FIG. 1.
Fluorescence images of PLF in macrophage-like U937 cells (A to G) and L. pneumophila stained with PI (H to J). (A to G) U937 cells were incubated with Rh-dextran (70,000 MW, lysine fixable) for 40 h, washed, and then incubated for 1.0 h in RPMI medium to chase the Rh-dextran into lysosomes. U937 cells were then infected with various strains of FL-labeled L. pneumophila at a final concentration of 5 × 107 per ml for 0.5 h at 37°C. The differentiated U937 cells were washed again, covered with agarose, and then fixed with 4% Para in PBS immediately or after an additional 5.5 h of incubation at 37°C. Shown are confocal microscopy images of infected U937 cells. (A) Wild-type strain JR32 after 6 h in a phagosome which has not fused with host lysosomes (R575/530 = 0.178, compared to FL-labeled L. pneumophila RFL = 0.319 ± 0.058); (B) LELA2955 (icmX) after 0.5 h in one phagosome which has fused (R575/530 = 1.18 at the edge of one Mφ-like cell) while another phagosome has not fused (R575/530 = 0.243) (compare both values to RFL = 0.299 ± 0.045); (C) LELA1984 (icmE) after 6 h in a phagosome that fused with lysosomes and whose contents have been degraded and dispersed to form Mv-Mφ in which the FL is colocalized with Rh (of 22 defined spots, all had R575/530 values of >0.88, compared to FL-labeled L. pneumophila RFL = 0.264 ± 0.049); (D) LELA3118 (dotA) after 6 h in phagosomes that have fused with lysosomes and whose contents have been degraded and dispersed to form Mv Mφs whose FL was occasionally colocalized with Rh (greater than one-half of the punctate green vesicles in each cell have associated R575/530 values of less than the τ value [RFL + four SDs]); (E) LELA3118 (dotA) after 6 h in a phagosome which has fused and whose contents have been degraded to form an Mv Mφ containing discrete green punctate vesicles (R575/530 values were less than the τ value) among Rh-dextran-containing lysosomes; (F) LELA1984 (icmE) after 6 h in a phagosome which has fused with a lysosome and is completely filled with Rh-dextran (R575/530 = 1.80, compared to RFL = 0.264 ± 0.049); and (G) wild-type JR32 after 6 h in a phagosome which has not fused (R575/530 = 0.317, compared to RFL = 0.235 ± 0.053) and in a typical field of sparsely infected macrophages. Red indicates lysosomes containing Rh-dextran, green indicates FI-labeled whole bacteria or their remnants, and yellow indicates colocalization of the two markers. Panels A through D display single images of cross-talk-corrected I575-B (R) and I530-B (G) as well as their composite images (RG). Composite images of cross-talk-corrected I575-B and I530-B are shown in panels E through G. (H to J) L. pneumophila JR32 was grown for 2 days on ABCYE medium, suspended in phosphate buffer, and either not labeled (H), FL labeled and incubated overnight on ice (I), or FL labeled, incubated overnight on ice, and then incubated for 5 min in phosphate buffer containing 25% ethanol and 25% acetone (J). Bacteria were then incubated with PI, washed, and photographed under epifluorescence and phase-contrast microscopy with a Nikon Labophot UFX camera system. Shown are double exposures of L. pneumophila, which appears as dark rods. Red indicates nonviable L. pneumophila that stains positively with PI and represents 2, 2, and 100% of the bacteria in panels H, I, and J, respectively.