幽门螺杆菌尿素酶的提取与纯化

采用2种方法分离纯化了幽门螺杆菌尿素酶。第1种方法是以 DEAE-sepharose FF离子交换剂和Superdex 200凝胶为分离介质,用 NaCl盐浓度梯度洗脱法从幽门螺杆菌超声上清液中提取纯化尿素酶抗原,所得到的纯化尿素酶经SDS-PAGE电泳分析,表明它具有很高的纯度,只含有分子质量为64 000 u和31 000 u两种蛋白成分,分别对应于幽门螺杆菌尿素酶的A、B两种亚基。第2种方法是以 DEAE-sepharose FF离子交换剂为分离介质,采用先去掉部分杂蛋白的方法纯化尿素酶,其中的去杂步骤操作简单、快捷且处理样品量大,大大减轻了后面纯化步骤的难度,适合于尿素酶的大量制备。纯化的尿素酶作为抗原采用ELISA间接法检测抗HP蛋黄免疫球蛋白(IgY),实验表明,其仍具有良好的抗原性,是HP主要抗原物质。应用纯化尿素酶抗原或纯化尿素酶抗原与超声粉碎抗原的混合抗原刺激蛋鸡应该能够产生抗HP效价更高的蛋黄免疫球蛋白,可以将其应用于保健或各种与HP相关的胃病的抗菌免疫治疗。     

Helicobacter pylori,Protected from Antibiotics and Stresses Inside Candida albicans Vacuoles,Cause Gastritis in Mice

Due to (i) the simultaneous presence of Helicobacter pylori (ulcer-induced bacteria) and Candida albicans in the stomach and (ii) the possibility of prokaryotic–eukaryotic endosymbiosis (intravacuolar H. pylori in the yeast cells) under stresses, we tested this symbiosis in vitro and in vivo. To that end, intravacuolar H. pylori were induced by the co-incubation of C. albicans with H. pylori under several stresses (acidic pH, non-H. pylori-enrichment media, and aerobic environments); the results were detectable by direct microscopy (wet mount) and real-time polymerase chain reaction (PCR). Indeed, intravacuolar H. pylori were predominant under all stresses, especially the lower pH level (pH 2–3). Interestingly, the H. pylori (an amoxicillin-sensitive strain) inside C. albicans were protected from the antibiotic (amoxicillin), while extracellular H. pylori were neutralizable, as indicated by the culture. In parallel, the oral administration of intravacuolar H. pylori in mice caused H. pylori colonization in the stomach resulting in gastritis, as indicated by gastric histopathology and tissue cytokines, similar to the administration of free H. pylori (extra-Candida bacteria). In conclusion, Candida protected H. pylori from stresses and antibiotics, and the intravacuolar H. pylori were able to be released from the yeast cells, causing gastric inflammation with neutrophil accumulations.     

Phototherapy and Mechanism Exploration of Biofilm and Multidrug-Resistant Helicobacter pylori by Bacteria-Targeted NIR Photosensitizer

Helicobacter pylori (H. pylori) infection has been the leading cause of gastric cancer development. In recent years, the resistance of H. pylori against antibiotic treatment has been a great challenge for most countries worldwide. Since biofilm formation is one of the reasons for the antibiotic resistance of H. pylori, and phototherapy has emerged as a promisingly alternative antibacterial treatment, herein the bacteria-targeted near-infrared (NIR) photosensitizer (T780T-Gu) by combining positively-charged guanidinium (Gu) with an efficient phototherapeutic agent T780T is developed. The proposed molecule T780T-Gu exhibits synergistic photothermal therapy/photodynamic therapy effect against both H. pylori biofilms and multidrug-resistant (MDR) clinical strains. More importantly, the phototherapy mechanism of T780T-Gu acquired by the RNA-seq analysis indicates that structural deficiency as well as a decrease in metabolism and defense activity are the possible reasons for the efficient H. pylori phototherapy.     

Synthesis and Biological Evaluation of Dithiobisacetamides as Novel Urease Inhibitors

Thirty-eight disulfides containing N-arylacetamide were designed and synthesized in an effort to develop novel urease inhibitors. Biological evaluation revealed that some of the synthetic compounds exhibited strong inhibitory potency against both cell-free urease and urease in intact cell with low cytotoxicity to mammalian cells even at concentration up to 250 μM. Of note, 2,2′-dithiobis(N-(2-fluorophenyl)acetamide) ( d7 ), 2,2′-dithiobis(N-(3,5-difluorophenyl)acetamide) ( d24 ), and 2,2′-dithiobis(N-(3-fluorophenyl)acetamide) ( d8 ) were here identified as the most active inhibitors with IC₅₀ of 0.074, 0.44, and 0.81 μM, showing 32- to 355-fold higher potency than the positive control acetohydroxamic acid. These disulfides were confirmed to bind urease without covalent modification of the cysteine residue and to inhibit urease reversibly with a mixed inhibition mechanism. They also showed very good anti-Helicobacter pylori activities with d8 showing a comparable potency to the clinical used drug amoxicillin. The impressive in vitro biological profile indicated their immense potential as therapeutic agents to tackle H. pylori caused infections.