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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Intracellular, pathogenic Salmonella typhimurium avoids phago-lysosome fusion, and exists within a unique vacuolar niche that resembles a late endosome. This model has emerged from studying the trafficking of host proteins to the Salmonella-containing vacuole (SCV). Very little is known about the role of major host lipids during infection. Here, we show using biochemical analyses as well as fluorescence microscopy, that intracellular infection perturbs the host sterol biosynthetic pathway and induces cholesterol accumulation in the SCV. Cholesterol accumulation is seen in both macrophages and epithelial cells: at the terminal stages of infection, as much as 30% of the total cellular cholesterol resides in the SCV. We find that accumulation of cholesterol in the SCV is linked to intracellular bacterial replication and may be dependent on Salmonella pathogenicity island 2 (SPI-2). Furthermore, the construction of a three-dimensional space-filling model yields novel insights into the structure of the SCV: bacteria embedded in cholesterol-rich membranes. Finally, we show that the glycosylphosphatidylinositol (GPI)-anchored protein CD55 is recruited to the SCV. These data suggest that, in contrast to prevailing models, the SCV accumulates components of cholesterol-rich early endocytic pathways during intracellular bacterial replication.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Microorganisms have evolved a variety of strategies for survival and proliferation in mammalian host cells. For example, bacteria such as Mycobacterium, Listeria, Legionella and Salmonella evade normal host defence mechanisms, and cause significant morbidity and mortality. Salmonella typhimurium is a major cause of gastroenteritis in humans and has been used as a model for typhoid fever in the mouse. During the last 10–15 years S. typhimurium has greatly contributed to our knowledge of how intracellular pathogens cause disease. Many of the bacterial virulence determinants have been identified (Behlau and Miller, 1993; Hensel et al., 1995; Shea et al., 1996; Valdivia and Falkow, 1997; Hensel et al., 1998; Lee et al., 2000), and the recent publication of the S. typhimurium and S. typhi genomes (McClelland et al., 2001; Parkhill et al., 2001) will accelerate the identification of further virulence factors, both in Salmonella and other intracellular pathogens.

S. typhimurium infections most commonly occur by oral ingestion of contaminated food or water. The bacteria pass to the small intestine, where they invade both epithelial cells and professional phagocytes. Invasion of epithelial cells is a dynamic interaction between bacteria and host, and dependent on a type III secretion system (TTSS) found on Salmonella pathogenicity island 1 (SPI-1) (Galan and Curtiss, 1989; Galan, 1996; Eichelberg and Galan, 1999). Contact with a host cell induces the secretion of bacterial effector proteins into the host cell cytosol resulting in actin cytoskeletal rearrangement, membrane ruffling and subsequent uptake of bacteria into membrane bound phagosomes (Francis et al., 1992; Zhou et al., 1999). While S. typhimurium can survive in epithelial cells, its ability to disseminate and cause systemic infection depends on its survival within phagocytes (Buchmeier and Heffron, 1989; Vazquez-Torres et al., 1999). In vitro experiments indicate that S. typhimurium begins to replicate after a 3–5 h lag period, and continues up to 18– 24 h, when the bacteria exit the cell. A second pathogenicity island, SPI-2, which encodes a second TTSS, is known to be required for intracellular survival in macrophages and for virulence in the mouse model (Penheiter et al., 1997; Cirillo et al., 1998; Hensel et al., 1998). SPI-2 is not required for invasion of macrophages, and is activated by the vacuolar environment within the host cell (Valdivia and Falkow, 1996; Beuzon et al., 1999; Deiwick et al., 1999).

It was originally thought that S. typhimurium resides in lysosomes (Oh et al., 1996), but subsequent studies have shown that it may be a unique late endosomal compartment. Studies on vacuole biogenesis suggest that in the first 10 min, the SCV briefly interacts with the transferrin receptor and rab5, markers of the early endosomal pathway, but by 60 min, quickly diverges from this pathway and becomes an acidic compartment that contains lysosomal associated membrane proteins (LAMPs) (Steele-Mortimer et al., 1999). However, the SCV is not associated with typical late endosomal markers such as cathepsin D and mannose 6-phosphate receptor (Garcia-del Portillo and Finlay, 1995; Rathman et al., 1997), supporting the hypothesis that it does not undergo conventional interactions with the host endocytic pathway.

Because of its unique collection of host protein markers, the SCV is an area of active study, but very little is known regarding the lipid environment of the vacuole and how it changes during intracellular development. Cholesterol is a major lipid species and an important membrane component in mammalian cells; its roles in structure, signalling and trafficking are well documented (Stulnig et al., 1997; Mayor et al., 1998; Green et al., 1999; Harder and Simons, 1999). It is also known to play a critical role in vacuolar infection of multiple bacterial and protozoan pathogens in a variety of different host cells (Coppens et al., 2000; Gatfield and Pieters, 2000; Samuel et al., 2001). Brumell et al. (2001) recently demonstrated that the 6 h SCV in HeLa cells is prominently labelled by filipin, a polyene antibiotic which binds to 3β-hydroxysterols to emit a blue fluorescence that can be detected in the UV range. However, the effects of S. typhimurium infection on host sterol metabolism were not examined, and thus it remains unclear whether filipin association reflects the presence of cholesterol or other 3β-hydroxysterol precursors (see Fig. 1). In addition, the mechanisms of sterol recruitment to the SCV have not been addressed.

