Preliminary Analysis Of Host Chromatin Openness During Pasteurella Infection And The Expression Of ThiB Protein

Pasteurella multocida（Pm,Pasteurella multocida）is pathogenic to humans and a variety of animals.Infecting goats can cause acute or chronic pneumonia,with a low fatality rate but a high positive infection rate.During the interaction between Pm and the host,the transcriptome and proteome of the host cell change.At present,the research on these transcriptional regulation mechanisms is relatively blank,because the open chromatin of eukaryotes can allow trans-acting factors binding and participate in the transcriptional regulation of eukaryotes.Therefore,this study used ATAC sequencing technology to detect the chromatin open region of the whole genome of goat bronchial epithelial cells during Pm infection,and explore the transcriptome regulation mechanism of the disease process;The ability of pathogenic bacteria to obtain nutrients in suitable host niches is also an important component of virulence.Thiamine（VB₁）participates in the essential metabolic processes of the living body,and as a coenzyme,it participates in glycolysis,the citric acid cycle,and the pentose phosphate pathway and other metabolic processes.Previous studies in our laboratory found that as the time of Pm infection prolonged,the transcription levels of the metabolic pathway-related genes involved in VB₁ were significantly down-regulated.thiB is the Pm thiamine transporter substrate binding protein,so thiB protein expression and analysis was performed.The specific research is as follows:Experiment 1:Prepare F3 generation goat bronchial epithelial cell infection solution with HN01 bacterial solution,MOT:100,infect the cells for 4 hours,digest and freeze the cells then send the cell samples to the company for ATAC-seq（Assay for Transposase-Accessible Chromatin with high throughput sequencing）to obtain all open DNA sequences in the genome under HN01 infection for 4 hours.Results:（1）The growth curve of HN01 was drawn,and the relationship equation between colony number and OD600nm was established.（2）28,722 peaks were detected in the infected group and 13,079 peaks in the control group.GO and Pathway analysis of the unique peaks-related genes of the infected group showed that GO biological processes are significantly enriched in protein modification,cell metabolism,cell component organization,and phosphorus metabolism;metabolic pathways which in Fc gamma R-mediated phagocytosis,Endocytosis,Bacterial invasion of epithelial cells,Adhesion junctions,MAPK signaling pathway,Focal adhesion,Leukocyte transendothelial migration,etc.are significantly enriched.（3）The unique peak of infected group predicted 49 transcription factor binding sites,most of which belonged to the Fosl and JUN transcription factor families.（4）Compared with the control group,the infected group had 863 up-regulated peaks and 11 down-regulated peaks;differential peak-related genes were enriched in the MAPK signaling pathway and tryptophan metabolism pathway;and 56 transcription factors have been predicted,these transcription factors were basically From the AP-1 family.（5）Joint analysis of ATAC-Seq data and mRNA-seq data completed in this laboratory.Converting the peak-related reads count of ATAC-Seq data into the gene open value.Results:1)Gene openness affects gene expression,the greater the openness,the higher mean expression level.2)There are 785 genes that are potentially up-regulated by open up-regulation of proximal peaks and 11 genes are potentially up-regulated by open up-regulation of distal peaks,and the mean expression level of the latter is higher than that former（3）There are 49 genes in the overlap between the difference peak and the difference gene.Experiment 2,using HN01（D-type Pm derived from goat）as the ThiB gene amplification template,introducing the expression plasmid pET2 8a to construct the recombinant plasmid pET28a-ThiB to express the tbiB protein.Transform pET28a-ThiB into Escherichia coli and add inducer（IPTG）to induce protein expression.The collected protein samples were subjected to SDS-PAGE and Western Blot to detect the expression of thiB protein.Use online tools to analyze the biological information of thiB protein.Results:A band with a size of 1005 bp was amplified.SDS-PAGE identified a protein with a size of about 42 ku.Western Blot test detected a specific band of about 42 ku.The thiB protein is a secreted protein distributed in the cytoplasmic membrane and mainly hydrophilic.thiB highly conserved within the Pm species and has a apparent genetic distance from other pathogens.