Kahrs A F, Bihlmaier A, Facius D, Meyer T F
Max-Planck-Institut für Biologie, Abteilung Infektionsbiologie, Tübingen, Germany.
Mol Microbiol. 1994 Jun;12(5):819-31. doi: 10.1111/j.1365-2958.1994.tb01068.x.
One requirement for the invasion of, and tight adherence to, human epithelial cells by Neisseria gonorrhoeae is the synthesis of distinct opacity (Opa) outer membrane proteins, encoded by a family of phase-variable chromosomal genes. However, cloning and surface expression of invasion-promoting Opas in Escherichia coli is not sufficient for the efficient invasion of epithelial cells: additional factors besides Opa may be involved in this process. Using the phoA mini-transposon TnMax4, a library of gonococcal mutants affected in the expression of genes encoding exported proteins was generated through shuttle mutagenesis. Of a total of 608 PhoA+ plasmid clones identified in E. coli E145 approximately 40% were used successfully in transforming N. gonorrhoeae and in activating the corresponding chromosomal genes. Gonococci producing the invasion-promoting Opa50 served as the genetic background to identify 51 mutants unable to enter Chang human epithelial cells. We expect some of these mutations affect the interaction of N. gonorrhoeae with epithelial cells directly, while other mutants may carry defects in general house-keeping, secretory and/or regulatory determinants. In some mutants the loss of invasiveness appears to be due to a negative dominant effect of the PhoA+ fusions produced in these mutants. Some of the identified genes display a phase-variation phenomenon in E. coli and several genes are found in multiple copies in N. gonorrhoeae and/or present only in pathogenic Neisseria species.
淋病奈瑟菌侵袭并紧密黏附人上皮细胞的一个必要条件是合成由一组相变染色体基因编码的独特的不透明（Opa）外膜蛋白。然而，在大肠杆菌中克隆并在表面表达促进侵袭的Opa蛋白并不足以实现上皮细胞的有效侵袭：除了Opa蛋白之外，可能还有其他因素参与这一过程。利用phoA微型转座子TnMax4，通过穿梭诱变产生了一个受编码输出蛋白的基因表达影响的淋球菌突变体文库。在大肠杆菌E145中鉴定出的总共608个PhoA+质粒克隆中，约40%成功用于转化淋病奈瑟菌并激活相应的染色体基因。产生促进侵袭的Opa50的淋球菌作为遗传背景，以鉴定51个无法进入Chang人上皮细胞的突变体。我们预计其中一些突变直接影响淋病奈瑟菌与上皮细胞的相互作用，而其他突变体可能在一般的管家、分泌和/或调节决定因素方面存在缺陷。在一些突变体中，侵袭性的丧失似乎是由于这些突变体中产生的PhoA+融合蛋白的负显性效应。一些已鉴定的基因在大肠杆菌中表现出相变现象，并且在淋病奈瑟菌中发现几个基因有多个拷贝和/或仅存在于致病性奈瑟菌属物种中。