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Figure 1.  Filipin staining of Salmonella-infected macrophages. RAW 264.7 macrophages were infected with Salmonella (SL1344) (see Experimental procedures). At 2 h (A–C), 8 h (D–F), 13 h (G–I), and 20 h (J–L) post infection, cells were fixed, stained with filipin, and visualized by Deltavision deconvolution microscopy. GFP-expressing bacteria are shown in green, filipin is shown in red, and the Golgi is labelled with an asterisk. White arrows indicate the SCV. Single optical sections of each time point are shown.

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In this study, we show that in murine macrophages S. typhimurium infection affects the host sterol biosynthetic pathway and induces the accumulation of sterol pre-cursors and cholesterol. By selectively blocking the formation of precursors, we show that the major sterol associated with the SCV is cholesterol. This association is detected very early during infection and increases after multiple rounds of intracellular replication, such that the SCV becomes a major site of intracellular cholesterol ~12–20 h after invasion. This suggests that the SCV intercepts cholesterol transport pathways in infected macrophages. Using filipin staining data from wild-type bacteria and mutants in intracellular replication, we established a three-dimensional space-filling model that provides insight into cholesterol distribution in the SCV. Finally, detection of a new protein marker in the SCV, the GPI-anchored protein CD55, suggests persistent vacuolar interactions with early endosomal, cholesterol-rich membranes during intracellular replication.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Distribution of filipin staining in S. typhimurium -infected macrophages

The antibiotic filipin has been widely used to visualize cholesterol in mammalian cells (Watari et al., 1999; Leventhal et al., 2001). Filipin predominantly labels the plasma membrane, the major site of cellular cholesterol, as well as the Golgi compartment. To determine whether filipin labelled the SCV in macrophages, RAW 264.7 macrophages were infected with wild-type S. typhimurium SL1344, fixed at various times, labelled with filipin and examined by DeltaVision deconvolution, fluorescence microscopy (Fig. 1).  A S. typhimurium-specific lipo-polysaccharide (LPS) antibody was used to distinguish extracellular bacteria from intracellular bacteria at early times of infection (prior to bacterial replication). As the cells were not permeabilized, the antibody only recognized extracellular bacteria. Intracellular bacteria appear as green, because of their expression of GFP. As shown in Fig. 1A–C, single optical sections revealed that at 2 h post infection, filipin staining was associated with intracellular S. typhimurium, and localized around the bacterium. At 8 h post infection intracellular vacuoles containing multiple bacteria still were labelled by filipin, which again localized around the bacteria (Fig. 1D-F). At this time, deposits of sterol at the SCV become prominent in infected cells. Filipin staining of the Golgi in the infected cells is marked by an asterisk in Fig. 1E and H. SCV staining was never seen in association with the Golgi at any time post infection. By 12 and 20 h post infection, increased bacterial replication had taken place in infected macrophages, and more intense filipin staining of the SCV was observed (Fig. 1G–I and J–L). At 12 and 20 h post infection the bacteria occupied a greater cell volume, and the infected cells could be identified based on their filipin stain alone (Fig. 1H and K). The accumulation of cholesterol in the SCV was also seen from filipin staining of another wild-type strain, S. typhimurium 12023 (data not shown), suggesting that the phenomenon is not strain dependent.

Biochemical analysis of sterol accumulation

Although filipin has been used extensively as a marker for cellular cholesterol, it binds with a high degree of specificity to other 3β-hydroxysterols (Schroeder et al., 1973; Bittman et al., 1974a,b; Clejan and Bittman, 1984), such as intermediates in the sterol biosynthetic pathway. Figure 2A depicts the mammalian sterol biosynthetic pathway. Squalene is the first step committed solely to the biosynthesis of sterols, and lanosterol is the first 3β-hydroxysterol in the pathway. As filipin labels the SCV throughout infection, we investigated the levels of 3β-hydroxysterols in infected and uninfected cultures; 1 × 107 RAW 264.7 murine macrophages were infected with bacteria, such that 25–30% of the cells were infected. At 16 and 20 h post infection, lipids were extracted from infected and uninfected cultures and processed for HPLC (see Experimental procedures). HPLC analysis of the lipid fractions revealed that the mass of cholesterol as well as that of a product that co-migrated with lanosterol were increased in infected monolayers, compared with uninfected controls (Table 1). Levels of other 3β-hydroxysterols were not increased (data not shown). The increase in cholesterol mass of 4–5 μg (30% of the total cellular cholesterol) in infected monolayers was striking because cellular cholesterol is tightly regulated. Furthermore, because a maximum of only 30% of the cells were infected, the cholesterol mass in the infected cells must have increased by considerably more than 30%. The protein content of the infected cells also increased (see Table 1). This is probably the result of bacterial contribution because control experiments showed that bacterial infection does not stimulate host protein synthesis (data not shown). At 16 h infected cultures show 18.62 μg of cholesterol per mg of protein whereas uninfected cells show 16.85 μg of cholesterol per mg of protein.

