Abstract
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.