Fig 4.

The pilU mutant is delayed in microcolony formation on human epithelial cells. (A) Live-cell phase-contrast and fluorescence imaging of pharyngeal epithelial FaDu cells infected with wild-type FAM20 and the ΔpilU, ΔpilU_C, and ΔpilT mutants during a total time of 12 h. Representative time points were chosen to show when the wild type and the pilU mutant reached their respective maximal microcolony sizes. Prior to infection, bacteria were passed through a 5-μm-pore-size filter to break apart bacterial aggregates. The initial inoculum of bacteria was stained with the fluorescent DyLight488 N-hydroxysuccinimide ester, visible in red. Microcolonies are highlighted with a yellow dashed line around the periphery. The ΔpilT mutant was included as a positive control for aggregation. Bar = 10 μm. Quantification of microcolony formation (B) and microcolony dispersal (C) was done for the wild type and the ΔpilU mutant. Microcolony formation was determined as the time point when one persistent microcolony with a size of 10 μm first became visible. Microcolony dispersal was defined as the time point when tight bacterial aggregates visibly started to dissolve into single bacteria. Values are means ± standard deviations for the experiment depicted in panel A and are representative of all assays performed. Significant differences from the wild type are indicated with asterisks (P < 0.05; two-tailed Student's t test).