RESEARCH ARTICLE. Lewis G. Tilney 1 *, Omar S. Harb 1, Patricia S. Connelly 1, Camenzind G. Robinson 2, and Craig R. Roy 2 SUMMARY

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1 RESEARCH ARTICLE 4637 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 Lewis G. Tilney 1 *, Omar S. Harb 1, Patricia S. Connelly 1, Camenzind G. Robinson 2, and Craig R. Roy 2 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 *Author for correspondence ( kvranich@sas.upenn.edu) Accepted 13 September 2001 Journal of Cell Science 114, (2001) The Company of Biologists Ltd SUMMARY Within five minutes of macrophage infection by Legionella pneumophila, the bacterium responsible for Legionnaires disease, elements of the rough endoplasmic reticulum (RER) and mitochondria attach to the surface of the bacteria-enclosed phagosome. Connecting these abutting membranes are tiny hairs, which are frequently periodic like the rungs of a ladder. These connections are stable and of high affinity - phagosomes from infected macrophages remain connected to the ER and mitochondria (as they were in situ) even after infected macrophages are homogenized. Thin sections through the plasma and phagosomal membranes show that the phagosomal membrane is thicker (72±2 Å) than the ER and mitochondrial membranes (60±2 Å), presumably owing to the lack of cholesterol, sphingolipids and glycolipids in the ER. Interestingly, within 15 minutes of infection, the phagosomal membrane changes thickness to resemble that of the attached ER vesicles. Only later (e.g. after six hours) does the ER-phagosome association become less frequent. Instead ribosomes stud the former phagosomal membrane and L. pneumophila reside directly in the rough ER. Examination of phagosomes of various L. pneumophila mutants suggests that this membrane conversion is a fourstage process used by L. pneumophila to establish itself in the RER and to survive intracellularly. But what is particularly interesting is that L. pneumophila is exploiting a poorly characterized naturally occuring cellular process. Key words: L. pneumophila, Macrophage, Endoplasmic reticulum, Ribosomes, Membrane, Intracellular survival INTRODUCTION Following entry by phagocytosis/endocytosis, intracellular pathogens have evolved a variety of strategies for evading proteolysis by the host (Finlay and Falkow, 1997). Some, such as Listeria monocytogenes (Portnoy and Jones, 1994), Shigella flexneri (High et al., 1992), the protozoa Theileria parva (Dobbelaere and Heussler, 1999) and Trypanosoma cruzi (Hall, 1993) dissolve the phagosomal membrane and reproduce in the cytoplasm. Others, such as Mycobacterium tuberculosis, Chlamydia trachomatis (Finlay and Falkow, 1997) and the protozoa Toxoplasma gondii (Sibley, 1993) inhibit fusion of the phagosome/endosome by lysosomes and grow within an expanding permeablized endosome. And others, such as Coxiella burnetti (Heinzen et al., 1996) and the protozoan Leishmania survive and grow within the inhospitable confines of a phagolysosome. Perhaps the most bizarre and interesting solution is for the pathogen to enter the cell in an endosome and from there somehow move to the endoplasmic reticulum (ER) and its interconnected perinuclear space. Here it is safe from lysosomal degradation. At least three organisms have followed this last strategy, the virus SV40 and two bacteria, Brucella abortus (Pizarro-Cerda et al., 1998) and L. pneumophila (Swanson and Isberg, 1995). In the case of SV40, this virus is taken up in small uncoated vesicles or caveoli that become continuous with a complex tubular network of smooth membranes generated as extensions of the ER (Pelkmans et al., 2001; Kartenbeck et al., 1989). In the case of Legionella pneumophila, the causative agent of Legionnaires disease, the bacterium enters the cell in a phagosome that becomes surrounded by vesicles and mitochondria (Horwitz, 1983). This vacuole provides an intracellular sanctuary for L. pneumophila where these bacteria are protected from lysosomal degradation. This remarkable behavior of L. pneumophila, as first described by Horwitz (Horwitz, 1983), severely puzzled us. By what mechanisms does L. pneumophila induce vesicles to surround the phagosome? Do they protect it from fusion with lysosomes, and how does a former plasma membrane become studded with ribosomes? After all, the lipid composition and thickness of the plasma membrane and its unusual protein composition is very different from the ER. Specifically, the plasma membrane is rich in cholesterol, in amounts roughly equimolar to the sum of all the phospholipids in the membrane, and sphingolipids (such sphingomyelin and glycolipids), whereas the ER membranes lack or have extremely low concentrations of both.

