Characterization of Haemophilus ducreyi cdtA, cdtB, and cdtC mutants in in vitro and in vivo systems
Characterization of Haemophilus ducreyi cdtA, cdtB, and cdtC mutants in in vitro and in vivo systems
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DOI：
10.1128/iai.69.9.5626-5634.2001
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发表时间：
2001-09-01
影响因子：
3.1
通讯作者：
Hansen, EJ
中科院分区：
文献类型：
--
作者：
Lewis, DA;Stevens, MK;Hansen, EJ
杜氏嗜血杆菌表达一种由cdtABC基因簇编码的可溶性细胞致死性膨胀毒素(CDT)，可在培养上清液中检测到该毒素对HeLa细胞的杀伤作用。将杜氏嗜血杆菌的cdtA、cdtB和cdtC基因分别克隆到载体上，并在大肠杆菌背景下以单一或多种组合的形式表达其编码蛋白。所有这三种基因产物都必须表达，才能使大肠杆菌产生的培养上清液证明对HeLa细胞具有细胞毒性。构建了等基因的H.ducreyi cdtA和CDtB突变体，并将其与野生型亲本菌株和先前描述的H.ducreyi cdtC突变体(M.K.Stevens，J.L.Latimer，S.R.Lumbley，C.K.Ward，L.D.Cope，T.Lagergard和E.J.Hansen，Infect)相结合使用。伊蒙。67：3900-3908,1999)以确定CDtA、CDtB和CDtC蛋白对CDT活性的相对贡献。表达CDtA、CDtB和CDtC似乎是杜氏嗜血杆菌培养上清液对HeLa细胞显示细胞毒性所必需的。CdtB和cdtC突变体的全细胞超声波和周质提取物对HeLa细胞没有影响，而cdtA突变体的这些相同部分对这些相同的人类细胞有非常轻微的细胞毒性作用。CDtA似乎主要与杜氏梭菌细胞膜有关，而CDtB和CDtC主要存在于超声处理细胞的可溶性部分中。在实验性下巴温度依赖兔模型中，cdtA突变体和cdtB突变体都被发现是完全毒力的。
Haemophilus ducreyi expresses a soluble cytolethal distending toxin (CDT) that is encoded by the cdtABC gene cluster and can be detected in culture supernatant fluid by its ability to kill HeLa cells. The cdtA, cdtB, and cdtC genes of H. ducreyi were cloned independently into plasmid vectors, and their encoded proteins expressed singly or in various combinations in an Escherichia coli background. All three gene products had to be expressed in order for E. coli-derived culture supernatant fluids to demonstrate cytotoxicity for HeLa cells. Isogenic H. ducreyi cdtA and cdtB mutants were constructed and used in combination with the wild-type parent strain and a previously described H. ducreyi cdtC mutant (M. K. Stevens, J. L. Latimer, S. R. Lumbley, C. K. Ward, L. D. Cope, T. Lagergard, and E. J. Hansen, Infect. Immun. 67:3900-3908, 1999) to determine the relative contributions of the CdtA, CdtB, and CdtC proteins to CDT activity. Expression of CdtA, CdtB, and CdtC appeared necessary for H. ducreyi-derived culture supernatant fluid to exhibit cytotoxicity for HeLa cells. Whole-cell sonicates and periplasmic extracts from the cdtB and cdtC mutants had no effect on HeLa cells, whereas these same fractions from a cdtA mutant had a very modest cytotoxic effect on these same human cells. CdtA appeared to be primarily associated with the H. ducreyi cell envelope, whereas both CdtB and CdtC were present primarily in the soluble fraction from sonicated cells. Both the cdtA mutant and the cdtB mutant were found to be fully virulent in the temperature-dependent rabbit model for experimental chancroid.