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Figure 2.  Effect of TMD on cholesterol biosynthesis and accumulation in macrophages infected by Salmonella.

A. The mammalian sterol biosynthetic pathway. 3-Hydroxy-3-Methylglutaryl-Coenzyme A (HMG-CoA) and 4,4,10β-trimethyl-trans-decal-3β-ol (TMD) are shown.

B–C. Two cultures of RAW 264.7 macrophages were treated with TMD for 4 h, and then one was infected with S. typhimurium (SL1344) (see Experimental procedures). Both uninfected (B) and infected (C) cultures were mock treated (Bi,Ci) or continuously maintained in TMD (Bii, Cii) for the next 12 h. [3H]-Mevalonate was added at 2 h post infection to label the sterol biosynthetic pathway. At 12 h post infection, cells were harvested, their lipids extracted, and biosynthetic intermediates analysed. TMD inhibits conversion of squalene oxide to lanosterol in both control (Bii) and Salmonella-infected (Cii) macrophages. Squalene oxide (SqO) elutes in fractions 8–11, desmosterol (des) in 13–16, 7-dehydrocholesterol (7DC) in 17–20, lanosterol (lan) in 21–24, and cholesterol (chol) in 25–28.

D–F. TMD-treated cultures at 20 h post infection were fixed and stained for filipin. GFP-expressing bacteria are shown in green and filipin in red. Single optical sections are shown.

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Table 1.   Detection of sterols in Salmonella-infected macrophages.
Sampleμg Lanosterolμg Cholesterolmg Proteinμg Lanosterol:mg Proteinμg Cholesterol:mg Protein
  1. RAW 264.7 macrophages were infected with Salmonella (SL1344) (see Experimental Procedures). At the indicated times post infection, cells were harvested, the lipids extracted, and analysed by reverse phase HPLC. Total mass for lanosterol and cholesterol are shown. The numbers are averages of duplicate samples which varied by less than 20%.

Uninfected 16 h0.10 ± 0.0214.49 ± 1.820.86 ± 0.120.1216.85
Infected 16 h0.73 ± 0.0319.74 ± 1.701.06 ± 0.060.6918.62
Infected 20 h0.89 ± 0.1518.58 ± 1.281.15 ± 0.140.7716.16

The HPLC profiles of lipids from infected cells showed the presence of significant mass of a species that co-migrated with authentic lanosterol, a biosynthetic precursor. The lanosterol content of uninfected cells was found to be 0.12 μg per mg of protein, less than 1% of total sterol mass in macrophages (see Table 1). To confirm that the lipid peak, which co-migrated with lanosterol in infected cells, was indeed a biosynthetic precursor, control and S. typhimurium-infected macrophages were labelled with [3H]-mevalonate starting at 2 h post infection. At 12 h post infection, lipids were extracted and fractionated by HPLC. The [3H]-lanosterol peak co-migrated with authentic lanosterol at two different temperatures (30°C and 60°C), strongly supporting that it was, in fact, lanosterol. That biosynthetic label was incorporated into the unknown mass is consistent with the hypothesis that it lies along the sterol biosynthetic pathway. The approximate sevenfold increase in the amount of lanosterol mass, and the increase in the lanosterol: protein ratio in infected cells, suggest that S. typhimurium alters the flux of precursors through the biosynthetic pathway in macrophages. Nonetheless, the total mass of cholesterol in infected cultures (16–18 μg mg-1 protein) was 23-fold greater than the mass of lanosterol (0.7–0.8 μg mg-1 protein).

Inhibition of lanosterol synthesis does not prevent filipin labelling of the SCV in macrophages

Even though its mass was small, we tested the possibility that lanosterol contributed to the intense filipin staining of the SCV. For this purpose we used 4,4,10β-trimethyl-trans-decal-3β-ol (TMD), a specific inhibitor of squalene epoxidase, the enzyme that catalyses the conversion of squalene to 2,3(S)-oxidosqualene. Thus, TMD inhibits the incorporation of [3H]-mevalonate into intermediates beyond squalene and blocks the biosynthesis of lanosterol (Fig. 2A). The agent had the anticipated effect in both control and S. typhimurium-infected macrophages, causing radiolabel to accumulate in squalene oxide and diminish in lanosterol and cholesterol (Fig. 2B–C). In addition, lanosterol mass decreased in TMD-treated cells. However, filipin staining of the SCV in infected cells treated with TMD was indistinguishable from that of untreated infected cells (Fig. 2D–F). These data suggest that all of the sterol that accumulates in the SCV is cholesterol and that it is not biosynthetic in origin. As S. typhimurium does not have the capacity to synthesize sterols, the cholesterol in the SCV is probably derived from other host cell sources.

