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How the parasitic bacterium Legionella pneumophila modifies its phagosome and transforms it into rough ER: implications for conversion of plasma membrane to the ER membrane

¹ Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA
² Yale University School of Medicine, Section of Microbial Pathogenesis, New Haven, CT 06511, USA

View larger version (181K): [in a new window]   Fig. 1. Thin section cut near the surface of a U937 macrophage fixed after five minutes of infection, with L. pneumophila infected at an MOI of 20. Within the phagosome is a L. pneumophila bacterium. Attached to the basal surface of the phagosomal membrane are a series of vesicles of the ER. The bracketed region in (a) is shown at higher magnification in (b). The endosomal membrane is 70 Å thick, whereas the attached ER membranes are only 60 Å thick.

View larger version (179K): [in a new window]   Fig. 2. (a) Longitudinal section through a L. pneumophila bacterium residing in a phagosome after 15 minutes of infection at an MOI of 20. The phagosome is surrounded by attached ER vesicles, many of which are the rough ER type (arrowheads point to attached ribosomes). The surface of the ER vesicles is flattened and smooth where it is attached to the phagosomal membrane but irregular in contour on its unattached surface. Even at this low magnification it is possible to detect tiny osmiophilic hairs at the attachment surface between the phagosomal and ER membranes. (b) Higher magnification view of the area outlined in (a). The hairs can be seen better in this micrograph (arrows). Note that by 15 minutes, the thickness of the phagosomal and ER membranes is the same, about 60 Å each.

View larger version (133K): [in a new window]   Fig. 3. (a) Transverse section through a phagosome containing L. pneumophila after a 15 minute infection period. As in (Fig. 2), vesicles of ER, both studded with ribosomes and without, are attached to the phagosome. The region indicated is shown at higher magnification in (b). Both the phagosomal membrane and the ER membranes have the same thickness. (c) Longitudinal section through a phagosome containing L. pneumophila after a two hour infection period at an MOI of 1 applied for 30 minutes. The phagosome is surrounded by attached ER vesicles. The area boxed in (c) is shown at a higher magnification in (d).

View larger version (115K): [in a new window]   Fig. 4. (a) Transverse section through a L. pneumophila bacterium enclosed in a vacuole. The U937 cells were exposed to L. pneumophila for 30 minutes, then washed free of unattached bacteria and incubated for an additional 5.5 hours before fixation. The surface of the phagosome has reduced numbers of attached ER vesicles by this time. In their place are ribosomes that are directly attached to the phagosome. (b) Thin sections through a vacuole in a U937 cell containing two L. pneumophila. This U937 cell had been exposed to L. pneumophila for 30 minutes and after removal of unattached bacteria incubated for 19.5 hours. By this time, the L. pneumophila had replicated in the rough ER – identified as such by ribosomes attached to the membrane of the vacuole.

View larger version (93K): [in a new window]   Fig. 5. U937 cells were incubated with L. pneumophila for two hours, then washed, scrapped off the petri plates and homogenized. A low-speed pellet of unbroken cells or large cell fragments was removed by centrifugation. The supernatant was then centrifuged on a tabletop centrifuge and the pellet fixed. It contains L. pneumophila enclosed in its phagosome, and still attached to the surface of the phagosome are mitochondria and ER vesicles. (a) Since the ER is continuous with the outer nuclear membrane (NM), L. pneumophila-containing phagosomes can also be found attached to the nucleus (N) as well as to the outer ER vesicles (b).

View larger version (108K): [in a new window]   Fig. 6. Thin sections through a portion of the plasma membrane of three U937 macrophages. All are printed at the same magnification. All three sections show ER vesicles attached to the plasma membrane. In the areas indicated by the arrows we can see the hairs that attach these membranes together. (a) This macrophage was exposed to L. pneumophila for 15 minutes at an MOI of 20 before fixation. Extracellular L. pneumophila were present near this section as well as internalized L. pneumophila. (b) and (c) Uninfected U937 cells. As in (a). ER vesicles are attached to the plasma membrane.

View larger version (106K): [in a new window]   Fig. 7. Co-infection of U937 cells with two bacteria; L. pneumophila, a Gram-negative bacterium and a second Gram-positive bacterium are depicted here in this thin section of a phagosome. No ER vesicles attach themselves to the phagosome of the Gram-positive species. The thickness of the phagosomal membrane remains the same as the plasma membrane, 70 Å. This bacterium will be killed subsequently in a lysosome.

View larger version (148K): [in a new window]   Fig. 8. Thin section through a U937 cell with a phagosome containing a dotA mutant. This U937 cell was infected for 45 minutes prior to fixation. The phagosomal membrane does not have attached ER vesicles. Furthermore, the thickness of the phagosomal membrane remains as thick as the plasma membrane (70 Å thick (b)). As in Fig. 7, phagosomes containing dotA mutants fuse with lysosomes.

View larger version (125K): [in a new window]   Fig. 9. U937 cells were infected with L. pneumophila icmW mutants for 45 minutes, then unattached bacteria were washed away and incubation was continued for an additional 45 min before fixation. As is the case with wild-type L. pneumophila, ER vesicles become attached by tiny hairs (arrows in b) to the phagosomal membrane surrounding icmW mutants. Unlike the membrane surrounding wild-type L. pneumophila, which decreases from 70 Å to 60 Å within 15 minutes, there is no observable decrease in the thickness of the membrane surrounding icmW mutants at 1.5 hours (b). (c) and (d) U937 cells were infected with icmW mutants for 45 minutes, then washed and incubated for an additional 7.25 hours. Replicating bacteria could be found at this time and the phagosome membrane surrounding them had attached ER vesicles and mitochondria. However, unlike the situation after 1.5 hours (a), the thickness of the phagosomal membrane surrounding these replicating icmW mutants had decreased from 70 to 60 Å. (d) The membrane surrounding replicating icmW mutants is the same thickness as the ER membrane.

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