| Helicobacter pylori(H.pylori)is a gram-negative bacterium colonized in human gastric mucosa and the main pathogen causing chronic gastritis,gastric cancer and peptic ulcer.Because of the close relationship with gastric cancer,H.pylori is categorized as a strong carcinogen(class I carcinogen)by the International Cancer Research Institution.The prevalence of H.pylori in Chinese population is about 50%,and most people are infected in childhood.H.pylori colonized in gastric mucosa evade the immune response through molecular simulation and exist in the body for a lifetime without treatment.Antibiotic therapy is the main method for the eradication of H.pylori.However,the eradication rate has been decreasing with the increase of antibiotic resistance in H.pylori.Although the cure rate was improved for a period of time by changing the combination of antibiotics,increasing the dosage and treatment course of antibiotics,the downward trend of eradication rate caused by antibiotic resistance could not be effectively curbed.In addition,the increased use of antibiotics may alter the normal intestinal flora and cause changed resistance of intestinal flora.Therefore,it is of great value to explore new methods for the eradication of H.pylori.It is found that the inhibition of the FimH adhesin could reduce the colonization of uropathogenic Escherichia coli in the bladder of mouse and improve the cure rate of conventional therapy for antibiotic-resistant bacteria,suggesting that inhibiting the bacterial attachment is a promising approach.Attachment of H.pylori to gastric mucosa is the premise of its pathogenesis.A variety of outer membrane proteins are known to be involved in the adhesion of H.pylori to gastric epithelial cells,including BabA,SabA,AlpA/AlpB,OipA.These adhesins and their receptors are important targets for the development of drugs against H.pylori.BabA is one of the most important adhesins of H.pylori,which can bind to fucosylated blood group antigens including Lewis b and the H antigen.Exploring the distribution of BabA receptors is helpful for a better understanding of the adhesion mechanism of H.pylori and provides support for the research and development of drugs to inhibit H.pylori attachment.In this study,tissue microarray was used to analyze the distribution of BabA receptors in gastric mucosa of patients with gastric cancer.The subtotal gastrectomy specimens of 80 patients with gastric cancer were collected,and the gastric cancer,paracancerous and normal gastric mucosa of each patient were collected to make tissue microarrays.The specimens of 60 patients whose gastric cancer,paracancerous and normal tissues could be read normally were involved in the result analysis.Immunohistochemical results showed that 98.3%(59/60)of the gastric mucosa of gastric cancer patients were BabA receptor positive,suggesting that adhesion inhibitors based on BabA may have a promising application for the treatment of H.pylori infection.The positive rate of BabA receptor in gastric cancer was 56.7%(34/60),which was significantly lower than that in paracancerous(98.3%)and normal tissues(95%).The strong positive rate(SPR)of BabA receptor in gastric cancer tissues was only 2.9%(1/34),while the SPR in paracancerous and normal tissues were 61.0%(36/59)and 66.7%(38/57),suggesting that BabA receptors were mainly distributed in paracancerous and normal gastric mucosa.At low magnification,the staining of the epithelium near the gastric cavity was light,and the closer to the fundus of the gastric mucosa,the stronger the staining was.At high magnification,the epithelium was mainly composed of surface mucous cells with less staining.There were more parietal cells in the middle part of gastric mucosa and the staining of these cells was light and evenly distributed.The chief cells were mainly at the fundus of the gastric gland and was stained dark,and the staining was significantly darker at the top of the chief cells near the lumen of gastric gland,suggesting that the chief cells might express more BabA receptors.The binding sites of BabA to receptors are in the crown motif,which is mainly composed of four reverse parallel β-sheets.The crown motif is relatively independent and stable,and the binding site is located on the ring structure on side of the β-sheets.The analysis of the structure and function of the crown motif of BabA is of significance for the development of H.pylori adhesion inhibitors.Amino acids and hydrogen bonds in the β-sheets of the crown motif were analyzed to get a smaller protein preserving the spatial structure associated with adhesion.Three proteins have been cloned and expressed in vitro:BabA(extracellular domain of BabA protein,which is the full-length protein without signal peptides),Crown(the crown motif of BabA)and C51(the truncated crown motif).Compared with the Crown,23 amino acids were reduced in the C51 protein,of which 11 amino acids were in the β-sheets and 12 amino acids in the ring structure on side of the β-sheets.The BLI technique(Bio-layer interferometry,BLI)was used to determine affinity dissociation constants(Kd)of three proteins(BabA,Crown,C51)with Lewis b.The results showed that the Kd values of Lewis b with BabA protein,Crown and C51 were 12.1 μM,213μM and 241μM,respectively,suggesting that all three proteins could bind to Lewis b.However,the binding of BabA protein with Lewis b was stronger than that of the Crown and C51 protein.Flow cytometry was used to detect the effects of the three proteins on the adhesion of H.pylori J99 strain to AGS cells,showing that BabA and Crown reduced the adhesion of J99 strain to AGS cells by 24.8%(P=0.009)and 11.1%(P=0.024),while C51 increased the adhesion by 28.0%(P=0.001).The effects of three proteins on the agglutination of J99 strain were detected and found that J99 strain were uniformly distributed in natural state and with the addition of BabA and Crown,but J99 strain obviously agglutinated into clusters with the addition of C51 protein.Bacterial agglutination effect may be the reason for the increased adhesion of J99 to AGS cells caused by the C51.Cryopreserved and subcultured H.pylori strains may have changes in adhesion and amoxicillin resistance.Transcriptome analysis of cryopreserved H.pylori strains showed that down-regulated genes were significantly enriched in integral component of membrane(GO:0016021)and plasma membrane(GO:0005886),indicating that the change of genes related to membrane may be the main reason of decreased amoxicillin resistance of cryopreserved H.pylori strains.The expression of some adhesins changed in cryopreserved strains,such as SabA,AlpB,but the expression of BabA did not change significantly,suggesting that the selection of H.pylori strains may be not affected by cryopreservation factors in the study of anti-adhesion of H.pylori targeting BabA.Tissue microarrays were used to determine the distribution of BabA receptors in gastric mucosa of patients with gastric cancer.The Crown(the crown motif of BabA)and C51(the truncated crown motif)was cloned and expressed in vitro for the first time.The binding abilities of three proteins with different molecular weights(BabA,Crown,C51)to the Lewis b were detected and effects of these three proteins on adhesion of H.pylori to AGS cells were evaluated.These results are of valuable for understanding the adhesion mechanism of H.pylori and developing H.pylori attachment inhibitors.The effect of cryopreservation on H.pylori was analyzed by transcriptome,and found that it significantly affected the amoxicillin-resistance and the expression of several adhesins.However,the expression of BabA was not affected by cryopreservation,which indicated that the H.pylori-cell interaction model is feasible in the anti-adhesion study based on BabA protein. |
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