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中华口腔医学研究杂志(电子版) ›› 2009, Vol. 3 ›› Issue (06) : 581 -587. doi: 10.3877/cma.j.issn.1674-1366.2009-06-002

基础研究

口腔副溶血链球菌Fap1 非依赖性菌毛的电镜观察及相关体外黏附实验
彭志翔1,(), Teresa Ruiz2, Paula Fives-Taylor3, Hui Wu4   
  1. 1.510055 广州, 中山大学光华口腔医学院·附属口腔医院·口腔医学研究所
    2.Molecular Physiology and Biophysics,University of Vermont, Burlington, VT 05405, USA
    3.Department of Microbiology and Molecular Genetics, University of Vermont,Burlington,VT 05405,US
    4.Departments of Pediatric Dentistry and Microbiology, Schools of Dentistry and Medicine,University of Alabama, Birmingham, AL 35294, USA
  • 收稿日期:2009-09-17 出版日期:2009-12-01
  • 通信作者: 彭志翔

TEM observation of a non-Fap1 dependent fimbriae in Streptococcus parasanguinis and relevant in vitro binding assay

Zhi-xiang PENG1,(), Ruiz Teresa1, Fives-Taylor Paula1, Wu Hui1   

  1. 1.Guanghua School of Stomatology, Institute of Stomatological Research, Sun Yat-sen University,Guangzhou 510055, China
  • Received:2009-09-17 Published:2009-12-01
  • Corresponding author: Zhi-xiang PENG
引用本文:

彭志翔, Teresa Ruiz, Paula Fives-Taylor, Hui Wu. 口腔副溶血链球菌Fap1 非依赖性菌毛的电镜观察及相关体外黏附实验[J/OL]. 中华口腔医学研究杂志(电子版), 2009, 3(06): 581-587.

Zhi-xiang PENG, Ruiz Teresa, Fives-Taylor Paula, Wu Hui. TEM observation of a non-Fap1 dependent fimbriae in Streptococcus parasanguinis and relevant in vitro binding assay[J/OL]. Chinese Journal of Stomatological Research(Electronic Edition), 2009, 3(06): 581-587.

目的

探讨副溶血链球菌表面Fap1 非依赖性短菌毛结构的形态学表现及其在细菌黏附中的作用。

方法

通过插入置换法构建Fap1 蛋白系列缺陷株以去除Fap1 依赖性长菌毛结构,电镜下观察细菌表面结构的形态学变化;系列缺陷株的黏附功能通过唾液包被的羟基磷灰石体外黏附实验加以评价。

结果

所有Fap1 蛋白形成缺陷株的菌体表面存在一种短菌毛结构,该种Fap1 非依赖性菌毛垂直于细胞表面呈稀疏分布,长度不大于100 nm;体外黏附实验表明仅存在短菌毛结构的系列缺陷株的黏附功能与野生株相比有明显下降,但仍保持19% ~27%的黏附能力。

结论

副溶血链球菌表面Fap1 非依赖性短菌毛在细菌的黏附活动中仅起一定辅助作用。

Objective

To investigate the S.parasanguinis cell surface morphological characteristics of non-Fap1 dependent short fimbriae and its possible role in bacterial adhesion.

Methods

An insertional replacement was carried out to construct a series of Fap1 defect mutants, thus the Fap1 dependent long fimbriae structure was removed. The bacterial surface morphological change was observed with transmission electron microscope (TEM); The adhesion capacities of the mutants were tested by using an in vitro saliva-coated hydroxyapatite (SHA)model.

Results

The non-Fap1 dependent short fimbriae exist on the cell surface of all Fap1 defect mutants. This kind of short fimbriae were perpendicular to cell surface and distributed sparsely with length less than 100 nm. In vitro SHA binding assay showed the mutants with short fimbriae had an obvious decrease in adhesion ability while comparing with that of the wild type strain. But they still remained 19% ~ 27% binding functions.

Conclusion

The non-Fap1 dependent short fimbriae merely play some minor role in S.parasanguinis bacterial adhesion activity.

表1 研究所用菌株及质粒
图1 副溶血链球菌Fap1 蛋白合成相关基因等位置换突变株构建 副溶血链球菌fap1 基因突变株及糖基化相关蛋白编码基因gap1,gap2 及gap3 突变株通过卡那霉素抗性基因aphA-3 分别等位置换入相关靶基因加以构建。 靶基因分别从副溶血链球菌总DNA 中扩增,PCR 片段连接入pGEM-T easy 形成重组体,运用反向PCR 技术删除重组体中的靶基因。重组DNA 与卡那霉素抗性基因aphA-3 分别以相同的两个核酸内切酶消化后连接为新的重组质粒,删除原有的AmpR 基因后,将质粒转化入副溶血链球菌野生株FW213,抗生素平板筛选出带卡那霉素抗性的单菌落而得到相关的基因突变株
图2 副溶血链球菌野生株及突变株透射电镜图像 A:副溶血链球菌野生株FW213; B:fap1 突变株VT1393; C:gap1 突变株VT1617; D:gap2 突变株VT1618; E:gap3 突变株VT1619。 黑箭头指示长菌毛结构,白箭头指示短菌毛结构,图2A 中的比例尺标为100 nm
图3 羟基磷灰石体外黏附实验 所有结果得自2 次独立的实验,每个样品准备3 份,数据以±s 表示。 fap1-为fap1 基因突变株,gap1-,gap2-及gap3-分别为糖基化相关蛋白编码基因gap1,gap2 及gap3 突变株
表2 副溶血链球菌野生株及突变株的表面结构及黏附能力
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