Department of Veterinary Sciences, Ludwig-Maximilians-Universität München, Oberschleissheim, Germany.
PLoS One. 2013 Apr 30;8(4):e63104. doi: 10.1371/journal.pone.0063104. Print 2013.
The nonhemolytic enterotoxin (Nhe) is known as a major pathogenicity factor for the diarrheal type of food poisoning caused by Bacillus cereus. The Nhe complex consists of NheA, NheB and NheC, all of them required to reach maximum cytotoxicity following a specific binding order on cell membranes. Here we show that complexes, formed between NheB and NheC under natural conditions before targeting the host cells, are essential for toxicity in Vero cells. To enable detection of NheC and its interaction with NheB, monoclonal antibodies against NheC were established and characterized. The antibodies allowed detection of recombinant NheC in a sandwich immunoassay at levels below 10 ng ml⁻¹, but no or only minor amounts of NheC were detectable in natural culture supernatants of B. cereus strains. When NheB- and NheC-specific monoclonal antibodies were combined in a sandwich immunoassay, complexes between NheB and NheC could be demonstrated. The level of these complexes was directly correlated with the relative concentrations of NheB and NheC. Toxicity, however, showed a bell-shaped dose-response curve with a plateau at ratios of NheB and NheC between 50:1 and 5:1. Both lower and higher ratios between NheB and NheC strongly reduced cytotoxicity. When the ratio approached an equimolar ratio, complex formation reached its maximum resulting in decreased binding of NheB to Vero cells. These data indicate that a defined level of NheB-NheC complexes as well as a sufficient amount of free NheB is necessary for efficient cell binding and toxicity. Altogether, the results of this study provide evidence that the interaction of NheB and NheC is a balanced process, necessary to induce, but also able to limit the toxic action of Nhe.
非溶血肠毒素（Nhe）是导致蜡样芽孢杆菌引起的腹泻型食物中毒的主要致病性因素之一。Nhe 复合物由 NheA、NheB 和 NheC 组成，它们都需要按照特定的结合顺序在细胞膜上结合才能达到最大的细胞毒性。在这里，我们表明，在靶向宿主细胞之前，NheB 和 NheC 之间在自然条件下形成的复合物对于 Vero 细胞的毒性是必不可少的。为了能够检测 NheC 及其与 NheB 的相互作用，建立并表征了针对 NheC 的单克隆抗体。这些抗体允许在夹心免疫测定中检测到低于 10ngml⁻¹的重组 NheC，但在蜡样芽孢杆菌菌株的天然培养上清液中不能或只能检测到少量的 NheC。当 NheB 和 NheC 特异性单克隆抗体在夹心免疫测定中组合使用时，可以证明 NheB 和 NheC 之间的复合物。这些复合物的水平与 NheB 和 NheC 的相对浓度直接相关。然而，毒性表现出钟形剂量反应曲线，在 NheB 和 NheC 的相对浓度为 50:1 和 5:1 之间达到平台。NheB 和 NheC 之间的比例较低和较高都会强烈降低细胞毒性。当比例接近等摩尔比时，复合物形成达到最大值，导致 NheB 与 Vero 细胞的结合减少。这些数据表明，一定水平的 NheB-NheC 复合物以及足够数量的游离 NheB 对于有效的细胞结合和毒性是必要的。总之，这项研究的结果提供了证据，表明 NheB 和 NheC 的相互作用是一个平衡的过程，对于诱导但也能够限制 Nhe 的毒性作用是必要的。