Salmonella typhimurium induces a phenotype of delayed cell death in macrophages. This is dominant in primary macrophages as well as J774 cells, and has also been reported in RAW macrophages (Monack et al., 1996; Monack et al., 2001). We therefore determined the viability of control and infected macrophages at 10 and 20 h by measuring their ability to exclude Trypan blue. GFP-expressing bacteria were used for the infections, so that Trypan blue dye exclusion and infection could be monitored simultaneously in alternating brightfield and fluorescence images. At least 100 cells were counted and scored in four categories: uninfected, Trypan blue ±; or infected, Trypan blue ±. In infected cultures, even after 20 h, 97% of uninfected cells and 91% of infected cells remain viable (data not shown), suggesting that filipin staining and cholesterol accumulation is not a consequence of cell death. Recruitment of host cholesterol to the SCV is also seen in HEp-2 cells infected with S. typhimurium (data not shown). This suggests that cholesterol accumulation is not a consequence of apoptosis as, unlike macrophages, epithelial cells do not undergo apoptosis in response to S. typhimurium infection. Cholesterol accumulation was also seen in cells infected with non-opsonized bacteria, indicating that the recruitment of cholesterol is not linked to the route of entry.

Redistribution of host cholesterol is dependent on intracellular bacterial replication

Examination of single optical sections in Fig. 1 suggested that cholesterol accumulation in the SCV increased with time after infection. Figure 3 displays the ratios of SCV cholesterol: total cellular cholesterol (Fig. 3A) and SCV cholesterol: SCV volume (Fig. 3B) at 2 and 20 h post infection, estimated using DeltaVision software (see Experimental procedures). For the SCV cholesterol: total cellular cholesterol ratios, the software polygon function was used to draw a polygon around the SCV in 0° projections of infected macrophages. These polygons were then superimposed onto the corresponding filipin wavelength and used to quantify the filipin signal asso-ciated with the SCV. For quantification of total cellular cholesterol, polygons were drawn around 0° projections of entire macrophages. By 20 h, 20–40% of the total cellular filipin stain resides in the SCV. Filipin staining has shown that the Golgi membranes contain only small amounts of cholesterol (Orci et al., 1981 and data not shown). Thus, the amount of cholesterol in the SCV exceeds that in the Golgi at late time points, and becomes the major site of intracellular cholesterol accumulation in infected cells. In contrast, at 2 h, only 1–3% of filipin stain associates with the SCV, suggesting that the accumulation of SCV cholesterol is the result of intracellular bacterial replication.

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Figure 3.  Quantification of cellular cholesterol and SCV-associated cholesterol in Salmonella-infected macrophages. RAW 264.7 macrophages were infected with Salmonella (SL1344), fixed and stained with filipin at 2 and 20 h post infection, and visualized by Deltavision deconvolution microscopy. SoftworX quantitation software was used to determine total cellular filipin intensity, SCV-associated filipin intensity, and GFP volumes (see Experimental procedures). The ratios of SCV filipin to total cellular filipin, and SCV filipin to GFP volume, are shown.

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Although cholesterol associates with the SCV from the earliest times of intracellular infection, we were interested in determining if the net accumulation of cholesterol was altered over the course of intracellular replication. We therefore examined the density of filipin associated with the SCV over the time course of infection (Fig. 3B). As 0° projections do not yield volume data, we used a ‘field’ approach where polygons were drawn to outline GFP-labelled bacteria over single optical sections and the total filipin staining associated with the SCV was determined by integrating signal associated with all polygons over the volume of the SCV (see Experimental procedures). At 2 h, this ratio was 2.2 × 104, while at 20 h it increased to 4.7 × 104. These values were significantly different (P < 0.0001), suggesting that net accumulation of cholesterol associated with the SCV may increase at later times of intracellular replication.

Cholesterol accumulation in the SCV of Salmonella mutants

Next, we used filipin staining to examine cholesterol in cells infected with mutants having defects in intracellular replication. The ompR and phoP mutants contain mutations in two-component regulatory systems necessary for the ability of S. typhimurium to sense various intracellular environmental cues, such as low magnesium, low pH, etc. (Bajaj et al., 1996; Lee et al., 2000). These regulatory mutants have been previously shown to be avirulent in mice and exhibit a tenfold defect in intracellular replication in macrophages (Lee et al., 2000 and data not shown). The ssaT mutant contains a mutation in a structural component of the intracellular-specific TTSS found on SPI-2, and was also shown to be defective in intracellular replication (Gallois et al., 2001). In contrast to previous reports that ompR and phoP mutants can sustain large vacuoles (Garvis et al., 2001), we found that 92% of ompR and 94% of phoP vacuoles contained only one to five bacteria (Table 2). Five to six per cent of ompR and phoP infections sustained intermediate vacuoles of five to 15 bacteria, while only one to two per cent sustained large vacuoles (more than 20 bacteria). In contrast, a significant fraction of ssaT mutants contained both small (one to five bacteria) and intermediate (five to 15 bacteria), with 8% large (more than 20 bacteria) vacuoles. For comparison, in wild-type infections, 66% of vacuoles contained more than 20 bacteria (Table 2). Thus, compared to wild type, by 20 h all three of the mutants showed defects in intracellular replication as measured by the number of bacteria in vacuoles. Because the majority of the ompR and phoP mutants failed to achieve even intermediate vacuoles, we did not analyse them further. In contrast, as many as 45% of the ssaT vacuoles showed an intermediate or late phenotype, and were examined further. As shown in Fig. 4A–C, single optical sections taken through a large ssaT vacuole (containing ~25 bacteria: see corresponding 0° projection shown in Fig. 4D–F for total number of bacteria in SCV) indicate the presence of a low level of filipin stain in association with some of the intracellular bacteria. However, a 0° projection that shows a flat composite of all of the optical sections and intracellular bacteria in the cell (Fig. 4D–F) failed to reveal a high intensity of filipin accumulation at the SCV. This is because cholesterol accumulation is not detected at every optical section taken through the SCV. In contrast, vacuoles infected with wild-type S. typhimurium, whether viewed as single optical sections (see Fig. 1) or 0° projections (Fig. 4G-I) show high levels of filipin accumulation. These data suggest that ssaT mutants fail to create an SCV that accumulates intracellular cholesterol. As the ssaT mutants are defective in the apparatus for SPI-2 dependent protein secretion into the host cell, effectors of this transport system may underlie the accumulation of cholesterol in the SCV. However, because single optical sections do reveal low levels of filipin stain in association with ssaT mutants, it is possible that sterol accumulation in the SCV is regulated by multiple factors.

