FIG. 4.

To determine if gonococcal PLD plays a role in the cytoskeletal rearrangements leading to membrane ruffling of the cervical epithelium, we performed scanning electron microscopy. Scanning electron microscopy analysis demonstrates that aberrant cytoskeletal rearrangements occur upon infection of cervical epithelia with PLD mutant gonococci compared to infection with wild-type gonococci. Endocytosis mediated by CR3 requires receptor clustering. The absence of bacterial clusters in electron micrographs taken of mutant gonococci at 3 h postinfection (upper panel) may be reflective of the inability of these bacteria to elicit up-regulation of CR3 or of their inability to initiate signaling cascades required for CR3 clustering. Similarly, the absence of membrane ruffles (lower panel) in PLD-infected cells suggests that gonococcal PLD may be required to potentiate the cytoskeletal rearrangements required to form membrane ruffles. These processes are restored when assays are performed with PLD mutant gonococci in the presence of primed wild-type supernatants. No observable differences between mutant or wild-type gonococci were noted in the ability of gonococci to interact with each other or with cervical cells at earlier points of infection. Electron micrographs shown in the lower panel correspond to the respective boxed areas shown in the upper panel. Magnifications: A, ×1,000; B, ×1.1,000; C, ×800,000; D, ×9,000; E, ×10,000; and F, ×15,000.