Grassmé H U, Ireland R M, van Putten J P
Max-Planck-Institut für Biologie, Abteilung Infektionsbiologie, Tübingen, Germany.
Infect Immun. 1996 May;64(5):1621-30. doi: 10.1128/iai.64.5.1621-1630.1996.
Neisseria gonorrhoeae enters cultured human mucosal cells following binding of a distinct gonococcal opacity (Opa) outer membrane protein to cell surface proteoglycan receptors. We examined the route of internalization that is activated by Opa-expressing gonococci (strain VP1). Microscopy of infected Chang epithelial cells showed that gonococcal uptake was insensitive to monodansylcadaverine (150 microM), which interferes with clathrin-mediated endocytosis. Similarly, indirect immunofluorescence staining for clathrin in infected cells showed distribution of cellular clathrin unaltered from the distribution in noninfected cells. The microtubule inhibitors colchicine (50 microM) and nocodazole (20 microM) but not the microtubule-stabilizing agent taxol (10 microM) caused a moderate (30 to 50%) reduction in gonococcal entry without affecting bacterial adherence. The most dramatic effects were obtained with the microfilament-disrupting agent cytochalasin D (3 microM), which totally blocked bacterial entry into the cells. Double immunofluorescence staining of gonococci and actin filaments in infected cells demonstrated bacterium-associated accumulations of F-actin as an early signal of bacterial entry. The recruitment of F-actin was transient and disappeared once the bacteria were inside the cells. Cytochalasin D disrupted the actin cytoskeleton architecture but did not prevent the recruitment of F-actin by the bacteria. Adherent, noninvasive gonococcal Opa variants lacked the ability to mobilize F-actin. Recombinant Escherichia coli expressing the gonococcal invasion-promoting Opa of gonococcal strain MS11 (Opa50) adhered to the epithelial cells in an Opa-dependent fashion but was not internalized and did not recruit detectable amounts of F-actin. Coinfection with the E. coli recombinant strain and gonococci resulted in specific entry of the diplococci, despite the presence of large numbers of adherent E. coli cells. Together, our results indicate that Opa-mediated gonococcal entry into Chang cells resembles phagocytosis rather than macropinocytosis reported for Salmonella spp. and sequentially involves gonococcal adherence to the cell surface, Opa-dependent and cytochalasin-insensitive recruitment of F-actin, and cytochalasin D-sensitive bacterial internalization.
淋病奈瑟菌通过一种独特的淋病奈瑟菌不透明（Opa）外膜蛋白与细胞表面蛋白聚糖受体结合后进入培养的人黏膜细胞。我们研究了由表达Opa的淋病奈瑟菌（菌株VP1）激活的内化途径。对感染的Chang上皮细胞进行显微镜观察发现，淋病奈瑟菌的摄取对单丹磺酰尸胺（150微摩尔）不敏感，单丹磺酰尸胺会干扰网格蛋白介导的内吞作用。同样，对感染细胞中的网格蛋白进行间接免疫荧光染色显示，细胞内网格蛋白的分布与未感染细胞中的分布没有改变。微管抑制剂秋水仙碱（50微摩尔）和诺考达唑（20微摩尔），但不是微管稳定剂紫杉醇（10微摩尔），使淋病奈瑟菌的进入适度减少（30%至50%），而不影响细菌的黏附。用微丝破坏剂细胞松弛素D（3微摩尔）获得了最显著的效果，它完全阻止了细菌进入细胞。对感染细胞中的淋病奈瑟菌和肌动蛋白丝进行双重免疫荧光染色显示，与细菌相关的F-肌动蛋白积累是细菌进入的早期信号。F-肌动蛋白的募集是短暂的，一旦细菌进入细胞内就会消失。细胞松弛素D破坏了肌动蛋白细胞骨架结构，但没有阻止细菌对F-肌动蛋白的募集。黏附性、非侵袭性的淋病奈瑟菌Opa变体缺乏动员F-肌动蛋白的能力。表达淋病奈瑟菌菌株MS11促进侵袭的Opa（Opa50）的重组大肠杆菌以Opa依赖的方式黏附于上皮细胞，但未被内化，也未募集到可检测量的F-肌动蛋白。尽管存在大量黏附的大肠杆菌细胞，但与大肠杆菌重组菌株和淋病奈瑟菌共感染导致双球菌特异性进入。总之，我们的结果表明，Opa介导的淋病奈瑟菌进入Chang细胞类似于吞噬作用，而不是报道的沙门氏菌属的巨吞饮作用，并且依次涉及淋病奈瑟菌黏附于细胞表面、Opa依赖且对细胞松弛素不敏感的F-肌动蛋白募集以及对细胞松弛素D敏感的细菌内化。
