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Summary

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

Autophagy is an important constitutive cellular process involved in size regulation, protein turnover and the removal of malformed or superfluous subcellular components. The process involves the sequestration of cytoplasm and organelles into double-membrane autophagic vacuoles for subsequent breakdown within lysosomes. In this work, we demonstrate that the intracellular pathogen Listeria monocytogenes can also be a target for autophagy. If infected macrophages are treated with chloramphenicol after phagosome lysis, the bacteria are internalized from the cell cytoplasm into autophagic vacuoles. The autophagic vacuoles appear to form by fusion of small cytoplasmic vesicles around the bacteria. These vesicular structures immunolabel with antibodies to protein disulphide isomerase, a marker for the rough ER. Internalization of metabolically arrested cytoplasmic L. monocytogenes represents an autophagic process as the vacuoles have double membranes and the process can be inhibited by the autophagy inhibitors 3-methyladenine and wortmannin. Additionally, the rate of internalization can be accelerated under starvation conditions and the vacuoles fuse with the endocytic pathway. Metabolic inhibition of cytoplasmic bacteria prevents them from adapting to the intracellular niche and reveals a host mechanism utilizing the autophagic pathway as a defence against invading pathogens by providing a route for their removal from the cytoplasm and subsequent delivery to the endocytic pathway for degradation.


Introduction

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

Many bacteria have evolved mechanisms for intracellular survival after invasion of mammalian cells. Following phagocytosis, bacteria become sequestered in membrane-bound organelles, called phagosomes, which undergo a process of maturation that involves acidification and several fusion events. The phagocytic pathway is recognized to be an important component of host defence against microorganisms and many microbes are killed and degraded in this compartment. However, some intracellular pathogens block or alter the maturation of the phagosome and remain in vacuoles that neither acidify nor fuse with lysosomes, while others escape into the cytoplasm (Sinai and Joiner, 1997). The outcome of an infection is determined both by the invading organism and the defence mechanisms of the host cell. Multiple signalling events in the host cell can be triggered or modulated at different stages of infection, and can include stress and pro-inflammatory responses, even cell death.

Intracellular pathogens are known to modify their environment in multiple ways to avoid destruction by innate host cell defence mechanisms, while exploiting the natural cellular physiology in their favour. The most successful pathogens are those that strongly interfere with host cell functions. Less virulent variants fail to thrive and are presumably overcome by host cell defence mechanisms. A subgroup of vacuolar pathogens, including Legionella pneumophila (Swanson and Isberg, 1995), Brucella abortus (Pizarro-Cerda et al., 1998a, b), and Porphyromonas gingivalis (Dorn et al., 2001), appear to infiltrate the autophagic pathway in order to avoid host cell defences and in some instances, to gain access to the ER. Once within this compartment, these bacteria may modify the pathway – perhaps by preventing maturation of autophagosomes, in order to establish an environment necessary for replication and survival. The intracellular route of these organisms is difficult to study however, and recent evidence suggests that L. pneumophila may be utilizing alternative pathways to gain eventual access to the ER (Tilney et al., 2001).

Autophagy is a bulk protein degradation process where cytoplasmic components, including organelles, become enclosed by double-membrane structures termed autophagic vacuoles (AVs). These fuse with the endocytic pathway (Dunn, 1990; Seglen and Bohley, 1992) and their contents are eventually degraded in lysosomes. Autophagy is essential for cells to survive during nutrient limitation and may be crucial for cellular remodelling during development and differentiation (Bursch 2001). By keeping autophagy under control, cells are able to modulate their protein mass (Canuto et al., 1993) as well as remove malformed subcellular components (Lardeux and Mortimore, 1987; Luiken et al., 1992). Dysfunction of autophagy has been linked to the aetiology of cancer (Kisen et al., 1993; Bursch et al., 1996; Liang et al., 1999), and an increasing number of genetic (Notterpek et al., 1997; Kalimo et al., 1988), and prion-related (Boellaard et al., 1989; 1991) diseases. Recent molecular genetic studies in yeast have identified at least 15 autophagy-specific genes and provided a detailed understanding of the molecular components involved in the induction of autophagy and AV formation, docking and fusion; mammalian homologues of these genes are in the process of being identified (see Klionsky and Emr 2000; Abeliovich and Klionsky 2001;Reggiori and Klionsky 2002 for reviews).

