肠毒素大肠杆菌CS3定居因子抗原和融合肠毒素基因在减毒痢疾杆菌中的共表达

肠毒素和定居因子抗原 (CFAs)是肠毒素大肠杆菌 (ETEC)两种主要的致病因素。有效的ETEC疫苗应包括这两种成分。采用基因重组技术 ,将定居因子CFA/II的共有抗原成分CS3菌毛抗原和LT B/ST融合肠毒素基因克隆至以asd基因为选择标记的重组质粒上 ,与asd基因剔除的弗氏志贺氏菌Fwl0 1构成了宿主 载体平衡致死系统。结果表明 ,在无抗生素条件选择的情况下 ,该重组菌可稳定表达CS3菌毛抗原和LT B/ST融合肠毒素抗原。通过口服和鼻饲方式免疫小鼠 ,可诱生相应的血清IgG抗体 ,同时能够检测到分泌型IgA的产生 ,表明该重组菌可以有效的诱导产生粘膜免疫。     

肠毒素C2蛋白C,N末端对其活性的影响

目的:分析金黄色葡萄球菌肠毒素C2(SEC2)中N,C末端对其超抗原活性和可溶性表达能力的影响。方法:应用基因工程技术对SEC2的N,C末端进行部分删除,获得三种突变蛋白,并对其进行体外超抗原活性和可溶性表达能力的比较。结果:对SEC2的N,C末端的删除都在一定程度上影响其超抗原活性和可溶性表达能力,其中,N末端的两个删除突变体的超抗原活性分别降低40%和48%,而删除C末端则使其可溶性表达水平下降到野生型的20%左右。结论:SEC2蛋白分子的N末端对其超抗原活性起主要作用,C末端对其可溶性表达具有显著影响,而完整的SEC2分子对于其发挥最大生物学活性是必要的。     

A monoclonal antibody against staphylococcal enterotoxin B superantigen inhibits SARS-CoV-2 entry in vitro

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Mechanism of action of a cytotonic enterotoxin produced by Aeromonas hydrophila

In this study, we describe the mechanism of action of a cytotonic enterotoxin produced by two isolates of Aeromonas hydrophila. Isolates SSU and Ah65 are of different origin and both are capable of producing either a cytotoxic enterotoxin or aerolysin. A cytotonic enterotoxin produced by diarrheal isolate SSU, which was purified and characterized in our laboratory, elevated intracellular cAMP and PgE2 levels in cultured Chinese hamster ovary (CHO) cells. Likewise, enterotoxic activity expressed by a cytotonic enterotoxin was detected in the culture filtrate of a fish isolate (Ah65) after cytotoxic activity was neutralized with homologous aerolysin monoclonal antibodies. This cytotonic enterotoxin also elevated intracellular cAMP and PgE2 levels in CHO cells, suggesting a cholera toxin-like mechanism of action for Aeromonas cytotonic enterotoxins.     

Isolation of a heat-labile enterotoxin produced by a human strain of Escherichia coli by wheat-germ agglutinin affinity chromatography

Abstract A chromatographic method, wheat-germ agglutinin affinity chromatography, was developed to isolate Escherichia coli heat-labile enterotoxin from human source. Isolated LT enterotoxin showed potent activity in the rabbit jejunal loop assay, and immunological and structural analogies with cholera enterotoxin in the radial immuno-hemolysis test and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively.     

AlphaLISA检测金黄色葡萄球菌肠毒素E方法的建立

目的:利用AlphaLISA技术,建立新型均相检测方法,对金黄色葡萄球菌肠毒素E(SEE)进行检测。方法:采用2种SEE抗体建立AlphaLISA检测方法,羊抗肠毒多克隆抗体同受体微球偶联,SEE小鼠单克隆抗体标记生物素,并同偶联了亲和素的供体微球连接,3种组分与待测抗原形成双抗体夹心结构,680nm的光激发供体微球产生能量,将周围反应体系中的氧分子转变为激发态,并将能量传递给受体微球,产生520~620nm的荧光,荧光信号与抗原浓度正相关。对该方法的反应体系进行优化,验证其重复性、敏感性和特异性,并采用该方法检测菌液上清和食品模拟样本。结果:所建立的AlphaLISA检测方法重复性较好,批间变异系数和批内变异系数皆小于10%;检测SEE的灵敏度可以达到100pg/mL,并与其他几种毒素均无交叉反应,具有较好的特异性。该方法也能够准确地对金黄色葡萄球菌菌液上清中的SEE进行检测,对食品模拟样本的检测也有较好的灵敏度。结论:建立了检测SEE的AlphaLISA方法。该方法均相免洗,操作便捷,用时短,灵敏度更高,可对菌液上清和不同食品模拟样本中的SEE进行检测。     

