Bish Samuel E, Song Wenxia, Stein Daniel C
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA.
Microbes Infect. 2008 Aug-Sep;10(10-11):1182-91. doi: 10.1016/j.micinf.2008.06.014. Epub 2008 Jul 17.
Neisseria gonorrhoeae can invade into cervical epithelial cells to overcome this host defense barrier. We developed a beta-lactamase reporter system that allowed us to quantify at the single cell level if a host cell internalized a viable or nonviable microorganism. We autodisplayed beta-lactamase on the surface of FA1090 [FA1090Phi(bla-iga')] and demonstrated by confocal fluorescence microscopy and flow cytometry that FA1090Phi(bla-iga') cleaved the beta-lactamase substrate CCF2-AM loaded into host cells only when gonococci were internalized by these host cells. While FA1090Phi(bla-iga') adhered to almost all ME180 cells, viable N. gonorrhoeae were internalized by only a subset of cells during infection. Nonviable gonococci adhered to, but were not internalized by ME180 cells, and failed to recruit F-actin to sites of adherent bacteria. Overall, we show that epithelial cell invasion is a dynamic process that requires viable N. gonorrhoeae. We demonstrate the advantages of the beta-lactamase reporter system over the gentamicin protection assay in quantifying bacterial invasion. The reporter system that we have developed can be adapted to studying the internalization of any bacterial species into any host cell.
淋病奈瑟菌可侵入宫颈上皮细胞以突破这种宿主防御屏障。我们开发了一种β-内酰胺酶报告系统，该系统使我们能够在单细胞水平上量化宿主细胞是否内化了活的或非活的微生物。我们将β-内酰胺酶自动展示在FA1090 [FA1090Phi(bla-iga')]的表面，并通过共聚焦荧光显微镜和流式细胞术证明，只有当淋球菌被这些宿主细胞内化时，FA1090Phi(bla-iga')才会切割加载到宿主细胞中的β-内酰胺酶底物CCF2-AM。虽然FA1090Phi(bla-iga')几乎能黏附到所有ME180细胞上，但在感染过程中，只有一部分细胞内化了活的淋病奈瑟菌。非活的淋球菌能黏附到ME180细胞上，但不会被内化，也无法将F-肌动蛋白募集到黏附细菌的部位。总体而言，我们表明上皮细胞侵袭是一个需要活的淋病奈瑟菌的动态过程。我们证明了β-内酰胺酶报告系统在量化细菌侵袭方面优于庆大霉素保护试验。我们开发的报告系统可适用于研究任何细菌物种进入任何宿主细胞的内化过程。