Morphological and biochemical studies have shown that autophagy is a multistep process. The first step is the sequestration of various organelles (including mitochondria, peroxisomes) and cytoplasmic components into a double-membrane AV. This sequestration can be inhibited by 3-methyladenine (Seglen and Gordon, 1982; Blommaart et al., 1997a), which has been shown to inhibit the class III phosphoinositide 3-kinase (PI3-kinase), and by wortmannin, a general inhibitor of PI3-kinases (Petiot et al., 2000). The AVs then fuse with endocytic organelles, the inner membrane is degraded and finally the sequestered cellular components are degraded in an autolysosome (Punnonen et al., 1993). The autophagic pathway is understood to be ATP dependent and is tightly regulated by amino acids and hormones, being dramatically stimulated by starvation (Blommaart et al., 1997b).

However, prominent gaps remain in our understanding of autophagy in mammalian cells. For example, the origin of the membranes used to assemble AVs is still unresolved. Various sources have been proposed, including the rough ER (Dunn, 1990), a post-Golgi compartment ( Yamamoto et al., 1990), or a novel compartment (Stromhaug et al., 1998). In addition, there is no definitive information on how cells recognize specific cargo as targets for autophagy, partially a result of the difficulty in identifying newly forming AVs in the cell cytoplasm. The process by which cells are able to identify targets for autophagy was once thought to be a non-selective process. However, it is clear that in some instances, cells can selectively target superfluous molecules or organelles for degradation (Dunn, 1994; Kim and Klionsky, 2000; Stromhaug et al., 2001). Indeed, autophagy is the only mechanism for the turnover of organelles including mitochondria and peroxisomes. As autophagy is a degradative pathway, it is possible that cells might also use this pathway as a defensive mechanism against invading pathogens, in addition to the well-characterized phago-lysosomal route.

A group of bacteria that include Listeria, Shigella and Rickettsiae are known to enter cells by phagocytosis and then break out from the phagosome into the cytoplasm where they replicate (Andrews and Webster, 1991; Meresse et al., 1999). Avirulent mutants of these bacteria do not generally escape from the phagosome, and are degraded by the phago-lysosomal pathway. One member of this group, Listeria monocytogenes, is a Gram-positive bacterium with a well-documented invasion process into mammalian cells. After entering cells in a phagosome, the bacteria use a thiol-activated haemolysin to disrupt the phagosome membrane and enter the cytoplasm. In the cytoplasm, the bacteria use a protein product of the actA gene to recruit host actin and form comet-shaped tails (Tilney and Portnoy, 1989;Beauregard et al., 1997). This allows them to move throughout the host cell and eventually into adjacent cells. Mutant forms of L. monocytogenes, lacking the actA gene, have the ability to escape from the phagosome and can multiply within host cells. However, they are defective in intra- and intercell spread and have reduced virulence in vivo (Brundage et al., 1993).

Once the Listeria escape into the cytoplasm after disrupting the phagosome membrane, they counteract antimicrobial host cell defences and begin to grow and divide using the host cell's nutrients. Tilney et al. (1990) found that treating Listeria-infected J774 cells with chloramphenicol (a specific inhibitor of bacterial protein synthesis) could inhibit bacterial growth and the process of actin filament polymerization. While examining cytoplasmic L. monocytogenes treated with the antibiotic chloramphenicol, we discovered that, over time they became re-enclosed within vacuoles. We demonstrate that this internalization of metabolically arrested cytoplasmic L. monocytogenes into vacuoles represents an autophagic process because the vacuoles have double membranes and the process can be inhibited by classic autophagy inhibitors. In addition, these vacuoles fuse with the endocytic pathway and the time course of internalization is accelerated under starvation conditions. Thus preventing cytoplasmic bacteria from adapting to the intracellular niche may enable the host cell to utilize the autophagic pathway for their eventual destruction.