2 4638 JOURNAL OF CELL SCIENCE 114 (24) This results in the endoplasmic reticulum membrane being thinner than the plasma membrane, a feature that may influence differences in accumulation of transmembrane proteins (Bretscher and Munro, 1993). In this current study, we amplify and extend the morphological description of Horwitz (Horwitz, 1983), revealing several previously undescribed phenomena associated with the establishment of the L. pneumophila phagosome. More specifically, we illustrate morphologically the existence of physical connections between the ER vesicles and mitochondria, and the L. pneumophila phagosome. Furthermore, we show that the thickness of the phagosomal membrane containing L. pneumophila changes to resemble an ER membrane. This is followed some hours later by the attachment of ribosomes directly to the newly thinned phagosomal membrane. We then describe the behavior of five L. pneumophila mutants that amplify our morphological description of the wild-type L. pneumophila by emphasizing some of the changes that occur as L. pneumophila adapts to life within the macrophage. Overall, the results presented in this study support the idea that L. pneumophila subverts normal cellular processes to protect itself from proteolysis. Thus, studying the interaction of L. pneumophila with its host cell gives us the opportunity to understand additional features of basic cell biological phenomena that occur in eukaryotic cells. Study of these basic processes, which include the attachment of ER vesicles to the plasma membrane and changes in membrane composition, are some of our goals for the future. Ultimately we must determine why uninfected host macrophages behave in this fashion and how pathogens orchestrate this behavior. This report then, we hope, will stimulate others to investigate what we assume is so far an undescribed or poorly described pathway in eukaryotic cells in which the plasma membrane is converted to the ER. MATERIALS AND METHODS Cell lines and bacterial strains U937 cells (ATCC#: CRL ) are a human monocyte lymphoma cell line (Sandstrom and Nilsson, 1976) that exhibit macrophage-like characteristics when stimulated with phorbol myristic acid (PMA). Two srains of L. pneumophila, (AA100 and CR39), both of which are serogroup 1 strains, were utilized in this study. U937 cell maintenance and differentiation U937 cells were maintained in suspension in RPMI-1640 (Gibco) supplemented with 10% heat-inactivated fetal bovine/calf serum (FBS) and glutamine. Cells can be differentiated by the addition of 50 ng/ml PMA (5 µl of a 1 mg/ml solution of PMA per 100 ml of cells). For the purpose of electron microscopy U937 cells were either differentiated in six-well culture plates or in 12.5 cm 2 flasks. For sixwell culture plates cells/well were seeded and incubated at 37 C with 5% CO 2 for 48 hours in the presence of PMA. For 12.5 cm 2 flasks, U937 cells were treated with PMA in a 175 cm 2 flask for two days. Adherent macrophages were then removed and replated into 12.5 cm 2 flasks and incubated at 37 C with 5% CO 2 for hours. Before infection U937 cells were washed three times in warm culture media. L. pneumophila strains and growth conditions L. pneumophila strain AA100 (graciously provided by Dr Yousef Abu Kwaik at the University of Kentucky, Lexington, KY) was grown on buffered charcoal yeast extract (BCYE) agar plates for 48 hours at 37 C. L. pneumophila has previously been shown to be most virulent following logarithmic growth (Byrne and Swanson, 1998). In order to achieve this growth phase, a loop full of the plate-grown bacteria was inoculated into 5 ml of pre-warmed buffered yeast extract (BYE) media in a 50 ml conical and grown at 37 C with shaking for ~18 hours as described previously (Harb and Abu Kwaik, 2000). Bacterial growth was monitored spectophotometrically and bacteria were harvested once the culture OD 550 reached (Gao et al., 1999). An OD 550 of 1 is equal ~ /ml. L. pneumophila strain CR39 and its isogenic mutants were grown for 48 hours at 37 C on BCYE agar plates, then resuspended in PBS to an OD 600 of Bacteria from this suspension were then diluted into 12.5 cm 2 flasks containing U937 cells to achieve the appropriate multiplicity of infection (MOI). Infection of U937 cells with L. pneumophila Bacteria were suspended to the appropriate MOI in U937 culture media, in this case MOI of 20. This culture was added to the differentiated macrophages in six-well plates and spun down at 150 g for 10 minutes to synchronize the infection. Plates were transferred to 37 C in air supplemented with 5% CO 2 for the appropriate time. Extracellular bacteria were removed by washing the macrophages three times with prewarmed culture media. For longer time points, extracellular bacteria were killed using 50 µg/ml gentamicin for one hour as described previously (Harb and Abu Kwaik, 1998). Preparation of L. pneumophila vacuoles U937 cells in 50 ml of RPMI 1640 (10% FBS) were differentiated with PMA for 48 hours. L. pneumophila were grown to an OD 550 of (~18 hours) in BYE media, and U937 cells were infected at an MOI of 5. Infection was allowed to proceed for two hours following which cells were removed from flasks using a cell scraper in 6 ml of RPMI. 1 ml of the resuspended cells was not homogenized. The rest of the cells were homogenized in a dounce homogenizer (in/out up to five times; after each time 1 ml of cells was removed into a microfuge tube and left on ice until homogenization was complete). Microfuge tubes were spun down at 400 g for three minutes to remove large fragments and/or unbroken cells (4 C). The supernatant was removed and spun down at 2000 g for one minute (4 C). The pellet was resuspended in 0.5 ml fixative and spun down at 12,000 g for three minutes at 4 C. Fresh fixative was added to the pellets, which were prepared for electron microscopy. Electron microscope techniques U937 cells were grown to confluence on either six-well culture plates or 12.5 cm 2 plastic tissue culture flasks. L. pneumophila that had reached the post-exponential growth phase were suspended at the appropriate MOI and added to the culture plates for the allotted time interval. At the appropriate time, for example, after 0.5, 1 or 1.5 hours, the extracellular bacteria were washed off three times with warmed culture media and the plates incubated with warmed culture media until fixation. The media was removed and the U937 cells fixed in situ with a freshly made solution of 1% glutaraldehyde (from an 8% stock from Electron Microscopy Sciences (EMS), Fort Washington, PA) 1% OsO 4 in 0.05 M phosphate buffer at ph 6.2 for 45 minutes. After fixation, the cells in petri plates were rinsed three times with cold distilled water and en bloc stained with uranyl acetate overnight. The petri plates were then dehydrated in ethanol then placed into hydroxypropyl methacrylate (EMS), which does not react with the plastic in the petri dish, and embedded in L 112, an epon substitute (Ladd, Burlington, VT). Following polymerization of the epon, the block was cut out and mounted and thin sections were cut through their exposed surfaces. Thin sections were collected on naked grids stained with uranyl acetate and lead citrate and examined in a Philips 200 electron microscope.

