A Preliminary Study On The Damaging Effect Of Adenylate Cyclase Of Escherichia Coli From Sheep Lung On Alveolar Macrophages In Mice

Objective: Extraintestinal pathogenic E.coli(Ex PEC)is an important pathogen of zoonosis,which can cause human and animal pneumonia,neonatal meningitis,septicemia and other diseases.Adenylate cyclase(Adenylate cyclase,AC)is a kind of virulence factors closely related to bacterial virulence.Its coding gene cya A is one of the most important genes regulating virulence in some bacterial genomes,which can regulate a variety of physiological functions of bacteria.In order to study how the AC acts of Ex PEC XJ10 causing sheep pneumonia on alveolar macrophages(MH-S)in the significant barrier of respiratory tract,the MH-S were infected by Ex PEC XJ10 wild strain(XJ10WT)and cya A gene deleted strains(XJ10Δcya A),the cells were collected for transcriptome and proteomic analysis,the effect of AC of sheep lung derived Ex PEC XJ10 on MH-S in respiratory infection was systematically analyzed to provide meaningful data for clarifying the mechanism of sheep lung derived E.coli infection.Methods:(1)Taking the XJ10 WT as a control,the m RNA expression levels of adhesion and invasionrelated genes of XJ10Δcya A were detected by q PCR;the adhesion of XJ10Δcya A to MH-S cells was analyzed by co-culture of cells and bacteria invasion ability and CCK-8 method were used to analyze the inhibition of bacterial proliferation on MH-S cells;Flow Cytometry was used to detect the apoptosis rate of cells co-cultured with bacteria.(2)The XJ10 WT and XJ10Δcya A were co-cultured with MH-S cells respectively,and the cells were collected for m RNA transcriptome analysis,through the difference analysis of the data,the transcripts and signal pathways with significant differences were selected and verified by q PCR.(3)XJ10WT and XJ10Δcya A were co-cultured with MH-S cells respectively,and the cells were collected for TMT proteomics measurement.After the difference analysis of the data,the proteins and signal pathways with significant differences were selected and verified by Western blot.At the same time,IL-1β,Il-6 and TNF-α in the cell supernatant was detected by ELISA.Results:(1)Compared with the XJ10 WT,the transcription levels of 6 genes related to adhesion and invasion were down-regulated in the XJ10Δcya A,among which crl,csg A,flu,pap,and mot A genes were significantly different.When MH-S cells were infected at a multiplicity of infection of 100,the amount of adherent bacteria of the XJ10Δcya A and XJ10 WT reached saturation in 2h and lasted to 5h,and the amount of invasive bacteria increased from 1h to 5h and reached a peak,and then continued to decline.MH-S cells were infected at a multiplicity of infection of 10,the number of adherent bacteria began to decrease after reaching the peak at 5h,and the number of invasive cells gradually decreased after reaching the peak at about 7h.In different multiplicity of infection and different time periods,the amount of adherent and invasive bacteria of the XJ10Δcya A was less than that of the wild strain.When the multiplicity of infection of the 2 strains was 100,the inhibition rate of cell proliferation of the XJ10Δcya A was significantly lower than that of the wild strain at 3h-6h;The cell proliferation inhibition rate of the amount of bacteria with multiplicity of infection of 100 was significantly higher than that of the infection ratio of 10 at 1h-6h.(2)m RNA transcriptome sequencing results showed that a total of 73 differentially expressed genes were screened in the XJ10 WT infection group,of which 56 were significantly up-regulated and 17 were significantly down-regulated;2571,271 and 357 GO terms were enriched in molecular functions,cell components and biological processes;131 pathways including JAK-STAT signaling pathway,necroptosis,NF-κB signaling were enriched with KEGG analysis,among them,the m RNA expression of genes involved in the enrichment of acute inflammatory response were significantly up-regulated compared with the XJ10 WT group.q PCR verification results were consistent with transcriptome sequencing results.(3)TMT proteomic sequencing results showed that compared with the XJ10 WT infection group,a total of 123 differentially expressed proteins were screened out of the 7070 proteins identified in the XJ10Δcya A infection group,of which 54 were significantly up-regulated and 54 were significantly down-regulated.499,369 and 2948 GO term were enriched in the molecular function,cell composition and biological processes of GO analysis,and 87 pathway including cytokine-cytokine receptor interaction,NF-κB signal pathway and TNF signal pathway were enriched in KEGG analysis.Combined analysis omics analysis showed that Bcl2a1 and Il1 b were mainly involved in NF-κB signal pathway and apoptosis.Western blot results showed that the proteome sequencing results were consistent;ELISA method was used to detect the contents of IL-1β,Il-6 and TNF-α in the cell supernatant,and it was found that XJ10Δcya A infection group could stimulate the secretion of MH-S cells to produce more IL-1β,IL-6 and TNF-α.Conclusion:(1)The deletion of cya A gene resulted in the down-regulation of transcription level of adhesion and invasion related genes of XJ10,the decrease of adhesion and invasion ability to MH-S cells,the decrease of cell proliferation inhibition rate,and the decrease of apoptosis rate.(2)AC of XJ10 can infect host cells through the NF-κB signaling pathway,induce host cell apoptosis and inhibit the secretion of inflammatory factors.(3)Bcl2a1 and Il1 b are two significantly up-regulated data from transcriptome and proteomic association screening,which are involved in NF-κB signaling pathway and apoptosis.