Results

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

Growth of wild-type and ΔactA L. monocytogenes in J774 cells

The mouse macrophage-like J774 cell line is a convenient host for the intracellular pathogen L. monocytogenes. The bacteria are rapidly phagocytosed and, commencing within 20 min after initial exposure, the phagosomal membrane is dissolved by secreted Listeriolysin. The bacteria then enter the cytoplasm where they recruit host actin to gain motility and move from cell to cell (Tilney and Portnoy, 1989). For these studies, we have used both wild-type and a mutant form of L. monocytogenes (ΔactA) that can grow and multiply normally within host cells but is defective in intra-and intercellular spread (Brundage et al., 1993). The mutant bacteria dissolve the phagosomal membrane and enter the cell cytoplasm just like the wild type, but are immobilized in the cell cytoplasm by their inability to polymerize host actin.

Quantitative examination by light microscopy of J774 cells exposed to L. monocytogenes suggested that there was no difference in binding and uptake between wild-type and ΔactA bacteria during the early stages of association. In these first experiments, gentamicin was used to eliminate extracellular bacteria but the cultures were not exposed to any other antibiotics. Approximately 10% of the cells were infected after a 1 h exposure to either wild-type or ΔactA bacteria (Table 1). Differences between the wild type and ΔactA bacteria were observed only when cells were incubated with viable bacteria for longer periods. For wild-type bacteria, the percentage of infected J774 cells increased to approximately 30% after 6 h incubation, while the percentage of cells containing ΔactA bacteria remained unchanged at 10% (Table 1). The distinct growth patterns of the wild type and ΔactA bacteria within J774 cells were also revealed by examination of Giemsa-stained preparations. At 6 h after infection, wild-type bacteria were observed scattered throughout the cell and at the ends of thin processes extending out of and away from the cell (Fig. 1A). Frequently, groups of adjacent cells contained bacteria. In contrast, although numerous ΔactA bacteria could be seen clustered together within a single cell, this was usually only in one part of the cell (Fig. 1B), adjacent cells rarely contained bacteria. This data confirms the inability of ΔactA mutant bacteria to spread from cell to cell.

Table 1. . Comparison of cell-to-cell spread between wild type and ΔactA Listeria monocytogenes.
BacteriaTime (h)Infected cells (%)
  1. J774 macrophage-like cells were exposed to wild-type or ΔactA mutant bacteria for 1 h. Cells were either fixed immediately or incubated for a further 5 h without the addition of chloramphenicol (= 6 h after infection). The percentage of cells with associated bacteria (one or more) was quantified in Giemsa-stained preparations. For the 6 h time point, data from two separate experiments are presented.

ΔactA1 8.4
ΔactA6 8.3
ΔactA6 9.1
wt110.8
wt628.6
wt632.0
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Figure 1. Wild-type and ΔactA L. monocytogenes exhibit distinct growth patterns in J774 cells. Cells were exposed to bacteria for 1 h and then incubated for a further 5 h (without the addition of chloramphenicol) (= 6 h after infection) and fixed for microscopy. A and B. In Giemsa-stained preparations, the wild-type bacteria (A) can be seen scattered throughout the cell and in adjacent cells, whereas the ΔactA bacteria (B) are clustered together within a few cells. Wild-type bacteria infect neighbouring cells via cytoplasmic projections, but are not extracellular. C and D. F-actin was visualized by staining with rhodamine-conjugated phalloidin (in red) and bacteria were detected by indirect immunofluorescence using polyclonal anti-L. monocytogenes primary antibodies followed by FITC-conjugated secondary antibodies (in green). Cells infected with wild-type bacteria contained comet tail-like structures of polymerized actin that co-localized with bacteria (C, arrowheads). In contrast, cells infected with ΔactA mutant bacteria were aggregated within single cells (arrow) and no co-localization with F-actin is observed (D). Bar, 10 µm.

