Activation of Shiga-like toxins by mouse and human intestinal mucus correlates with virulence of enterohemorrhagic Escherichia coli O91:H21 isolates in orally infected, streptomycin-treated mice
Activation of Shiga-like toxins by mouse and human intestinal mucus correlates with virulence of enterohemorrhagic Escherichia coli O91:H21 isolates in orally infected, streptomycin-treated mice
复制标题
DOI：
10.1128/iai.64.5.1569-1576.1996
复制
发表时间：
1996-05-01
影响因子：
3.1
通讯作者：
OBrien, AD
中科院分区：
文献类型：
--
作者：
MeltonCelsa, AR;Darnell, SC;OBrien, AD
肠出血性大肠杆菌（EHEC）091：H21分离株B2 F1和H414-36/89在经口感染链霉素处理的小鼠模型中具有毒性。先前的研究表明，B2 F1和H414-36/89在从小鼠小肠和结肠分离的粘液中生长到高水平，并且小肠粘液中的生长与毒力有关。我们通过测定培养上清液对Vero细胞的细胞毒性，测量了B2 F1在补充有小鼠肠粘液的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。在小鼠肠粘液中生长的B2 F1的培养物上清液（而不是产生SLT-II或SLT-IIc的EHEC菌株）对Vero细胞的毒性比在LB肉汤中生长的B2 F1的培养物上清液高约35- 350倍。这种增加的毒性并没有通过伴随的抗原含量的增加来反映。此外，当将携带编码从H414-36/89克隆的SLT或从B2 F1纯化的SLT-IIvhb的质粒的B2 F1或K-12菌株的培养物上清液与小鼠肠粘液孵育时，样品显示出比它们单独与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。这些毒素制剂在与人结肠粘液孵育后也显示出增加的细胞毒性。相比之下，LB生长的EHEC分离株的培养上清液产生的SLT-I，SLT-II，SLT-IIc，或SLT-IIe没有显示出增加的细胞毒性后，与小鼠或人的肠粘液孵育。用小鼠肠粘液或胰蛋白酶处理的纯化的SLT-II和SLT-IIvhb的A亚基被粘液切割成A（1）片段，但与用小鼠肠粘液处理不同，胰蛋白酶介导的切割不会导致Vero细胞细胞毒性活性增加。这一发现表明，与粘液孵育后检测到的SLT-IIvhb的细胞毒性增加可能不是由于A亚基裂解为A（1）和A（2）片段。总之，这些结果表明，小鼠或人的肠粘液直接激活B2 F1和H414-36/89的SLT-II相关毒素，并表明毒素激活可能解释了链霉素处理小鼠中B2 F1和H414-36/89的低50%致死剂量。
The enterohemorrhagic Escherichia coli (EHEC) 091: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 the mouse small intestine and colon and that growth in small-intestinal 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 A(1) fragments by the mucus, but trypsin-mediated cleavage, unlike treatment with mouse intestinal mucus, did not result in increased Vero cell cytotoxic 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 A(1) and A(2) 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.