Table 2.  . Numbers of bacteria in macrophages infected with Salmonella mutants.
Strain1–5 Bacteria (%)5–15 Bacteria (%)20+ Bacteria (%)
  1. RAW 264.7 macrophages were infected with SL1344 wild type or with Salmonella mutants ompR, phoP or ssaT (see Experimental procedures). At 20 h post infection, cells were fixed, stained with filipin and examined by Deltavision deconvolution microscopy. For each mutant, 100 infected cells were counted and the number of bacteria in each cell recorded and assigned to one of three categories: 1–5 bacteria, 5–15 bacteria or more than 20 bacteria.

ompR92 6 2
phoP94 5 1
ssaT5636 8
SL1344181666
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Figure 4.  Distribution of filipin in macrophages infected with the Salmonella mutant ssaT (A–F) or wild-type SL1344 (G–I). Infected cells at 20 h were fixed, stained with filipin, and visualized by Deltavision deconvolution microscopy.

A–C. A single optical section of a ssaT vacuole.

D–F. All of the optical sections of this vacuole stacked in a 0° projection.

G–I. A 0° projection from a wild-type SL1344 vacuole. White arrows indicate the SCV. GFP-expressing bacteria are shown in green, and filipin is shown in red.

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A high-resolution model of the distribution of cholesterol and intracellular S. typhimurium

To improve our understanding of the distribution of cholesterol in the SCV, we examined a series of consecutive optical sections (Fig. 5). To simplify the analysis, we selected a smaller vacuole containing 13 bacteria (a subset of the bacteria are labelled #1–5). As shown in the first optical section (Ai), bacterium #1 is surrounded by cholesterol at its top end. Movement through the optical sections indicates that cholesterol tightly surrounds the bacterium throughout its length, suggesting that it is completely encapsulated in cholesterol. In contrast, bacteria #2–5 are not surrounded by cholesterol in every optical section, suggesting that cholesterol does not uniformly envelop all intracellular bacteria. However, in a subset of optical sections, high levels of cholesterol are seen in close proximity with the bacteria. This suggests that the distribution of cholesterol in the SCV is complex.

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Figure 5.  Single optical section series showing distribution of cholesterol in a wild-type SCV and a three-dimensional space-filling model. RAW 264.7 macrophages were infected with Salmonella (SL1344) (see Experimental procedures). Infected cells were fixed, stained with filipin, and visualized by Deltavision deconvolution microscopy. Serial sections were taken through a vacuole containing 13 bacteria (A), and used to construct a space filling model (B) with the SoftworX modelling software. The plasma membrane (PM) and selected bacteria (#1–5) are indicated. GFP-expressing bacteria are shown in green, and filipin, indicating cholesterol, is shown in red.

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In order to further understand the spatial relationship between the bacteria and cholesterol, the serial optical sections were used to create a three-dimensional space-filling model. The resulting three-dimensional model is shown in Fig. 5B. Two distinct perspectives are presented: the view in Bi was rotated forward by 90° and 180° to the right to create the view shown in Bii; bacteria #4 and #5 cannot be seen in Bi and #3 cannot be seen in panel Bii. The model highlights several important aspects of cholesterol association with the SCV. First, bacteria are always associated with cholesterol. Second, whereas some bacteria (such as #1) are completely surrounded, others (such as #2–5) are embedded to varying degrees in a ‘cloud’ of cholesterol. Importantly, cholesterol appears immediately proximal to large regions of the bacteria and spaces between the bacteria can be completely filled with cholesterol.

Recruitment of the GPI-anchored protein CD55 to the SCV

Cholesterol is important for the function of various protein receptors (Simons and Toomre, 2000). Glycosylphosphatidylinositol (GPI)-anchored proteins are a diverse family that share a conserved C-terminal post-translational lipid modification (Ferguson, 1999). Studies with folate receptor have shown that the GPI anchor is important for selective retention in cholesterol-rich endocytic compartments and for effective cellular uptake of folate (Chatterjee et al., 2001). Although delivery to the early endosome is not cholesterol sensitive, exit to the host plasma membrane from the recycling compartment is facilitated by cholesterol depletion (Mayor et al., 1998). Because proliferation of S. typhimurium results in intracellular cholesterol accumulation, we examined whether the GPI-anchored protein CD55, or the decay-accelerating factor (DAF), that resides in the macrophage plasma membrane associates with the SCV.

