Transporters HP0939, HP0497, and HP0471 participate in intrinsic multidrug resistance and biofilm formation in Helicobacter pylori by enhancing drug efflux.
转运蛋白 HP0939 、 HP0497 和 HP0471 通过增强药物外排参与幽门螺杆菌内在多药耐药和生物膜形成。
- 作者列表："Cai Y","Wang C","Chen Z","Xu Z","Li H","Li W","Sun Y
BACKGROUND:The multidrug resistance of Helicobacter pylori is becoming an increasingly serious issue. It is therefore necessary to study the mechanism of multidrug resistance of H pylori. We have previously identified that the HP0939, HP0497, and HP0471 transporters affect the efflux of drugs from H pylori. As efflux pumps participate in bacterial multidrug resistance and biofilm formation, we hypothesized that these transporters could be involved in the multidrug resistance and biofilm formation of H pylori. MATERIALS AND METHODS:We therefore constructed three knockout strains, Δhp0939, Δhp0497, and Δhp0471, and three high-expression strains, Hp0939he , Hp0497he , and Hp0471he , using the wild-type (WT) 26 695 strain of H pylori as the template. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of wild strains, knockout strains, and high-expression strains to amoxicillin, metronidazole, and other antibiotics were measured. The efflux capacity of high-expression strains and wild strains was compared by Hoechst 33 342 accumulation assay. RESULTS:Determination of the MIC and MBC of the antibiotics revealed that the knockout strains were more sensitive to antibiotics, while the high-expression strains were less sensitive to antibiotics, compared to the WT. The ability of the high-expression strains to efflux drugs was significantly higher than that of the WT. We also induced H pylori to form biofilms, and observed that the knockout strains could barely form biofilms and were more sensitive to several antibiotics, compared to the WT. The mRNA expression of hp0939, hp0497, and hp0471 in the clinically sensitive and multidrug-resistant strains was determined, and it was found that these genes were highly expressed in the multidrug-resistant strains that were isolated from the clinics. CONCLUSIONS:In this study, we found three transporters involved in intrinsic multidrug resistance of H pylori.
背景: 幽门螺杆菌的多药耐药问题日益严重。因此，有必要对幽门螺杆菌多药耐药机制进行研究。我们之前已经确定 HP0939 、 HP0497 和 HP0471 转运蛋白影响幽门螺杆菌药物的流出。由于外排泵参与细菌多药耐药和生物膜形成，我们假设这些转运蛋白可能参与 H.pylori 的多药耐药和生物膜形成。 材料和方法: 因此，我们使用野生型 (WT) 构建了三个基因敲除菌株 Δ hp0939 、 Δ hp0497 和 Δ hp0471，以及三个高表达菌株 Hp0939he 、 Hp0497he 和 Hp0471he 26 695 株 H.pylori 为模板。测定野生株、敲除株、高表达株对阿莫西林、甲硝唑等抗生素的最低抑菌浓度 (MIC) 和最低杀菌浓度 (MBC)。通过 Hoechst 33 342 积累试验比较高表达菌株和野生菌株的外排能力。 结果: 与 WT 相比，敲除菌株对抗生素更敏感，而高表达菌株对抗生素更不敏感。高表达菌株外排药物的能力明显高于 WT。我们还诱导 H.pylori 形成生物膜，观察到与 WT 相比，敲除菌株几乎不能形成生物膜，对几种抗生素更敏感。测定 hp0939 、 hp0497 和 hp0471 在临床敏感和多药耐药菌株中的 mRNA 表达,发现这些基因在临床分离的多药耐药菌株中高表达。 结论: 在本研究中，我们发现三种转运蛋白参与了幽门螺杆菌的内在多药耐药。
METHODS::Diffuse gastric cancer (DGC) is a lethal malignancy lacking effective systemic therapy. Among the most provocative recent results in DGC has been that the alter of the cellular cytoskeleton and intercellular adhesion. CD2-associated protein (CD2AP) is one of the critical proteins regulating cytoskeleton assembly and intercellular adhesion. However, no study has investigated the expression and biological significance of CD2AP in gastric cancer (GC) to date. Therefore, the aim of our study was to explore if the expression of CD2AP is associated with any clinical features of GC and to elucidate the underlying mechanism. Immunohistochemistry of 620 patient tissue samples indicated that the expression of CD2AP is downregulated in DGC. Moreover, a low CD2AP level was indicative of poor patient prognosis. In vitro, forced expression of CD2AP caused a significant decrease in the migration and invasion of GC cells, whereas depletion of CD2AP had the opposite effect. Immunofluorescence analysis indicated that CD2AP promoted cellular adhesion and influenced cell cytoskeleton assembly via interaction with the F-actin capping protein CAPZA1. Overall, the upregulation of CD2AP could attenuate GC metastasis, suggesting CD2AP as a novel biomarker for the prognosis and treatment of patients with GC.
METHODS::Aim: To identify the methylated-differentially expressed genes (MDEGs) that may serve as diagnostic markers and therapeutic targets in Epstein-Barr virus-associated gastric cancer (EBVaGC) and to explore the methylation-based pathways for elucidating biological mechanisms of EBVaGC. Materials & methods: Gene expression and methylation profiles were downloaded from GEO database. MDEGs were identified by GEO2R. Pathway enrichment analyses were conducted based on DAVID database. Hub genes were identified by Cytoscape, which were further verified by The Cancer Genome Atlas database. Results: A total of 367 hypermethylated, lowly expressed genes were enriched in specific patterns of cell differentiation. 31 hypomethylated, highly expressed genes demonstrated enrichment in regulation of immune system process. After validation using The Cancer Genome Atlas database, seven genes were confirmed to be significantly different hub genes in EBVaGC. Conclusion: EBVaGC-specific MDEGs and pathways can be served as potential biomarkers for precise diagnosis and treatment of EBVaGC and provide novel insights into the mechanisms involved.
METHODS:Gastric adenocarcinoma, like other cancers, is a multifactorial genetic disease, andmetastasis of cancer cells is one of the main features of this illness. The expressionlevels of the CFL1 gene have been modulated in this pathway. Using small interferingRNA (siRNA) in the treatment of gastric cancer is considered a hopeful genetherapeutic approach. The present study reported the level of CFL1 genes betweentumor and margin and healthy tissue of gastric cancer. Also, the features of a cationicnanoparticle with a polymer coating containing polyacrylic acid and polyethylenei-mine that were used in the delivery of CFL1 siRNA, were shown. Then thecytotoxicity, cellular uptake, and gene silencing efficiency of this nanoparticle wereevaluated with CFL1siRNA. Method:In this study, the CFL1 gene expression was measured in 40 gastricadenocarcinoma, marginal and 15 healthy biopsy samples by a real‐time polymerasechain reaction. Physicochemical characteristics, apoptosis, and inhibition of migrationof the delivery of CFL1 siRNA by nanoparticle and lipofectamine were investigated ingastric cancer cells. Result:The CFL1 expression was remarkably increased in gastric cancer tissues incomparison with the marginal samples and normal tissues (p< .05) and the biomarkerindex for CFL1 was obtained as 0.94, then this gene can be probably used as abiomarker for gastric cancer. After treatment of the AGS cell line by CFL1 siRNA, theCFL1 expression level of mRNA and migration in AGS cells were remarkablysuppressed after transfection. Furthermore, the amount of apoptosis increased(p< .05). Conclusion:Our results demonstrated that CFL1 downregulation in AGS cells caninterdict cell migration. Finally, our outcomes propose that CFL1 can function as anoncogenic gene in gastric cancer and would be considered as a potential purpose ofgene therapy for gastric cancer treatment