3 How Legionella pneumophila modifies its phagosome 4639 Measurements of membrane thickness and/or membrane separation from electron micrographs All our electron micrographs were photographed at 40,000 and printed at a magnification of 100,000. Individual prints were selected under an illuminated dissecting microscope at a magnification of 10. The membrane thickness was measured by placing an ocular micrometer disc on top of the segments of membranes that we wished to measure. We selected only those portions of the phagosomal membrane or the bound ER membranes where the membrane was cut transversely. Thus, after osmication one sees at one million magnification two clearly defined dense lines separating an intermediate space. If the dense lines are not clear and their margin not sharp but fuzzy, then the section is not cut perfectly normal to the membrane. Obviously these regions were not measured. On clearly defined transverse sections, we measured the width of the membrane as defined by the outer edges of the two dense lines that in electron micrographs define what we know is a membrane bilayer. To eliminate ambiguities in measurements, only one person (L.G.T.) measured all the membrane profiles. Each measurement included in Table 2 and Table 3 is of a separate ER vesicle attached to a phagosome containing a L. pneumophila bacterium. The number of separate phagosomes measured is also documented on all the tables. As shown in Table 2 and Table 3, the number of individual separate phagosomes measured varied from 7 13, and the total number of ER vesicles with its associated phagosomal regions measured ranges from The range here is due to the selection of only what L.G.T. considered to be perfect transverse section through the membrane. In all the tables, the measurements include the mean and standard deviations. Unpaired t-tests were performed on all the data in the tables so that we could compare the width of the phagosomal membrane and the ER membrane in the wild-type and mutants at varying times after infection. t-tests were also carried out by comparing the amount of coverage of the phagosome by ER vesicles at 15 minutes and 6 hours (Table 1). The P values for the comparisons are included in the Results section. Measurements of the separation of ER vesicles from the phagosomal membrane in regions where they are bound together by the osmiophilic hairs were made in the same way as measurements of the membrane thickness, for example prints at 100,000 magnification were examined under a dissecting microscope at 10. The values of membrane thickness of the phagosomal membrane and ER membranes varies, as documented in all the tables. For example, the mean values for the thickness of the ER membrane in the icm mutants (Table 3) varies from 57 to 64 Å, and in the 5 and 15 minute infections, they vary from 60 to 64 Å (Table 2). These differences are not statistically significant. For simplicity and to focus the reader s attention on the relative thickness of the ER and phagosomal membranes where they contact each other, we have placed on (Figs 1-3 and 7-9) values for these thicknesses as either 60 Å or 70 Å rather than the measured values here, for example, 61 Å or 57 Å. In showing our manuscript to other scientists they all felt this was appropriate labeling because (1) the actual values were not significantly different from the 60 or 70 Å values, and (2) it simplified the presentation of data. RESULTS L. pneumophila-containing phagosomes are encased rapidly by ER vesicles and mitochondria Five minute infection Using a multiplicity of infection (MOI) of 20 bacteria/ macrophage, a number of L. pneumophila were located within infoldings of the surface membrane. We have no way of knowing if these infoldings represent early stages in endocytosis, as serial sections were not cut. In addition to these infoldings, a few bacteria in this brief exposure were within phagosomes. A subset of these phagosomes were clearly 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.

4 4640 JOURNAL OF CELL SCIENCE 114 (24) associated with organelles even at this early time period following infection, although most of these phagosomes were not associated with vesicles or mitochondria (Fig. 1a). On the phagosome membrane opposite the cell surface, five vesicles were observed that have apparent connections to the phagosome as they approach within 70 Å of the phagosomal membrane. At points of attachment, tiny projections were observed connecting the abutting surfaces. The intimate association between the phagosomal membrane and two of the attached vesicles was more easily seen on higher magnification (Fig. 1b). These membranes were cut transversely and show the characteristic thin-section image of membranes, which consist of two dense lines separated by a less dense space. The thickness of these membranes will be important in what follows. In this micrograph, and others like it, the plasma membrane and the phagosomal membrane were approximately 70 Å thick, whereas the associated vesicle membrane was thinner and in appropriate regions, where measurements were possible, was 60 Å thick. Exact measurements are presented on the tables and will be discussed in greater detail subsequently. Table 1. Percentage of the phagosomal surface covered by attached ER and/or mitochondria Number of phagosomes measured Range (%) Average (%) 15 minutes ±4.5 2 hours ±2.9 6 hours ±13 Largest gaps between attached vesicles and phagosome 15 min 0.14 µm 2 hr 0.05 µm 6.5 hr 0.57 µm almost pancake shaped, as if these ER vesicles had flattened out as they zippered progressively to the phagosomal surface by the tiny hair-like connections (Fig. 3a,b). This leaves little free space between adjacent vesicles. We measured at 15 minutes, and at later infection times, the amount of phagosomal surface that was in direct contact with the cytoplasm or the phagosomal surface not covered by attached organelles (Table 1). We ignored all 15 and 30 minute infections Following 15 minutes of continuous exposure to L. pneumophila it was easy to find numerous examples of L. pneumophila within phagosomes. Out of 37 phagosomes each containing a single L. pneumophila bacterium, 15 did not have ER or mitochondria associated with them, 21 were completely surrounded by vesicles and/or mitochondria (Fig. 2a) and one was partially surrounded (similar to that shown in Fig. 1). Some of these vesicles had ribosomes associated with them, and thus could be identified as rough ER. The separation between the phagosomal membrane and the membrane lining an attached organelle, albeit vesicles of ER or mitochondria, was remarkably constant. We measured the distance between 30 phagosomes containing L. pneumophila and attached ER vesicles. The values measured were 67±11 Å. Thus in thin section, these two membranes look like parallel railroad tracks. Connecting these two membranes were tiny osmiophilic hairs that were periodic (Fig. 2b arrows). The phagosomal membrane closely follows the contours of the enclosed rod-shaped L. pneumophila bacterium, which in turn makes the phagosome rod shaped. If the hairs were bars or sheets they should appear differently, depending on the orientation of the bacterium. However, regardless of the plane of section through a phagosome [longitudinal (Fig. 2a), transverse (Fig. 3a) or an oblique section], these connections appeared as hairs, never as bars or sheets. As mentioned, in these early stages of infection we encountered L. pneumophila-containing phagosomes that have only a few ER vesicles connected to their surfaces, such as those depicted in (Fig. 1). Note that the region of attachment to the phagosome for most of these vesicles is small compared to their total surface area, which does not affect their spherical appearance significantly. By 15 minutes, however, many of the vesicles attached to these phagosomes were flattened, 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.

