Melton-Celsa A R, Darnell S C, O'Brien A D
Department of Microbiology and Immunology, F. Edward Hébert School of Medicine, Uniformed Services, University of the Health Sciences, Bethesda, Maryland 20814-4799, USA.
Infect Immun. 1996 May;64(5):1569-76. doi: 10.1128/iai.64.5.1569-1576.1996.
The enterohemorrhagic Escherichia coli (EHEC) O91:H21 isolates B2F1 and H414-36/89 are virulent in an orally infected streptomycin-treated mouse model. Previous studies demonstrated that B2F1 and H414-36/89 grow to high levels in mucus isolated from mouse small intestine and colon and that growth in small-intestine mucus is related to virulence. We measured the levels of Shiga-like toxins (SLTs) SLT-IIvha and SLT-IIvhb produced by B2F1 after growth in Luria-Bertani (LB) broth supplemented with mouse intestinal mucus by assaying the cytotoxicity of culture supernatants on Vero cells. Culture supernatants from B2F1 grown in mouse intestinal mucus, but not EHEC strains that produce SLT-II or SLT-IIc, were approximately 35- to 350-fold more toxic for Vero cells than supernatants from B2F1 grown in LB broth. This increased toxicity was not reflected by a concomitant increase in SLT antigen content. Furthermore, when culture supernatants from B2F1 or K-12 strains carrying plasmids encoding SLTs cloned from H414-36/89 or purified SLT-IIvhb from B2F1 were incubated with mouse intestinal mucus, the samples exhibited greater cytotoxicity than when they were incubated with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer alone. These toxin preparations also showed increased cytotoxicity after incubation with human colonic mucus. In contrast, culture supernatants from LB-grown EHEC isolates that produced SLT-I, SLT-II, SLT-IIc or SLT-IIe did not show increased cytotoxicity after incubation with mouse or human intestinal mucus. The A subunits of purified SLT-II and SLT-IIvhb that had been treated with mouse intestinal mucus or trypsin were cleaved to A1 fragments by the mucus, but trypsin-mediated cleavage, unlike treatment with mouse intestinal mucus, did not result in increased Vero cell cytotoxicity activity. This finding implies that the increased cytotoxicity of SLT-IIvhb detected after incubation with mucus is probably not due to cleavage of the A subunit into the A1 and A2 fragments. Taken together, these results indicate that mouse or human intestinal mucus directly activates SLT-II-related toxins from B2F1 and H414-36/89 and suggest that toxin activation may explain the low 50% lethal doses of B2F1 and H414-36/89 in streptomycin-treated mice.
肠出血性大肠杆菌（EHEC）O91:H21分离株B2F1和H414-36/89在经链霉素处理的口服感染小鼠模型中具有毒性。先前的研究表明，B2F1和H414-36/89在从小鼠小肠和结肠分离的黏液中能大量生长，且在小肠黏液中的生长与毒性相关。我们通过检测培养上清液对Vero细胞的细胞毒性，来测定B2F1在补充了小鼠肠道黏液的Luria-Bertani（LB）肉汤中生长后产生的志贺样毒素（SLTs）SLT-IIvha和SLT-IIvhb的水平。在小鼠肠道黏液中生长的B2F1的培养上清液，而非产生SLT-II或SLT-IIc的EHEC菌株的培养上清液，对Vero细胞的毒性比在LB肉汤中生长的B2F1的培养上清液高约35至350倍。这种毒性增加并未伴随SLT抗原含量的相应增加。此外，当携带从H414-36/89克隆的编码SLTs的质粒的B2F1或K-12菌株的培养上清液，或从B2F1纯化的SLT-IIvhb与小鼠肠道黏液一起孵育时，这些样品表现出比仅与N-2-羟乙基哌嗪-N'-2-乙磺酸（HEPES）缓冲液孵育时更大的细胞毒性。这些毒素制剂与人结肠黏液孵育后也显示出细胞毒性增加。相比之下，在LB中生长的产生SLT-I、SLT-II、SLT-IIc或SLT-IIe的EHEC分离株的培养上清液与小鼠或人肠道黏液孵育后未显示出细胞毒性增加。用小鼠肠道黏液或胰蛋白酶处理过的纯化SLT-II和SLT-IIvhb的A亚基被黏液切割成A1片段，但与用小鼠肠道黏液处理不同，胰蛋白酶介导的切割并未导致Vero细胞细胞毒性活性增加。这一发现表明，与黏液孵育后检测到的SLT-IIvhb细胞毒性增加可能不是由于A亚基切割成A1和A2片段所致。综上所述，这些结果表明小鼠或人肠道黏液直接激活了来自B2F1和H414-36/89的与SLT-II相关的毒素，并表明毒素激活可能解释了B2F1和H414-36/89在链霉素处理的小鼠中较低的50%致死剂量。