RAW 264.7 macrophages were infected with S. typhimurium SL1344, fixed in formaldehyde at 20 h post infection, and processed for immunofluorescence micro-scopy, using an antibody to murine CD55. As shown in Fig. 6A–C, CD55 was recruited to the SCV, suggesting that plasma membrane and/or early endosomal components whose trafficking is regulated by cholesterol may be recruited to the SCV. It is noteworthy that the transferrin receptor, which trafficks through the recycling endosome in a cholesterol-insensitive manner, does not associate with the SCV after the first 20 min of infection (Steele-Mortimer et al., 1999). SCVs containing the mutant ssaT fail to show prominent accumulation of CD55 (Fig. 6D–F). This suggests that the elevated association of CD55 with the SCV is linked to SPI-2 dependent intracellular replication, possibly due to a failure to accumulate elevated cholesterol in the SCV.

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Figure 6.  Distribution of CD55 in macrophages infected with wild-type Salmonella SL1344 (A–C) or mutant ssaT (D–F). At 20 h, infected cells were fixed and stained with anti-CD55 for study by Deltavision deconvolution microscopy (see Experimental procedures). A rhodamine-conjugated donkey anti-goat secondary antibody was used to visualize the anti-CD55 primary antibody. GFP-expressing bacteria are shown in green, CD55 is shown in red, and Hoechst-labelled cell nuclei are shown in blue. White arrows indicate CD55 staining. 0° projections are shown.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Despite the extensive characterization of various host proteins that interact with the Salmonella vacuole, relatively few studies have addressed the role of host lipids during intracellular infection. The filipin staining of the SCV in macrophages and HEp-2 cells that we observed is consistent with previous work by Brumell et al., (2001) showing that filipin stains the 6 h vacuole in HeLa cells. It is not clear why the sterol intermediate lanosterol accumulated, as blocking the synthesis of this precursor had no effect on intracellular survival (data not shown). However, the elevation of lanosterol may reflect Salmonella’s requirement for earlier precursors of the biosynthetic pathway (D. M., Y. Lange, and K. Haldar Catron unpublished). Although the mechanisms are still obscure, our findings suggest that Salmonella infection perturbs sterol metabolism in macrophages.

The accumulation of host cell cholesterol in the SCV is striking. At 20 h post infection the SCV cholesterol constitutes more than 30% of total cell cholesterol. Since ~90% of cell cholesterol normally resides in the plasma membrane (Lange et al., 1989), the intracellular accumulation caused by Salmonella infection suggests that the bacteria affect a major route of cholesterol transport. The major sources of cellular cholesterol are biosynthesis, and LDL-cholesterol released in the lysosomes. Intracellular cholesterol can also be derived from the large plasma membrane pool (Lange et al., 2002). These processes need to be evaluated in detail. Recent studies suggest that endocytic tracers added to cells post infection are not delivered to the SCV (Garcia-del Portillo and Finlay, 1995; Cuellar-Mata et al., 2002). This is consistent with evidence that an effector of the SPI-2 TTSS system, such as SpiC, induces a fusion block in cells (Uchiya et al., 1999). The ssaT mutant used in our studies is defective in the structure of the SPI-2 dependent TTSS and the delivery of effectors into the host cell. That the ssaT mutants do not cause accumulation of cholesterol in the SCV suggests that a SPI-2 TTSS effector may contribute to cholesterol transport in cells. This could be accomplished by directly deflecting host trafficking pathways or by altering aspects of vacuolar architecture that contribute to cholesterol retention in the SCV.

The mechanisms by which Salmonella induces redistribution of cellular cholesterol with accumulation in the SCV remain obscure. Unlike intracellular pathogens like M. tuberculosis (Gatfield and Pieters, 2000) and malaria parasites (Samuel et al., 2001), cholesterol is not required for the infection of Salmonella into host macrophages (Gatfield and Pieters, 2000 and D. M., and K. Haldar Catron unpublished). While this manuscript was in review, Garner et al., (2002) reported evidence for the requirement of cholesterol during infection of epithelial cells. Further, they showed SPI-1 dependent cholesterol redistribution in epithelial cells infected with S. typhimurium. We detect accumulation of cholesterol at 2 h in infected macrophages and have not determined the underlying genetic basis for this effect. Our studies focus on a later, SPI-2 dependent accumulation of cholesterol, and show that the Salmonella-containing vacuole becomes a major site of intracellular cholesterol accumulation and recruits a GPI-anchored protein. In macrophages, cholesterol accumulation is linked to intracellular bacterial replication and maturation of the SCV. The presence of elevated cholesterol in the SCV is unexpected because studies suggest that the vacuole resembles late endosomal compartments, which contain much less cholesterol than their early endosomal or recycling endosomal counterparts. Cholesterol may play a role in nutrient acquisition, as individual bacteria may be more effective at obtaining host metabolites when in close proximity to internal membranes. It may also play a role in bacterial exit, which has not been studied.