5 How Legionella pneumophila modifies its phagosome 4641 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). those which were either partially enclosed, similar to the situation in (Fig. 1), or completely lacking associated mitochondria and vesicles. Out of 10 L. pneumophila-containing phagosomes, 85 to 99% of the phagosomal surface was associated with vesicles/mitochondria (Table 1). The largest gap we found between vesicles on the phagosome was 0.14 µm. In most cases the gap was only 0.03 µm. Continuous exposure to L. pneumophila for an additional 15 minutes presents images similar to those we already described in the 15 minute sample. Some of the phagosomes were completely surrounded by organelles, others partly surrounded, and a few had no associated organelles, as the latter had probably been endocytosed just before fixation. Two hour infection Macrophages were exposed to L. pneumophila for 30 minutes, extracellular bacteria removed, and the macrophages incubated for an additional 1.5 hours before fixation. By this time, we could find no L. pneumophila in phagosomes that lacked associated ER vesicles and mitochondria. The only change from the 15 or 30 minute samples was that the enveloping ER vesicles, initially composed of the small ER vesicles, had fused to form long flattened structures connected to the phagosomal membrane by the tiny osmiophilic hairs (Fig. 3c,d). We measured 10 L. pneumophila-containing phagosomes and found that between 88 and 98% of the endosomal surface was covered by organelles (Table 1). The largest gap between attached vesicles of ER was 0.05 µm. As both longitudinal and transverse sections through the phagosome gave the same results, this means that the endosomal surface area not covered by organelles would be at worst a circle not larger than 500 Å in diameter. Thus, the associated vesicles may provide a barricade against fusion with tiny acidifying vesicles and/or lysosomal vesicles larger than 0.05 µm. Six hour infection Two changes from the 15 minute, 30 minute and 120 minute samples were apparent at this stage. First, by this time some of the L. pneumophila within an enclosed phagosome were elongating and undergoing binary fission (data not shown). Second, there were now regions on the former phagosomal membrane that lacked both small spherical ER vesicles and large flattened ER vesicles, and in their place ribosomes were attached (Fig. 4a). In other words, six hours after infecton, a portion of the former phagosomal membrane began to resemble bona fide RER, albeit with regions of attached vesicles (both rough and smooth ER). The percentage of the former phagosomal membrane containing L. pneumophila that had associated ER vesicles/ mitochondria was determined once again. The values from eight independent examples ranged from 41 to 70% (Table 1). In the cytoplasmically exposed regions, ribosomes were now attached. The largest gap between attached vesicles was 0.57 µm. Data in Table 1 indicate that there was significantly less coverage of the phagosome by ER vesicles after six hours compared to 15 minutes or 2 hours after infection (P<0.0001). 20 hour infection By 20 hours, numerous L. pneumophila were now located within a single vacuole, which indicates that replication had occurred within these compartments (Fig. 4b). One of the most significant changes observed at 20 hours was that the vacuolar membrane no longer had attached RER vesicles but was now completely covered with ribosomes, a clear indication that it had become RER. Host organelles remain attached to phagosomes containing L. pneumophila following cell fractionation Following a two hour infection with L. pneumophila, crude phagosomal fractions were prepared as described (see materials and methods). In all cases, the isolated phagosomes containing L. pneumophila were connected to vesicles of the ER, both rough and smooth, and also to mitochondria (Fig. 5a).

