Study On The Effect Of ProWX On The Expression Of Virulence Related Genes Of Helicobacter Pylori

Objective:Helicobacter pylori(Hp)is currently the only bacterium proven to survive long-term in the human stomach.It is the main cause of chronic gastritis,peptic gastric ulcer and other diseases.It is also a high risk factor for gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue(MALT)lymphoma.In complex microenvironment,Hp can rely on its unique physiological characteristics to maintain its survival,virulence and pathogenicity,which is closely related to the induction of microenvironment or the absorption and transport of nutrients from microenvironment.Periplasmic binding protein(PBP)is the key link of ATP binding cassettes systems or ABC transporter substrate entering the cell,which is responsible for the specific binding of transport substrate.This study focuses on the effect of HP0818 on the expression of virulence-related genes in Hp.Bioinformatics analysis suggests that the protein encoded by hp0818(proWX)may have osmotic protection.When the osmotic pressure of the external environment increases,in order to combat the loss of intracellular water,bacteria will accumulate compatible solutes(such as sugars,alcohols,amino acids and their derivatives,L-carnitine,etc.)to adapt to the environment with high osmotic pressure.Therefore,it is speculated that ProWX may play a very important role in the survival and virulence gene expression of Hp under specific conditions.This study will focus on the effect of ProWX on the expression of virulence-related genes in Hp,in order to provide new insights into the physiological characteristics and pathogenic mechanism of Hp and provide new ideas for drug target search.Methods:1.Construction of Hp26695 proWX gene knockout strain Hp26695ΔproWX.According to the whole NCBI Hp26695 genome sequence,primers were designed to amplify the upstream and downstream fragments of proWX gene and kanamycin resistance gene(kana ~R).Three fragments were cloned into p BluescriptⅡSK(-)linearized vector fragment by ligation-independent cloning,(LIC)method,and the recombinant knockout plasmid p BluescriptⅡSK(-)ΔproWX was constructed.The recombinant plasmid was transformed into Hp26695 by electric shock,and the positive clones were screened by blood plate containing kanamycin.Several positive clones were randomly selected and cultured,and the genomes were extracted for PCR verification.2.Bioinformatics analysis of proWX gene.Type HP0818 through the NCBI sub-search box Gene and compare and search the ProWX functional domains through the protein conserved domain search tool(NCBI conserved domain search).The phylogenetic tree was generated by NCBI Smart BLAST homology search and COBALT.3.ProWX gene is involved in Hp tolerance to salt stress.Hp26695 and Hp26695ΔproWX were densely crossed on common blood plates and cultured in micro-aerobic environment for 2 to 3 days.The growth rate and colony morphology of bacteria were observed.Then set up 0.5%(standard Brucella medium),1%,1.5%,2%salt concentration of Brucella medium,shake bacteria culture,and further observe the growth status of bacteria.qRT-PCR was used to detect the transcriptional expression under the stimulation of Hp26695 ProWX salt.4.Effects of proWX on Hp-related virulence,inflammation and adhesion.The expression of Cag A was detected by transcriptome study and qRT-PCR to further verify the expression of related genes.,Western blot was used to detect the expression of Cag A.After AGS cells were infected with Hp26695 and Hp26695ΔproWX,the expressions of IL-1β,IL-6,IL-8 and TNF-αin AGS cells were detected by qRT-PCR,and the expression of IL-8 was detected by ELISA.AGS cells were infected with Hp26695 and Hp26695ΔproWX respectively.After the planktonic Hp was washed out,the total RNA,of bacterial cells was extracted to detect the expression of Hp 16s r RNA and GAPDH in AGS cells in total RNA to compare the adhesion of Hp26695 and Hp26695ΔproWX to AGS cells.Results:1.Helicobacter pylori Hp26695 proWX knockout strain Hp26695ΔproWX was successfully constructed.2.Bioinformatics analysis suggests that hp0818 gene may encode osmotic protective agent ABC transporter/substrate binding protein ProWX,which accumulates osmotic protective agents(such as sugars,alcohols,amino acids and their derivatives,L-carnitine,etc.)in hypertonic environment to combat water loss.3.Hp26695ΔproWX grew well on the common Colombian blood plate,and the growth rate and colony morphology did not change significantly,indicating that proWX is not a necessary gene for Hp growth in the medium with normal salt concentration.When setting up the Brucella medium with 0.5%(standard Brucella medium),1%,1.5%and 2%salt concentration,it was observed that compared with the Hp26695 group,the growth of Hp26695ΔproWX group was significantly inhibited at 1.5%and 2%salt concentration.At the same time,qRT-PCR detection showed that the expression of ProWX increased with the increase of salt concentration,suggesting that ProWX may be involved in the tolerance of Hp to increased salt concentration.4.Transcriptomics analysis showed that,compared with Hp26695,in Hp26695ΔproWX,138 genes were significantly up-regulated and 113 genes were significantly down-regulated.Among the up-regulated genes including T4SS and adhesion-related genes,the down-regulation of Alp A,Alp B,Hop Q,and Lab A was obvious.qRT-PCR confirmed that the expression of Cag A decreased,and the expression of T4SS-related genes was up-regulated.ProWX could up-regulate the expression of Hp Cag A,inhibit the expression of T4SS-related genes,and thus inhibit the expression of most flagella-related genes of Hp.Most genes related to flagella synthesis are up-regulated;Western blot data show that the expression of Cag A decreases significantly at the protein level.5.Hp26695 and Hp26695ΔproWX respectively infect AGS cells,and the content of Hp26695,Hp26695ΔproWX infected cells IL-1β,IL-6,IL-8,TNF-αexpression were presented to varying degrees down;ELISA method to detect IL-8 expression and qRT-PCR results are basically the same.Compared with Hp26695,Hp26695ΔproWX significantly decreased AGS cells.The specific mechanism needs further study.Conclusion:1.ProWX participates in Hp tolerance to salt stress.2.ProWX promotes the expression of Hp Cag A and inhibits the expression of T4SS-related genes and most flagella-related genes.3.ProWX is related to the adhesion and inflammation of Hp.Knockout of ProWX can significantly reduce the adhesion level of Hp to AGS cells and the expression of related inflammatory cytokines.