Induction Of Inflammatory Response In Gastric Epithelial Cells By Janibacter Melonis From The Stomach And Its Machanism

Objective:The gastric microbiome,consisting of hundreds of bacterial genera normally,maintains homeostasis of gastric physiological processes.Dysbiosis of the gastric microbiome leads to mucosal inflammation and lesions of the stomach.Molecular analysis of gastric microbiota showed that Janibacter increased in gastric cancer.Its abundance is severalfold that of chronic gastritis,intestinal metaplasia,and intraepithelial neoplasia patients.In this study,to elucidate the pathogenic role of Janibacter in gastric mucosal lesions,Janibacter melonis PA32 was isolated from the stomach of a patient with gastric cancer,and its pathogenic properties were explored by in vitro.experiments and genomic analyses.Methods:PA32 strain was isolated from human gastric mucosa specimens by BHI medium.The strain was identified as J.melonis strain PA32,by typical colony appearances,Gram staining,BLAST alignments based on 16 S r RNA gene and phylogenetic tree construction.The acid resistance capacity of J.melonis was determined by acid resistance assay.Antimicrobial susceptibility testing was performed by disk diffusion method.The AGS cells co-cultured with J.melonis in vitro model were constructed.Adhesion ability was evaluated by microscopic examination and plate count method.The cytotoxicity was evaluated by LDH release assay.Transcriptomic analysis was conducted to find the upregulated expression genes of proinflammatory cytokines in AGS cells,and further verified by q RT-PCR and ELISA.The expression level of NF-κB in AGS cells infected with J.melonis was detected by q RT-PCR.Finally,through whole genome sequencing,the potential pathogenic genes such as adhesion genes and secretion systems were explored.Results:Acid survival assay showed that J.melonis survived at p H 2.0 after 2 h with a survival rate of 65%,suggesting it was highly resistant to acid.J.melonis was resistant to metronidazole,and susceptible to clarithromycin,tetracycline and levofloxacin.Attachment of the bacterium to the surface of AGS cells was observed with an adhesion index of 26.2 ± 3.1 cfu,suggestting its ability to adhere to gastric epithelial cells.Cytotoxicity assay showed that at 2 h of coincubation with J.melonis,the released LDH was more than 5 times that of AGS cells uninfected by PA32,suggesting its cytotoxicity.Transcriptome analyses showed that the expression levels of proinflammatory cytokines and chemokines IL-8,IL-6,CXCL-1 and CXCL-2 were significantly increased compared to uninfected AGS cells,and the fold change were 2.7,7.6,2.6 and 2.2,respectively.Increased m RNA levels of these genes in infected AGS cells were further validated using q RT-PCR,the fold changes were 3.3,2.7,2.3 and 2.5,respectively.Infection with J.melonis enhanced the expression of NF-κB in AGS cells.Genomic analyses revealed a number of virulence genes related to adhesion such as aid A and tuf.Conclusions:J.melonis,isolated from the stomach,may adhere to gastric epithelial cells through aid A,tuf and other adhesion related genes,and induce NF-κB-dependent inflammatory response in gastric epithelial cells,which was manifested in up-regulated expressions of IL-8,IL-6,CXCL-1 and CXCL-2.J.melonis may be involved in the occurrence and development of gastric mucosal inflammation.