Rhen M, Riikonen P, Taira S
Department of Biochemistry, University of Helsinki, Finland.
Mol Microbiol. 1993 Oct;10(1):45-56. doi: 10.1111/j.1365-2958.1993.tb00902.x.
The plasmid-carried spv genes promote virulence of salmonellae in mice by allowing bacterial growth in the reticuloendothelial tissue. When the bacteria are cultivated under normal laboratory conditions the spv genes appear dormant. This communication explores the transcriptional regulation of spv genes within murine macrophage-like J774-A.1 cells utilizing a new reporter system. Transcriptional fusions were constructed between promoter elements of the Salmonella enterica var. Typhimurium spv genes and the KS71A fimbrial gene cluster. The expression of KS71A fimbriae in fusion-carrying Escherichia coli strains was found to be under the control of the transcriptional activator gene spvR. In strains overproducing SpvR, KS71A fimbriae were assembled on the bacterial cell surface and could be detected by bacterial agglutination or immunofluorescence of intact bacteria; the reporter activity was quantified by estimating the percentage of fluorescent bacteria and by immunoblotting of cell lysates. The activity of the reporters, when transformed into the parent Typhimurium TML R66, was low and revealed less than 0.3% fimbriated cells under in vitro culture conditions. A 15-30-fold increase in fimbriation was observed when the bacteria were cultivated within J774-A.1 cells. No such increase occurred when the reporter fusions were transformed into TML R66 cured of the virulence plasmid. Insertional inactivation of the spvR gene of the virulence plasmid in Typhimurium TML R66 also abolished induction, whereas corresponding inactivation of spvA or spvB did not reduce induction. No increase in reporter activity was obtained in Typhimurium of line Q1, which is naturally avirulent for mice, although the strain was provided with virulence plasmid pEX102 of line TML R66. We conclude that the intracellular environment of J774-A.1 cells induces the spv genes and that this induction requires gene functions of both the bacterial chromosome and the virulence plasmid.
携带质粒的spv基因通过使细菌在网状内皮组织中生长来促进沙门氏菌在小鼠体内的毒力。当细菌在正常实验室条件下培养时，spv基因似乎处于休眠状态。本通讯利用一种新的报告系统探讨了鼠巨噬细胞样J774 - A.1细胞内spv基因的转录调控。构建了肠炎沙门氏菌鼠伤寒变种spv基因的启动子元件与KS71A菌毛基因簇之间的转录融合体。发现携带融合体的大肠杆菌菌株中KS71A菌毛的表达受转录激活基因spvR的控制。在过量表达SpvR的菌株中，KS71A菌毛组装在细菌细胞表面，可通过细菌凝集或完整细菌的免疫荧光检测；通过估计荧光细菌的百分比和细胞裂解物的免疫印迹来定量报告活性。当转化到亲本鼠伤寒TML R66中时，报告基因的活性很低，在体外培养条件下显示不到0.3%的菌毛化细胞。当细菌在J774 - A.1细胞内培养时，观察到菌毛化增加了15 - 30倍。当报告融合体转化到去除毒力质粒的TML R66中时，没有发生这种增加。鼠伤寒TML R66中毒力质粒的spvR基因的插入失活也消除了诱导作用，而spvA或spvB的相应失活并没有降低诱导作用。对于对小鼠天然无毒的Q1系鼠伤寒菌株，尽管该菌株带有TML R66系的毒力质粒pEX102，但报告基因活性没有增加。我们得出结论，J774 - A.1细胞的细胞内环境诱导spv基因，并且这种诱导需要细菌染色体和毒力质粒的基因功能。