Examination of the distribution of cholesterol associated with the SCV yielded a space-filling view of the intracellular bacteria. If the SCV is a vacuole bounded by a single membrane that surrounds intracellular bacteria suspended in the lumenal space, our data suggest that cholesterol deposits within membranes in the SCV. Beuzon et al. (2000) have recently argued that each intracellular bacterium is encapsulated by a tightly associated vacuolar membrane: each vacuolar bacterium then clusters with its nearest neighbours. In the context of this model our data would argue that cholesterol distributes unevenly through multiple SCVs, suggesting that the vacuolar environment is not the same for each bacterium. Alternatively, it is possible that as the SCVs proliferate, they induce cholesterol accumulation in host cell membranes proximal to a vacuolar cluster.

Since cholesterol is an important host component, its redistribution may come at some cost to the host cell. Cholesterol accumulation in mammalian cells has previously been implicated in apoptosis (Yao and Tabas, 2000; 2001). Salmonella infection is known to cause apoptosis in macrophages but not epithelial cells. Thus, our finding that cholesterol accumulates in the SCV in HEp-2 cells as well as in macrophages suggests that the phenomenon is probably not linked to apoptosis. Elevation of cholesterol also induces lipid microdomains, or ‘rafts’, in cells (Simons and Ikonen, 2000). Thus, recruitment of cholesterol may suggest a role for membrane microdomains in SCV signalling, or bringing of other host cell membranes and receptors to the SCV. In this regard, we find that CD55 is recruited to the SCV, providing a novel marker for this compartment. The glycosphingolipid GM1 is also recruited (data not shown), suggesting that multiple raft components may associate with the SCV. The accumulation of CD55 in the SCV is unexpected because the vacuole resembles late endosomal compartments. How-ever, recent studies suggest that association of early endosomal rabs such as 5 and 18 with the SCV occurs during intracellular replication (Hashim et al., 2000). Thus contrary to prevailing models, the SCV may provide insights into trafficking of earlier endocytic compartments. Finally, elevation of cholesterol and its esters induce foam cell formation of macrophages (Schaffner et al., 1980), and are thought to underlie neurodegenerative diseases (Puglielli et al., 2001), suggesting that Salmonella may prove to be a useful tool in dissecting major intracellular pathways of cholesterol transport in host cells.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Materials

4,4,10β-Trimethyl-trans-decal-3β-ol (TMD) was the generous gift of T. A. Spencer (Dartmouth College). Filipin and cholesterol were purchased from Sigma (St Louis, MO). Lanosterol was from Steraloids (Newport, RI). [3H]-mevalonolactone was purchased from PerkinElmer (Boston, MA). Trypan blue was from Life Technologies (Carlsbad, CA). Mouse monoclonal anti-Salmonella typhimurium LPS was from Advanced Immuno-Chemical (Long Beach, CA). Goat anti-CD55 was from Santa Cruz Biotechnologies (Santa Cruz, CA). Rhodamine-conjugated anti-mouse secondary antibody was from ICN Cappell (Aurora, OH) and rhodamine-conjugated donkey anti-goat antibody was from Research Diagnostics (Flanders, NJ).

Growth conditions of bacterial strains and cells

The Salmonella typhimurium wild-type strain SL1344 was used for these experiments. Visualization and selection of this strain was accomplished by the introduction of the plasmid pFPV25.1, which contains gfpmut3A under the control of a constitutive promoter, as well as an ampicillin resistance marker (Valdivia and Falkow, 1996). The phoP (Miller et al., 1989; Beuzon et al., 2001), ompR (Dorman et al., 1989), and ssaT (Hensel et al., 1997; Gallois et al., 2001) mutants have also been described previously. For macrophage infections, Salmonella cultures were grown in LB broth in a shaking incubator overnight, and then subcultured and grown to late log/early stationary phase. Bacteria were opsonized in media containing 20% mouse serum for 30 min before infection. For epithelial cell infections, bacteria were grown in LB containing 0.3 M NaCl without shaking to late log/early stationary phase. Ampicillin (50 μg ml–1), kanamycin (50 μg ml–1), tetracycline (25 μg ml–1) and streptomycin (50 μg ml–1) were used for selection of bacterial strains. RAW 264.7 murine macrophages were from American Type Culture Collection (ATCC) and were maintained in Dulbecco’s modified Eagle medium containing glutamine, glucose, sodium bicarbonate (ATCC), and 10% fetal bovine serum (Gibco) at 37°C and 5% CO2, without antibiotic. HEp-2 cells were maintained in minimal essential media with Earle’s salts (ATCC).

