幽门螺杆菌双组分系统耐酸相关蛋白ArsS的原核表达及纯化

目的原核表达并纯化幽门螺杆菌双组分系统耐酸相关蛋白ArsS,为深入研究其功能及其在幽门螺杆菌耐酸机制中的作用奠定基础。方法以幽门螺杆菌菌株26695基因组为模板,采用PCR法扩增ArsS基因,插入pET-22b(+)载体,构建重组原核表达质粒pET-22b(+)-ArsS,转化E.coliBL21(DE3),0.5mmol/LIPTG25℃诱导表达,并采用亲和层析与分子筛层析对重组蛋白进行纯化。结果重组原核表达质粒pET-22b(+)-ArsS经双酶切及测序鉴定,证明构建正确;重组蛋白以可溶性形式表达,表达量占菌体总蛋白的30%以上;分子筛层析图谱显示,在150mmol/LNaCl条件下,目的蛋白层析效果较好,纯化后的重组蛋白纯度可达95%以上,浓度约为10mg/ml。结论已成功原核表达并纯化获得了高纯度的ArsS蛋白。     

Helicobacter pylori Outer Membrane Vesicles and Extracellular Vesicles from Helicobacter pylori-Infected Cells in Gastric Disease Development

Extracellular vesicles (EVs) are cell-derived vesicles important in intercellular communication that play an essential role in host-pathogen interactions, spreading pathogen-derived as well as host-derived molecules during infection. Pathogens can induce changes in the composition of EVs derived from the infected cells and use them to manipulate their microenvironment and, for instance, modulate innate and adaptive inflammatory immune responses, both in a stimulatory or suppressive manner. Gastric cancer is one of the leading causes of cancer-related deaths worldwide and infection with Helicobacter pylori (H. pylori) is considered the main risk factor for developing this disease, which is characterized by a strong inflammatory component. EVs released by host cells infected with H. pylori contribute significantly to inflammation, and in doing so promote the development of disease. Additionally, H. pylori liberates vesicles, called outer membrane vesicles (H. pylori-OMVs), which contribute to atrophia and cell transformation in the gastric epithelium. In this review, the participation of both EVs from cells infected with H. pylori and H. pylori-OMVs associated with the development of gastric cancer will be discussed. By deciphering which functions of these external vesicles during H. pylori infection benefit the host or the pathogen, novel treatment strategies may become available to prevent disease.     

HpaA-VacA IgY对小鼠胃内感染幽门螺杆菌的预防作用

目的 研究HpaA-VacA IgY对小鼠幽门螺杆菌(Helicobacter pylori,H.pylori)胃内定植及引起炎症的预防作用,为制备集预防和治疗H.pylori感染为一体的IgY制剂奠定基础。方法将BALB/c小鼠随机分为阳性对照组、阴性对照组、感染预防组和炎症预防组,阳性对照组灌喂含108 cfu/ml H.pylori的菌液;阴性对照组灌喂布氏肉汤代替H.pylori菌液;感染预防组灌喂不同剂量的IgY后,再灌喂同等剂量的菌液;炎症预防组灌喂同等剂量的菌液后,再灌喂不同剂量的IgY。通过胃黏膜H.pylori培养和组织切片染色镜检观察H.pylori定植及炎症反应程度。结果 HpaA-VacA IgY剂量为6 mg/只时,对H.pylori感染的预防率为88.9%,预防效果较好,当HpaA-VacA IgY的剂量为8 mg/只时,能完全预防H.pylori感染及其所引起的胃黏膜炎症。结论小鼠口服HpaA-VacA IgY具有预防和治疗H.pylori感染的作用,可用于制备防治H.pylori感染的口服制剂。     

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.     

硫糖铝对抗幽门螺杆菌重组VacA卵黄抗体的保护作用

目的观察硫糖铝对抗幽门螺杆菌重组细胞空泡毒素抗原(VacA)卵黄抗体(IgY)的保护作用,为制备能耐受胃酸和蛋白酶的IgY制剂奠定基础。方法诱导重组菌DH5α-vacA-pQE30,大量表达并纯化VacA重组蛋白,免疫洛曼母鸡,制备VacAIgY,并进行纯化。在不同pH值的IgY液中加入不同浓度的硫糖铝和不同浓度的胃蛋白酶,将含不同浓度硫糖铝的IgY反复冻融7次,将含30%硫糖铝的IgY室温放置1d～4周,ELISA法测定各实验组IgY的相对抗体活性(AT/AC)。结果在pH1.5、2.0和3.0的条件下,含30%～60%硫糖铝的IgY的相对抗体活性高于不加硫糖铝的IgY;在pH1.5条件下,60%硫糖铝可使IgY的相对抗体活性保持68.7%;在pH2.0和3.0条件下,50%和40%以上的硫糖铝几乎可完全保持IgY的相对抗体活性。在pH值为2.0和3.0时,正常胃蛋白酶浓度(0.02mg/ml)下,30%以上的硫糖铝均可有效保护IgY的相对抗体活性。30%以上的硫糖铝可增强IgY的抗冻融能力。室温放置4周后,含30%硫糖铝的IgY仍能保持80%以上的相对抗体活性。结论30%以上的硫糖铝可增强VacAIgY对低pH和胃蛋白酶的耐受能力及抗冻融能力,是较理想的IgY保护剂。     