6 4642 JOURNAL OF CELL SCIENCE 114 (24) In no case did we find L. pneumophila-containing phagosomes that lacked an abutting population of ER vesicles, nor did we 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. find free L. pneumophila. Of particular importance, was the fact that although the ER vesicles appeared swollen, the surface abutting the endosomal membrane was separated by approximately 70 Å. In favorable regions the tiny hairs that connect the vesicles to the phagosomal membrane could be seen (data not shown). There were instances where phagosomes containing L. pneumophila had the nucleus of the host cell attached. As shown in Fig. 5b, the phagosomal membrane was connected to the outer nuclear envelope, and, as expected, the separation of these membranes was approximately 70 Å. In retrospect this connection could have been predicted since the outer nuclear envelope is contiguous with the ER. The thickness and thus the lipid composition of the phagosomal membrane surrounding L. pneumophila is altered upon association of host vesicles The lipid composition of cellular membranes dramatically affects how they appear in electronmicrographs. For instance, the fixation and staining procedures we have followed allow us to differentiate plasma membrane from membranes surrounding cellular organelles. In our micrographs, plasma membrane and the newly formed phagosomal membrane appear about 70 Å in diameter, whereas, the ER and/or mitochondrial membranes appear thinner (about 60 Å). Interestingly, we found that at early stages of infection, when ER vesicles and mitochondria first attach to phagosomes containing L. pneumophila, disparity in thickness was apparent in these abutting membranes (Fig. 1). However, 15 minutes after infection, when ER vesicles surround the phagosome, the former phagosomal membrane displayed the same thickness as the ER membrane (Fig. 2b; Fig. 3b). Thus, within 15 minutes there appears to be a dramatic decrease in thickness of the phagosomal membrane when the phagosome is surrounded by organelles. This conclusion is strengthened by the data presented in Table 2, which shows that the phagosomal membrane was significantly thicker than the ER membrane after five minutes of infection (P<0.0001). However, after 15 minutes, the phagosomal membranes had the same thickness. Since the thickness of a membrane is influenced by the presence of cholesterol and sphingolipids (Bretscher and Monro, 1993), by measuring the thickness of phagosomal and 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).

7 How Legionella pneumophila modifies its phagosome 4643 plasma membrane directly beneath the associated extracellular L. pneumophila had attached ER vesicles (data not shown). We concluded that these vesicles were attached to the plasma membrane because they were separated from each other by a constant distance of approximately 70 Å, and between the two abutting membranes were tiny hair-like connections. We also found ER vesicles attached to the plasma membrane (Fig. 6a) where extracellular L. pneumophila were not associated with the cell surface. One possible interpretation is that the L. pneumophila were attached to the surface at these locations but failed to remain connected during fixation, dehydration and embedding. Another possibility is that even in uninfected U937 cells, ER vesicles may be attached to the plasma membrane for purposes that have not been uncovered. To rule out the latter, we examined the plasma membrane of uninfected U937 cells. Much to our surprise we found ER vesicles (Fig. 6b,c), some complete with attached ribosomes (Fig. 6c), connected to the plasma membrane of these uninfected macrophages. These ER vesicles must be attached to the plasma membrane because 1) unattached vesicles were separated from the plasma membrane by a cortical layer of actin, 2) there was consistent spacing of approximately 70 Å between the two membrane bilayers, and 3) osmiophilic hairs were detectable at attachment sites (arrows). 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. ER membranes we conclude that L. pneumophila-containing phagosomes are altered in their lipid composition. This alteration occurs rapidly between five and 15 minutes after infection. L. pneumophila exploit a natural and as of yet uncharacterized host cellular process Through careful examination of the plasma membrane of infected U937 cells, we found instances where L. pneumophila were attached to host cells, probably as a prelude to phagocytosis. Interestingly, in these instances the cytoplasmic Table 2 Number of regions of Number of attached ER/ different phagosome phagosomes Thickness measured measured 5 min infection Phagosomal membrane 72±7 Å ER membrane 60±5 Å min infection Phagosomal membrane 64±3 Å ER membrane 64±3 Å Simultaneous infection of U937 macrophages with two bacteria, L. pneumophila, a Gram-negative bacterium and a second Gram-positive bacterium Although simultaneous infection of macrophages with two bacteria was done in error and was due to contamination with a second bacterium, the results proved interesting. The question we inadvertently addressed was, is the connection of the ER vesicles and mitochondria around endosomes restricted to vacuoles containing L. pneumophila or is this a global phenomenon occurring for other endosomes in L. pneumophilainfected cells? In other words, do individual endocytosed L. pneumophila induce the ER association for each endosome or does L. pneumophila induce, perhaps by transforming the host cell, a generalized response to all endosomes? Because the two infecting bacteria are morphologically distinct, L. pneumophila being Gram-negative and the other bacteria being Gram-positive, it was easy to identify the fate of each in thin sections. As was the case for L. pneumophila, the Gram-positive bacterium entered the cell surrounded by an endosomal membrane (Fig. 7a). However, unlike L. pneumophila-containing endosomes, ER vesicles were never seen connected to the endosomal membrane containing the Gram-positive bacterium. The thickness of endosomal membranes was measured for vacuoles containing these Grampositive bacteria. In all cases, the thickness of the membrane surrounding these Gram-positive bacteria matched that of lysosomes and the plasma membrane, never that of the ER vesicles situated nearby in the same micrograph (Fig. 7a,b). In the same macrophage, Gram-negative L. pneumophila were found in phagosomes whose membrane thickness was the same as the connected ER vesicles (data not shown). Recruitment of ER vesicles to phagosomes containing L. pneumophila requires the Dot/Icm transporter Genetic analysis of intracellular growth mutants has revealed

