Bacterial adherence to human PMNs. Fluorescent E. coli cells, nonexpressing control (pACYC184), Ag43-expressing (pPKL330), and AIDA-expressing (pLH44) cells, were mixed with PMNs. (A to C) Representative fluorescence micrographs of E. coli MG1655 Δflu (GFP), demonstrating that the majority of Ag43-expressing cells were taken up by PMNs in an opsonin-independent manner while neither control cells nor AIDA-expressing cells were taken up by PMNs. (D) E. coli MG1655 Δflu expressing the mutant Ag43 (pSF2; with RGD motif changed to KGE) was also taken up by PMNs. (E and F) Fluorescence micrographs of E. coli 83972 (YFP), nonexpressing control and Ag43-expressing cells, illustrating Ag43 adherence and uptake to PMNs. All images were obtained after coincubation of E. coli cells and PMNs for 60 min at 37°C. Bars, 40 μm (A, B, C, E, and F), 20 μm (D).

E. coli exposed to human blood and internalization of Ag43-expressing E. coli by PMNs. (A) E. coli strain MG1655 Δflu, harboring control plasmid (pACYC184) or Ag43-encoding plasmid (pPKL330), was incubated with blood or buffer for 15 min, and bacterial viability was determined. Results are presented as survival in blood compared with buffer and means from three independent experiments; error bars indicate standard deviations (values were 4.4 × 10⁸ and 6.0 × 10⁸ CFU/ml in buffer for control plasmid and Ag43, respectively). (B) Bacterial uptake by human PMNs monitored by flow cytometry to determine the percentage of phagocytic PMNs after bacterial incubation for 45 min at 37°C. Phagocytic green fluorescent PMNs were detected after incubation with GFP-expressing E. coli control (pACYC184) and Ag43-expressing (pPKL330) cells followed by a wash of external cells. Values are means of results from 10 samples, and error bars indicate standard deviations (unpaired t test, P < 0.0001). The experiment was performed in duplicate with separate cultures and blood donors. (C) Internalization of bacteria in PMNs was assessed by addition of gentamicin (18 μg/ml) to mixtures of PMNs with control or Ag43 E. coli; bacterial viability was determined after 30 min of incubation. Error bars indicate standard deviations of results from three independent experiments. (D) Internalization of Ag43-expressing bacteria in PMNs was blocked by treatment of PMNs with cytochalasin D, leading to lack of protection against gentamicin. Error bars indicate standard deviations of results from two separate experiments. (E) Survival of phagocytosed Ag43-expressing E. coli inside PMNs. Coincubation of bacteria and PMNs for 0 to 120 min at 37°C was followed by serial dilutions and plating. Bacterial viabilities are presented relative to survival in buffer and are means of results from three independent experiments, and error bars indicate standard deviations.