FIG 1.

The proteasome is required for optimal intracellular growth of L. longbeachae and Dot/Icm-dependent decoration of its phagosomes with polyubiqutinated proteins. (A) U937 cells pretreated with the indicated concentrations of MG-132 for 1 h were challenged with wild-type L. longbeachae or L. pneumophila at an MOI of 10 for 2 h. After removing extracellular bacteria, infections proceeded for 14 h. The number of bacteria in each vacuole was determined using a fluorescence microscope after immunostaining with the L. longbeachae or L. pneumophila antibody. At least 100 vacuoles were scored for each sample. (B) U937 cells were infected wild-type or the ΔdotB mutant L. longbeachae and L. pneumophila strains for the indicated durations. Fixed cells were then immunostained with anti-L. longbeachae, anti-L. pneumophila, and anti-ubiquitinated proteins (FK1). The nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) (dark blue). The association of polyubiquitinated species with the phagosomes was scored under a fluorescence microscope. Scale bar, 2 μm. (C) Percentages of phagosomes associated with polyubiquitinated proteins. At least 100 LCVs per coverslip were measured for each sample. Results in panels A and C are means ± standard derivations (SDs) calculated from three coverslips. Similar results were obtained in three independent experiments. Statistics analysis was performed by unpaired two-tailed Student t tests, and a P value of <0.05 represents a significant difference.