8 4644 JOURNAL OF CELL SCIENCE 114 (24) 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. that L. pneumophila requires a specialized transport apparatus for evading lysosome fusion (Andrews et al., 1998; Berger et al., 1994; Matthews and Roy, 2000; Roy et al., 1998; Sadosky et al., 1993; Segal and Shuman, 1997; Wiater et al., 1998). This transport apparatus is encoded by 24 dot and icm genes located on the L. pneumophila chromosome (Segal et al., 1998; Vogel et al., 1998) and is similar to type IV transporters found in a number of other bacteria (Christie and Vogel, 2000). To determine whether the Dot/Icm transport apparatus is required for the attachment of ER vesicles to phagosomes containing L. pneumophila, we studied the dota mutant, which fails to express virulence functions requiring the Dot/Icm transporter (Coers et al., 2000). L. pneumophila dota mutants do not grow and reproduce inside U937 cells, but instead reside in endosomes that fuse with lysosomes (Berger et al., 1994). We found that phagosomes containing L. pneumophila dota mutants did not have ER vesicles or mitochondria attached to their surface (Fig. 8a). Furthermore, the phagosomal membrane surrounding dota bacteria remained the same thickness as the plasma membrane and the lysosomal membrane (Fig. 8b). These data indicate that the attachment of ER vesicles to phagosomes containing L. pneumophila, as well as the lipid exchange that results in thinning of the phagosomal membrane, requires a functional Dot/Icm transport apparatus. 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. Distinct processes control evasion of lysosome fusion, recruitment of ER vesicles, lipid exchange and direct attachment of ribosomes to phagosomes containing L. pneumophila To determine which bacterial products are required for the attachment of ER vesicles to phagosomes containing L. pneumophila, we examined icm mutants that have phenotypes that are different from those mutants lacking a functional transporter, such as the dota mutants. The icmr, icms and icmw genes are essential for growth of L. pneumophila in primary macrophages; however, unlike the dota mutants, lossof-function mutations in these genes result in bacteria that have a limited capacity to survive and grow in U937 cells (Coers et al., 2000). Accordingly, these mutants may, by their phenotypes, be able to inform us about what the missing proteins in each mutant accomplishes for intracellular growth. icms mutant Macrophages were incubated for 45 minutes with L. pneumophila mutant CR393 ( icms). Extracellular L. pneumophila were washed away and endosomes containing mutant bacteria were allowed to develop for an additional 45 minutes. Like phagosomes containing wild-type L. pneumophila, those harboring icms mutants had rough and smooth ER vesicles and mitochondria attached by tiny hairs. Furthermore, by 90 minutes, the phagosomal membrane thickness resembled that of the attached ER vesicles (Table 3). Statistical analysis indicates that there was no significant

9 How Legionella pneumophila modifies its phagosome 4645 Table 3 Number of regions of Number of attached ER/ different phagosome phagosomes Thickness measured measured icmw 90 min infection Phagosomal membrane 68.4±5.2 Å ER membrane 56.8±5.8 Å icms 90 min infection Phagosomal membrane 64.7±3.1 Å 21 9 ER membrane 64±3.1 Å 21 9 icm S icmw 90 min infection Phagosomal membrane 69±3.9 Å 18 7 ER membrane 60±4.9 Å 18 7 icmr 90 min infection Phagosomal membrane 73.6±6.6 Å ER membrane None connected icmw 8 hr infection Phagosomal membrane 61.7±4.4 Å ER membrane 60±5 Å Table 4. Percentage of phagosomal surface covered by ER Number of phagosomes measured Range Mean Wild type ±10.6 icms ±7.8 icmw ±13.2 icms icmw ±15.5 Analysis of L. pneumophila icm mutants at 90 minutes. difference in the thickness of phagosomal membranes surrounding icms mutants and the ER (P=0.486). The only differences we found between this mutant and wild-type L. pneumophila was that the ER vesicles did not flatten along the surface of the phagosome but instead appeared as nearly spherical vesicles attached to the former phagosomal membrane (data not shown). Thus, the phagosomal surface was not covered as extensively by ER vesicles and/or mitochondria. The range was 62 to 88% with a mean of 75% versus 88% in the wild-type (Table 4, P=0.0104). In a few cases, there were no ER vesicles/mitochondria attached to phagosomes containing icms mutants. We also found some L. pneumophila in various stages of breakdown in lysosomes. In a further group of experiments, U937 cells were infected for 45 minutes then unattached L. pneumophila were washed away and the macrophages cultured for a further 7.25 hours for a total of 8 hours of infection. In these samples we found that many of the phagosomes contained multiple bacteria indicating that replication of L. pneumophila had occurred within these vacuoles. The thickness of the former endosomal membrane enclosing the now duplicated L. pneumophila was the same thickness as ER membranes, which was what we expected from the situation at 90 minutes after infection. Most of the endosomes still had ER vesicles attached to them by the tiny hairs and a few had ribosomes attached to them directly. icmw mutant As with the wild-type, the phagosomes enclosing L. pneumophila mutant CR157 ( icmw) had ER vesicles and mitochondria attached to their cytoplasmic surfaces by tiny hairs (Fig. 9a,b). The separation of abutting membranes was approximately 70 Å (Fig. 9b). We measured the percentage of the phagosome surface covered by ER/mitochondria in 12 different icmw-containing phagosomes in macrophages that had been exposed to L. pneumophila for 90 minutes (45 minute infection + 45 minutes of growth). The percentage varied from a minimum of 41% to a maximum of 87% with a mean of 74% (Table 4). Thus, these phagosomes were more naked than phagosomes containing wild-type bacteria (P=0.0203), a fact that may explain why icmw mutants end up in lysosomes. Of particular interest to us was the observation that the phagosomal membrane surrounding the icmw mutants was the same thickness as the plasma membrane, even though the phagosomal membrane was partially surrounded with attached ER and mitochondria (Fig. 9b). We measured 47 phagosomes (Table 3), and in all cases, the former phagosomal membrane was approximately 70 Å thick (68.4±5.2 Å), in contrast