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首页> 外文期刊>Infection and immunity >Characterization of Haemophilus ducreyi cdtA, cdtB, and cdtC Mutants in In Vitro and In Vivo Systems
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Characterization of Haemophilus ducreyi cdtA, cdtB, and cdtC Mutants in In Vitro and In Vivo Systems

机译：体外和体内系统中杜克嗜血杆菌cdtA，cdtB和cdtC突变体的表征

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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 cdtCgenes 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 describedH. 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 cdtAmutant and the cdtB mutant were found to be fully virulent in the temperature-dependent rabbit model for experimental chancroid.
机译：嗜血杆菌表达一种可溶性细胞致死性扩张毒素（CDT），该毒素由 cdtABC 基因簇编码，并且可以通过杀死HeLa细胞的能力在培养上清液中检测到。 H的 cdtA，cdtB 和 cdtC 基因。将ducreyi 独立克隆到质粒载体中，并在大肠杆菌背景中单独或以多种组合表达其编码的蛋白质。为了表达 E，必须表达所有三个基因产物。大肠杆菌来源的培养上清液证明对HeLa细胞具有细胞毒性。等基因H。构建了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结合使用。 ducreyi cdtC 突变体（MK Stevens，JL Latimer，SR Lumbley，CK Ward，LD Cope，T。Lagergard和EJ Hansen，Infect。Immun。67：3900-3908，1999）来确定CdtA，CdtB和CdtC蛋白具有CDT活性。 CdtA，CdtB和CdtC的表达似乎是H必需的。 ducreyi 来源的培养上清液对HeLa细胞具有细胞毒性。来自 cdtB 和 cdtC 突变体的全细胞超声提取物和周质提取物对HeLa细胞没有影响，而来自 cdtA 突变体的这些相同部分却具有对这些相同的人类细胞具有非常适度的细胞毒性作用。 CdtA似乎主要与 H相关。 ducreyi细胞包膜，而CdtB和CdtC都主要存在于超声处理细胞的可溶性部分中。发现 cdtA 突变体和 cdtB 突变体在实验性拟c虫的温度依赖性兔模型中均具有完全毒性。

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