Simon D, Rest R F
Department of Microbiology, Hahnemann University School of Medicine, Philadelphia, PA 19102-1192.
Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5512-6. doi: 10.1073/pnas.89.12.5512.
Members of the opacity-associated (Opa) outer membrane protein family of Neisseria gonorrhoeae have been proposed to mediate adherence to and invasion of cultured human epithelial cells. We transformed Escherichia coli with a plasmid containing a gonococcal opa gene fused in-frame to the leader sequence of the beta-lactamase gene as described by Palmer et al. [Palmer, L., Brooks, G. F. & Falkow, S. (1989) Mol. Microbiol. 3, 663-671]. These transformed E. coli [E. coli (opa)] expressed the heat-modifiable opa gene product (the Opa protein) in their outer membrane and adhered to and invaded ME-180 human endocervical epithelial cells. In a 2-h adherence assay, an average of 26.7 E. coli (opa) adhered per ME-180 cell, whereas the control E. coli carrying only the expression vector (pKT279) did not adhere at all (less than 0.15 bacterium per cell). We investigated the ability of the adherent E. coli (opa) to invade ME-180 epithelial cells by using a gentamicin selection assay. We recovered up to 1 x 10(6) gentamicin-resistant bacteria per monolayer when ME-180 cells were infected with E. coli (opa) compared to less than 10 bacteria when the epithelial cells were infected with the same number of control E. coli (pKT279). The kinetics and level of invasion by E. coli (opa) were similar to invasion by Opa+ N. gonorrhoeae. Maximum invasion occurred 4 h after infection with 4 x 10(7) bacteria. Transmission electron microscopy studies confirmed that E. coli (opa) invaded ME-180 cells. In comparative studies, the number of E. coli (opa) that invaded HEC-1-B human endometrial epithelial cells was about an order of magnitude less than the number that invaded ME-180 cells, and E. coli (opa) did not invade Chang human conjunctival epithelial cells at all. The observations that early (less than 4 h) invasion by E. coli (opa) was dramatically inhibited, in a dose-responsive manner, by the actin-disrupting reagent cytochalasin D but later invasion (8-24 h) was not suggest that invasion mediated by Opa proteins may occur by two mechanisms, only one of which is dependent upon microfilament function. Transmission electron microscopy also revealed that infected epithelial cells had a dramatically increased amount of cytoplasmic fibrillar material surrounding the nucleus. The function and genesis of this material remain unclear. These studies indicate that at least one gonococcal Opa protein is an invasin.
有人提出淋病奈瑟菌的不透明相关（Opa）外膜蛋白家族成员可介导对培养的人上皮细胞的黏附和侵袭。我们按照帕尔默等人的描述[帕尔默，L.，布鲁克斯，G.F.和法尔科夫，S.（1989年）《分子微生物学》3，663 - 671]，用一个含有与β-内酰胺酶基因前导序列框内融合的淋球菌opa基因的质粒转化大肠杆菌。这些转化后的大肠杆菌[大肠杆菌（opa）]在外膜表达热可修饰的opa基因产物（Opa蛋白），并黏附于和侵袭ME - 180人宫颈上皮细胞。在2小时的黏附试验中，每个ME - 180细胞平均黏附26.7个大肠杆菌（opa），而仅携带表达载体（pKT279）的对照大肠杆菌根本不黏附（每个细胞少于0.15个细菌）。我们通过庆大霉素筛选试验研究了黏附的大肠杆菌（opa）侵袭ME - 180上皮细胞的能力。当用大肠杆菌（opa）感染ME - 180细胞时，每单层可回收多达1×10⁶个耐庆大霉素的细菌，而当用相同数量的对照大肠杆菌（pKT279）感染上皮细胞时，回收的细菌少于10个。大肠杆菌（opa）的侵袭动力学和水平与Opa⁺淋病奈瑟菌的侵袭相似。用4×10⁷个细菌感染后4小时发生最大侵袭。透射电子显微镜研究证实大肠杆菌（opa）侵袭了ME - 180细胞。在比较研究中，侵袭HEC - 1 - B人子宫内膜上皮细胞的大肠杆菌（opa）数量比侵袭ME - 180细胞的数量少约一个数量级，并且大肠杆菌（opa）根本不侵袭Chang人结膜上皮细胞。用肌动蛋白破坏试剂细胞松弛素D以剂量反应方式显著抑制大肠杆菌（opa）的早期（少于4小时）侵袭，但后期侵袭（8 - 24小时）不受影响，这表明Opa蛋白介导的侵袭可能通过两种机制发生，其中只有一种依赖于微丝功能。透射电子显微镜还显示，被感染的上皮细胞核周围的细胞质纤维状物质数量显著增加。这种物质的功能和起源尚不清楚。这些研究表明至少一种淋球菌Opa蛋白是一种侵袭素。