to the membrane thickness of attached ER or mitochondria (56.8±5.8 Å). Statistical analysis indicates that this difference is highly significant (P<0.0001). In short, the membrane surrounding icmw mutants fails to change thickness rapidly, suggesting that the icmw gene product is an important determinant for lipid exchange (Table 3). At eight hour time points, we were able to find icmw mutants replicating in U937 cells. Like the wild-type L. pneumophila, replicating icmw mutants were identified in phagosomes that contained multiple bacteria (Fig. 9c). Generally, much of the phagosome surface remained at least partially covered with bound ER vesicles and mitochondria (Fig. 9c). However, by this stage there were a few areas in which ribosomes were attached directly to the former phagosomal membrane. We measured the thickness of 18 endosomes containing icmw mutants (Table 3). By eight hours, the membrane thickness was similar to attached ER vesicles and mitochondria membranes (Fig. 9d; Table 4). Thus, although the phagosome membrane thickness was approximately 70 Å at 90 minutes, by eight hours the thickness of the membrane surrounding these icmw mutants was no longer statistically different from that of the ER (P=0.2865). In short, these L. pneumophila were now residing in bona fide RER. icmw icms double mutant Previous studies did not reveal any phenotypic differences when an icmw mutant was compared to either an icms mutant or an icms icmw double mutant (Coers et al., 2000). In this study, however, we have observed that icmw mutants reside in phagosomes that do not change membrane thickness as rapidly as phagosomes containing icms mutants. These data suggest that the icmw mutation should be dominant over the icms mutation. Accordingly, one would predict that mutant L. pneumophila lacking both the IcmW and IcmS proteins would have a phenotype that resembles the icmw single mutant. To test this, U937 cells were infected with L. pneumophila CR503 ( icmw icms) for 45 minutes and were then incubated for an additional 45 minutes after extracellular bacteria were removed. Phagosomes containing the combined mutant had ER vesicles and mitochondria attached. Of the 18 phagosomes scored, coverage of the phagosome by mitochondria and ER

10 4646 JOURNAL OF CELL SCIENCE 114 (24) 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 wildtype 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. vesicles varied from 33% of the surface to 97% of the surface. On average, 78% of the phagosomal membrane surface was covered by ER and/or mitochondria (Table 4). These differences in phagosomal surface coverage are not statistically significant (P=0.1306). When we compared the membrane thickness of the phagosome (Table 3) to the attached ER vesicles, as predicted, the thickness of the phagosomal membrane was significantly thicker than the ER membrane (Table 3, P<0.0001). Like the icmw mutant, by eight hours the thickness of the phagosome membrane surrounding icmw icms double mutants was equal to that of the attached ER vesicles (data not shown). Thus, as predicted, the icmw allele of this mutant delays the thinning of the endosomal membrane that surrounds these bacteria to the thickness of the ER membrane (Table 3). icmr mutant In thin sections, the association of ER vesicles and mitochondria was not observed for most phagosomes enclosing the mutant L. pneumophila strain CR343 ( icmr) at time points taken either 90 minutes or eight hours post infection. We found two icmr-containing endosomes that had associated ER vesicles following eight hours of infection. The thickness of the phagosomal membrane surrounding the icmr mutant remains unchanged throughout the infection (Table 3). Interestingly, at both 90 minutes and eight hours infection times, we found that the membrane limiting the icmr phagosomes often exhibited signs of fracture, although the L. pneumophila remain at least partially enclosed by endosomal membrane. We seldom found broken phagosomal membranes with any of the other L. pneumophila mutants or with the wild-type infections. DISCUSSION The following new observations have been made by studying the morphology of phagosomes containing wild-type L. pneumophila: (1) within five minutes of infection, the phagosome containing L. pneumophila becomes surrounded by elements of the rough and smooth endoplasmic reticulum and mitochondria. The abutting membranes of these organelles were connected to the phagosome by fine connections or hairs with dimension of Å. These connections are stable as organelles such as the ER, mitochondria and even the cell nucleus remain connected to the L. pneumophila-containing phagosomes following phagosome isolation. This constitutes the first description of a physical connection between the L. pneumophila phagosome and its associated vesicles. (2) In cells that had internalized both L. pneumophila and a second bacterium, ER vesicles and mitochondria are found exclusively attached to the L. pneumophila-containing phagosomes. These data are consistent with previous results demonstrating that L. pneumophila infection does not affect the processing of heterologous endosomes (Coers et al., 1999). (3) At about 15 minutes after infection, the thickness of the endosomal membrane, a derivative of the plasma membrane, is reduced from approximately 70 Å, the thickness of the plasma membrane, to the thickness of ER or mitochondrial membranes. (4) 6.5 hours following infection ribosomes are found attached directly to the former endosomal membrane of L. pneumophila. As the number of ribosomes increases, the association of the ER and mitochondria with the former endosomal membrane decreases and eventually disappears, so by this stage L. pneumophila is growing inside bona fide RER. Does the attachment of the ER and mitochondria to the L. pneumophila-containing phagosome act as an effective barricade against lysosomal fusion? An obvious function for the attached organelles is to prevent the phagosome containing L. pneumophila from fusion with primary lysosomes and/or acidifying vesicles. As the attached