Selective Inhibition of Helicobacter pylori Carbonic Anhydrases by Carvacrol and Thymol Could Impair Biofilm Production and the Release of Outer Membrane Vesicles

Helicobacter pylori, a Gram-negative neutrophilic pathogen, is the cause of chronic gastritis, peptic ulcers, and gastric cancer in humans. Current therapeutic regimens suffer from an emerging bacterial resistance rate and poor patience compliance. To improve the discovery of compounds targeting bacterial alternative enzymes or essential pathways such as carbonic anhydrases (CAs), we assessed the anti-H. pylori activity of thymol and carvacrol in terms of CA inhibition, isoform selectivity, growth impairment, biofilm production, and release of associated outer membrane vesicles-eDNA. The microbiological results were correlated by the evaluation in vitro of H. pylori CA inhibition, in silico analysis of the structural requirements to display such isoform selectivity, and the assessment of their limited toxicity against three probiotic species with respect to amoxicillin. Carvacrol and thymol could thus be considered as new lead compounds as alternative H. pylori CA inhibitors or to be used in association with current drugs for the management of H. pylori infection and limiting the spread of antibiotic resistance.     

A Comparative Study on Nickel Binding to Hpn-like Polypeptides from Two Helicobacter pylori Strains

Combined potentiometric titration and isothermal titration calorimetry (ITC) methods were used to study the interactions of nickel(II) ions with the N-terminal fragments and histidine-rich fragments of Hpn-like protein from two Helicobacter pylori strains (11637 and 26695). The ITC measurements were performed at various temperatures and buffers in order to extract proton-independent reaction enthalpies of nickel binding to each of the studied protein fragments. We bring up the problem of ITC results of nickel binding to the Hpn-like protein being not always compatible with those from potentiometry and MS regarding the stoichiometry and affinity. The roles of the ATCUN motif and multiple His and Gln residues in Ni(II) binding are discussed. The results provided the possibility to compare the Ni(II) binding properties between N-terminal and histidine-rich part of Hpn-like protein and between N-terminal parts of two Hpn-like strains, which differ mainly in the number of glutamine residues.     

源于水杨酸的噻唑啉化合物的合成及杀菌活性研究

以水杨酸为原料衍生合成了一系列相应的噻唑啉化合物,均经NMR、IR等光谱手段确证结构,并对3e进行了X-衍射测定单晶结构。所有目标化合物都进行了杀菌活性测试,其中在50μg/mL浓度下,3b对芦笋茎枯的抑制率达到92%。     

Helicobacter pylori Neutrophil-Activating Protein Directly Interacts with and Activates Toll-like Receptor 2 to Induce the Secretion of Interleukin-8 from Neutrophils and ATRA-Induced Differentiated HL-60 Cells

Helicobacter pylori neutrophil-activating protein (HP-NAP)-induced production of reactive oxygen species (ROS) by neutrophils and monocytes is regulated by pertussis toxin (PTX)-sensitive G proteins, whereas HP-NAP-induced cytokine secretion by monocytes is mediated by Toll-like receptor 2 (TLR2). However, it is unclear whether TLR2 participates in HP-NAP-induced cytokine secretion by neutrophils. Here, all-trans retinoic acid (ATRA)-induced differentiated HL-60 cells were first employed as a neutrophil model to investigate the molecular mechanisms underlying neutrophil responses to HP-NAP. HP-NAP-induced ROS production in ATRA-induced differentiated HL-60 cells is mediated by the PTX-sensitive heterotrimeric G protein-dependent activation of extracellular signal-regulated kinase 1/2 and p38-mitogen-activated protein kinase, which is consistent with the findings reported for human neutrophils. Next, whether TLR2 participated in HP-NAP-induced secretion of interleukin-8 (IL-8) was investigated in neutrophils and ATRA-induced differentiated HL-60 cells. In both cells, TLR2 participated in HP-NAP-induced IL-8 secretion but not HP-NAP-induced ROS production. Interestingly, PTX-sensitive G proteins also contributed to the HP-NAP-induced secretion of IL-8 from neutrophils and the differentiated HL-60 cells. Our ELISA-based binding assay further revealed the competitive binding of Pam₃CSK₄, a TLR2 agonist, and HP-NAP to TLR2, which suggests the presence of specific and direct interactions between HP-NAP and TLR2. Thus, HP-NAP directly interacts with and activates TLR2 to induce IL-8 secretion in neutrophils and ATRA-induced differentiated HL-60 cells.