The B subunit of Escherichia coli heat‐labile toxin alters the development and antigen‐presenting capacity of dendritic cells

Escherichia coli's heat‐labile enterotoxin (Etx) and its non‐toxic B subunit (EtxB) have been characterized as adjuvants capable of enhancing T cell responses to co‐administered antigen. Here, we investigate the direct effect of intravenously administered EtxB on the size of the dendritic and myeloid cell populations in spleen. EtxB treatment appears to enhance the development and turnover of dendritic and myeloid cells from precursors within the spleen. EtxB treatment also gives a dendritic cell (DC) population with higher viability and lower activation status based on the reduced expression of MHC‐II, CD80 and CD86. In this respect, the in vivo effect of EtxB differs from that of the highly inflammatory mediator lipopolysaccharide. In in vitro bone marrow cultures, EtxB treatment was also found to enhance the development of DC from precursors dependent on Flt3L. In terms of the in vivo effect of EtxB on CD4 and CD8 T cell responses in mice, the interaction of EtxB directly with DC was demonstrated following conditional depletion of CD11c⁺ DC. In summary, all results are consistent with EtxB displaying adjuvant ability by enhancing the turnover of DC in spleen, leading to newly mature myeloid and DC in spleen, thereby increasing DC capacity to perform as antigen‐presenting cells on encounter with T cells.     

Determination of the toxic potential of Bacillus cereus isolates by quantitative enterotoxin analyses

Haemolysin BL (HBL) and non-haemolytic enterotoxin (Nhe), each consisting of three components, represent the major enterotoxins produced by Bacillus cereus. To evaluate the expression of these toxins, a set of 100 B. cereus strains was examined. Molecular biological characterization showed that 42% of the strains harboured the genes for HBL and 99% for Nhe. The production of all Nhe and HBL components were analyzed using specific antibodies and, in culture supernatants, detectable levels of HBL and Nhe were found for 100% of hbl-positive and 96% of nhe-positive strains. The concentrations of the HBL-L(2) and NheB component ranged from 0.02 to 5.6 microg mL(-1) and from 0.03 to 14.2 microg mL(-1), respectively. Comparison of the amount of NheB produced by food poisoning and food/environmental strains revealed that the median value for all food poisoning strains was significantly higher than for the food/environmental isolates. The data presented in this study provide evidence that specific and quantitative determination of the enterotoxins is necessary to evaluate the toxic potential of B. cereus. In particular, the level of Nhe seems to explain most of the cytotoxic activity of B. cereus isolates and may indicate a highly diarrheic potential.     

Role of <Emphasis Type="Italic">Yersinia enterocolitica</Emphasis> heat-stable enterotoxin (Y-STa) on differential regulation of nuclear and cytosolic calcium signaling in rat intestinal epithelial cells

The heat-stable enterotoxin (Y-STa) produced by the pathogenic strains of Yersinia enterocolitica is a causative agent of secretory diarrhea. We have reported earlier that Y-STa-induced inositol trisphosphate-mediated cytosolic calcium rise occurs in rat intestinal epithelial cells. In the present communication, the involvement of a nuclear calcium store in the action mechanism of Y-STa in rat intestinal epithelial cells has been shown. Calcium imaging with time series confocal microscopy shows that Y-STa stimulates both the nuclear and cytosolic calcium levels in rat intestinal epithelial cells where a rise in nuclear calcium precedes the cytosolic events. Moreover, Y-STa stimulates both cytosolic and nuclear inositol trisphosphate (IP₃) levels in a time-dependent manner. Western blot and immunocytochemical analysis reveal a higher density of IP₃ receptor type II in the nuclear membrane compared to the cytosol, which may be the cause of an early rise of the nuclear calcium level. Therefore, it is suggested that Y-STa regulates the nuclear and cytosolic calcium signals in a distinct temporal manner in rat intestinal epithelial cells.