11 How Legionella pneumophila modifies its phagosome 4647 organelles almost completely cover the endosomal surface (92% of the surface after 15 minutes and 95% of the surface after two hours), and since lysosomal enzymes, such as LAMP- 1, are not found in early L. pneumophila-containing vacuoles (Clemens and Horwitz, 1995; Roy et al., 1998; Swanson and Isberg, 1995), this is indeed an attractive hypothesis. Consistent with this hypothesis is the fact that dota mutants of L. pneumophila, and bacteria other than L. pneumophila, both of which do not enclose themselves in an ER barricade, are contained in phagosomes that fuse with lysosomes (Berger et al., 1994). However, this hypothesis cannot completely explain why L. pneumophila-containing phagosomes do not fuse with lysosomes because (1) a related Legionella species, Legionella micdadei, replicates in endosomes with no attached ER vesicles and/or ribosomes (Gao et al., 1999; Gerhardt et al., 2000). Furthermore, when L. pneumophila is phagocytosed by a protozoan, its natural host (Harb et al., 2000) ER vesicles and mitochondria take two hours to bind to the phagosome containing L. pneumophila, yet during this period the L. pneumophila are not killed by lysosomes (L.G.T and O.S.H, unpublished). (2) The icmr mutant of L. pneumophila does not attach ER vesicles and mitochondria to its endosomes, yet this mutant is still able to grow in U937 cells and has been shown to evade fusion with lysosomes more effectively than dota mutants (Coers et al., 2000). (3) Even though icms mutants and icmw mutants recruit ER vesicles, these mutants are defective in evading fusion with lysosomes (Coers et al., 2000). (4) Phagosomes containing dota mutants begin to accumulate the late endosomal protein LAMP-1 within five minutes of uptake, whereas phagosomes containing wild-type L. pneumophila evade this rapid endocytic maturation event (Roy et al., 1998; Wiater et al., 1998). As maximal coverage of ER vesicles on the surface of phagosomes containing wild-type L. pneumophila is not observed until minutes after uptake, it seems unlikely that these vesicles account for the complete evasion of LAMP-1 acquisition. Defining a four-stage process used by L. pneumophila to establish itself in the RER On the basis of the data presented here and that of others, we propose that L. pneumophila orchestrates a four-stage process that transforms a plasma-membrane-derived phagosome into an organelle that is very similar, if not identical, to RER. In the first stage, L. pneumophila inhibits the rapid fusion of host endosomes with the phagosome in which it resides. This process requires the Dot/Icm type IV secretion apparatus, as well as the icmw and icms gene products, as it has been shown previously that mutations in these genes severely affect the ability of L. pneumophila to avoid fusion with lysosomes (Coers et al., 2000). In the second stage, L. pneumophila directs the attachment of ER vesicles and mitochondria to the surface of the surrounding phagosome. Like the first stage, this second stage also requires the Dot/Icm secretion apparatus, indicating that this process is mediated by proteins injected into the phagosome by L. pneumophila. Our mutant analysis, however, indicates that the injected factors required at this stage are probably distinct from those that mediate the first stage. To be more specific, L. pneumophila icmw and icms mutants are defective in first stage events, but they still promote the attachment of ER vesicles and mitochondria to the phagosome surface, albeit at an efficiency that is slightly reduced when compared to wild-type bacteria (Table 4). In contrast, phagosomes containing icmr mutants can evade immediate fusion with lysosomes and do not attach ER vesicles or mitochondria to the phagosome rapidly. IcmR is a chaperone protein (Coers et al., 2000), which suggests that the L. pneumophila factor(s) directly responsible for promoting the attachment of ER vesicles to the phagosome may still be either low or of reduced quality. This explains why icmr mutations result in a severe second stage defect. This hypothesis predicts that, given enough time, some phagosomes containing icmr mutants should eventually convert to compartments resembling RER, as we observed, after eight hours. During the third stage, the thickness of the membrane surrounding L. pneumophila changes. This stage is probably concomitant with the second because it takes place between 15 and 30 minutes after phagocytosis. We have not identified a L. pneumophila mutant that is defective for this third stage of phagosome conversion. Although our data indicate that icmw mutations have a significant effect on the third-stage conversion events, this effect may be non-specific. For instance, this mutation could have slight pleiotropic effects on Dot/Icm transporter function that would perturb the normal flow of all factors secreted by this apparatus. In the fourth and final stage, ribosomes are found attached directly to the former phagosomal membrane enclosing L. pneumophila. This stage begins many hours after the membrane has changed thickness. In order to attach ribosomes to the former phagosomal membrane, translocans must be present. These in turn bind to the signal recognition particle on the ribosomes. Although we do not yet know precisely how these transmembrane channels appear in the former phagosomal membrane, it is possible that this occurs by the fusion of the attached RER vesicles with the phagosomal membrane at these later time periods. Consistent with this possibility is that the number of attached RER vesicles decreases as the number of ribosomes directly attached to the former phagosomal membrane increases. Furthermore, by this time the volume of the phagosome, which was constant from 15 minutes until two hours, increases. It has been reported recently that the viral pathogen SV40 directs its transport from the cell surface to ER by a two-step transport pathway (Pelkmans et al., 2001). Viral particles first enter cells through caveolae and then traffic to a novel sorting compartment called a caveosome. After several hours, viral particles exit the caveosome in tubular compartments that transit along microtubules and deposit SV40 into smooth ER. It is not known whether caveolae participate in uptake of L. pneumophila. There are, however, striking morphological differences in the early compartments in which these two pathogens reside. Most notably, ER is found to associate rapidly with phagosomes containing L. pneumophila, whereas ER markers are not observed on the early organelle in which SV40 resides. Thus, the process L. pneumophila use for trafficking to the ER appears to be distinct from that used by SV40. The attachment of ER vesicles to L. pneumophila endosomes cannot be related to autophagy Swanson and Isberg hypothesized that the association of the L. pneumophila with the macrophage and endoplasmic reticulum might be due to the L. pneumophila exploiting the autophagic