toxicity of HNAB01 strain was further demonstrated at the level of cellular infection.(2)RNA-sequencing was performed on bovine fibroblasts infected with bovine A.baumannii HNAB01 isolate 14 h to analyze the mechanisms involved in the immune response against A.baumannii infection by fibroblasts.The analysis showed that a total of3,014 differential expressed genes(DEGs).The DEGs though KEGG enrichment were mainly enriched in TNF,IL-17,NLR,MAPK,apoptosis,endocytosis,NF-κB and HIF-1signaling pathways.GSEA results also showed that the expression of related signaling pathways such as TLR,chemokines,Th17 differentiation was up-regulated.PPI analysis showed that all DEGs could be divided into an apoptosis functional cluster with JUN and CASP3 as core functional proteins and an innate immune functional cluster with IL-6,ERBB2,EGFR,mapk8,and CHUK as core functional proteins.(3)To verify the role of TLR4 receptor in recognition of A.baumannii infection,an LPS-free HNAB01 strain was successfully constructed using colistin induction method in this study,mice macrophages were successfully depleted using clodronate liposomes,and the respective models were constructed by infecting WT mice with HNAB01 wild-type strain,macrophage-depleted mice with hnab01 wild-type strain,and macrophage-depleted mice with LPS-free HNAB01 strain.The main results showed that lung bacterial load in all mice infected with the wild-type strain rose within 24 h post infection,whereas macrophage-depleted mice infected with the LPS-free HNAB01 strain had greater lung bacterial load at 12 h post infection than at 24 h post infection.Pathological sections revealed that upon infection with the wild-type strain,a significant inflammatory response occurred in the lungs of mice,with or without depletion of macrophages,whereas in the case of depletion of macrophages,deletion of LPS producing a small amount of A.baumannii only induced inflammatory changes in the lungs of mice.This result demonstrated that stromal cells were mainly dependent on the recognition of A.baumannii by TLR4 receptor. |
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