Gentamicin protection assays

RAW 264.7 macrophages were seeded at 3 × 105 per well in a 24-well tissue culture plate, and were allowed to grow for at least 15 h before infection. HEp-2 cells were seeded at 5 × 104 cells/well. Bacterial cultures were diluted in media to allow a multiplicity of infection of 10 bacteria per cell. Once bacteria were added, cells were centrifuged at 1000 g for 5 min and incubated for 20 min at 37°C and 5% CO2. The cells were washed three times with serum-free medium, incubated with 100 μg ml–1 of gentamicin for 90 min, and then maintained in 10 μg ml–1 of gentamicin for the remainder of the experiment. At various time points post infection, the cells were lysed with 1% Triton X-100, and intracellular bacteria were counted as colony-forming units (CFU). For TMD experiments, TMD (12 μg ml–1 in dimethyl sulphoxide) was added to the cells 4 h before infection in order to block host sterol biosynthesis, and was maintained for the duration of the experiment. As a control, dimethyl sulphoxide alone was added to a second set of wells.

Filipin staining

For fluorescence microscopy, cells were grown on 30 mm glass coverslips in 24-well plates. At various time points post infection, cells were washed three times in PBS and then fixed in 4% formaldehyde for 15 min at room temperature. Treatment with 50 mM ammonium chloride for 10 min was used to quench auto-fluorescence. Cells were stained with 100 μg ml–1 of filipin for 20 min, washed in phosphate-buffered saline (PBS), mounted on slides, and sealed with nail polish. Where indicated, anti-LPS (mouse) was used at 1:200 followed by a rhodamine-conjugated goat anti-mouse secondary antibody to distinguish extracellular bacteria.

Indirect immunofluorescence

Cells on glass coverslips were infected with bacteria, fixed and processed for immunofluorescence. Fixation was in 4% formaldehyde, followed by quenching with 50 mM ammonium chloride and permeabilization in PBS containing 0.5% Triton X-100. Blocking was done in PBS containing 0.4% fish skin gelatin. Infected cells were then stained with primary and secondary antibodies, or secondary alone. Primary antibodies were diluted in blocking solution at 1:200. Secondary antibodies, rhodamine-conjugated goat anti-mouse IgG, and rhodamine-conjugated donkey anti-goat IgG were diluted 1:200 in blocking solution. Hoechst staining was done at 10 μg ml–1. Coverslips were mounted onto glass slides using DABCO mounting media, and examined by Deltavision deconvolution fluorescence microscopy using standard UV, FITC and rhodamine filter sets.

Deconvolution fluorescence microscopy

Fluorescence microscopy and digital image collection were performed on an Olympus IX inverted fluorescence microscope and a Photometrix cooled CCD camera (CH350/LCCD) driven by DeltaVision software from Applied Precision (Seattle, WA) as described (Samuel et al., 2001). Briefly, 400 nm optical sections were taken through the depth of the cell, and DeltaVision software (softWoRx) was used to deconvolve these images (Hiraoka et al., 1991). For quantitative projections, the ‘additive’ method of data collection was used. Here, each ray collects and sums data from all of the voxels in its path and scales it down to an appropriate intensity. The data can be used for comparison of intensity in various structures within the image data. Fluorescence quantification was carried out with a 60×, NA 1.4 objective. Data from ten individual cells were analysed, and polygons were drawn to delineate the whole cell, the Golgi and the bacterial vacuole. Total fluorescence intensity, area and pixel numbers associated with the bacteria and cells were determined in 0° projections of Z-sections that spanned the entire thickness of each cell and the thickness of the associated vacuole. Single optical sections were chosen for presentation of images with filipin staining in infected cells. For large-scale analysis of vacuoles, a ‘volume’ method of quantification was used. For this method, a minimum threshold was set to construct polygons around the GFP signal in all optical sections of multiple fields. These polygons were then applied to the filipin signal in all optical sections and used to obtain intensities for SCV-associated filipin.

Radiolabelling, lipid extraction, and HPLC

Cells were labelled in six-well plates (4 × 106 cells per well) using [3H]-mevalonolactone. Radiolabel (25 μCi per well) was added to the cells at 2 h post infection. At various times thereafter, cells were harvested by scraping, pelleted at 1200 r.p.m. for 5 min, and then lysed in sterile water. Separate samples were resuspended in equal volumes of water, and the lipids extracted by the addition of chloroform:methanol (2:1), at a ratio of 5:1 (v/v). The samples were dried under nitrogen, and dissolved in acetonitrile/isopropyl alcohol (90:10 v/v) for analysis by HPLC.

Reverse phase HPLC of extracted lipids was carried out using a Beckman QC 11 system with a model 166 programmable detector and System Gold software. An Ultrasphere ODS reversed-phase column (4.6 × 150 mm) was used with acetonitrile/isopropyl alcohol as the mobile phase at a flow rate of 1.5 ml min–1. The effluent was monitored at 214 nm and fraction collected for the measurement of radioactivity. Samples were run at 30°C. Lanosterol and cholesterol standard retention times were measured at 12.4 and 14.3 s respectively. Protein was determined by the BCA method (Pierce).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We thank members of the Cianciotto and Haldar laboratories at Northwestern University for helpful discussions of the work. This work was supported by grants from the Northwestern University Department of Pathology (K.H.), a National Institutes of Health Cellular and Molecular Basis of Disease Predoctoral Training Grant 5T32GM08061 (D.C.), and a National Institutes of Health grant HL28448 (Y.L.).

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  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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