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To confirm that the ΔactA bacteria lacked the ability to polymerize actin in the cytoplasm of J774 cells, we visualized F-actin in cells containing the wild-type or ΔactA mutant bacteria using rhodamine-conjugated phalloidin. As expected, after 6 h incubation with wild-type bacteria, the J774 cells contained comet-tail structures that co-localized with approximately 30% of the bacteria labelled by an anti-Listeria antibody (Fig. 1C). These labelled bacteria were scattered throughout the J774 cells in a pattern similar to that observed in the Giemsa-stained preparations (Fig. 1C). By comparison, the ΔactA bacteria were aggregated within single cells and no evidence of cell-to-cell spread was observed (Fig. 1D). No association of phalloidin with the ΔactA bacteria was detected.

Escape of bacteria from phagosomes and re-entry of cytoplasmic bacteria into vacuoles after chloramphenicol treatment

Bacteria within J774 cells were easily identified in thin sections prepared for transmission electron microscopy (TEM). When cells were first exposed to L. monocytogenes, the bacteria were rapidly internalized and within 5 min were observed in phagosomes, a structure with a single enclosing membrane (Fig. 2A). After 3 h most of the bacteria had escaped from the phagosome and were found free in the cell cytoplasm (Fig. 2B).

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Figure 2. L. monocytogenes is phagocytosed by J774 cells, escapes into the cytoplasm, but re-enters vacuoles with double membranes after chloramphenicol treatment. Electron micrographs of cells containing internalized ΔactA bacteria. Scale bars, 0.5 µm. A. At 5 min after infection, individual bacteria (B) can be seen within phagosomes. The phagosome is formed from a single enclosing membrane (arrow). B. Three hours after infection, almost all of the bacteria (B) have escaped from the phagosome and are free in the cell cytoplasm. There is no evidence of actin polymerization around the mutant bacterium. Profiles of rough endoplasmic reticulum (RER, arrowhead) are in close proximity. C. Cells treated with chloramphenicol beginning at 3 h after infection and then incubated for an additional 3 h show intracellular bacteria (B) in vacuoles with double membranes (arrows). RER profiles are close to these membranes (arrowhead).

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We then found that if chloramphenicol was added to the cultures at 3 h after initial infection with wild-type or ΔactA bacteria (when the majority of bacteria were free in the cytoplasm), and incubated at 37°C for a further 3 h, many bacteria were observed to have re-entered vacuoles that had a double membrane (Fig. 2C). With longer periods of chloramphenicol treatment, increasing numbers of bacteria were seen in these vacuoles. As vacuoles with double membranes are a defining characteristic of autophagic vacuoles (Stromhaug et al., 1998), this prompted us to consider whether internalization of metabolically arrested Listeria might be a form of autophagy. Although this phenomenon was also observed with wild-type bacteria, to ensure that we were not examining the unrelated process of cell-to-cell spread, which involves structures that are morphologically similar to autophagosomes (Gedde et al., 2000), we chose to focus our studies on the ΔactA mutant. The remainder of the studies presented were obtained with the ΔactA mutant.

Quantification of the proportion of bacteria within vacuoles or in the cell cytoplasm, assessed by examination of thin sections in the TEM at different times after infection, showed that the percentage of bacteria found within vacuoles drops steadily from 1 to 3 h, such that at 3 h more than 95% of bacteria are free in the cytoplasm (Fig. 3). When examined at 1 h after initial infection, 40% of the intracellular bacteria were found in vacuoles. After 2 h, 20% of the bacteria were found in vacuoles and after 3 h, only 5% of the intracellular bacteria were in vacuoles; the remaining 95% were free in the cell cytoplasm. Thus while internalization is rapid, escape from the phagosome occurs gradually, but is essentially complete at 3 h. At 3 h after initial exposure (designated t = 0 h), when most of the bacteria were free within the cell cytoplasm, the cells were treated with chloramphenicol (indicated by an arrow on Fig. 3). Following treatment with chloramphenicol, a steady increase in the percentage of bacteria found within vacuoles was observed. After 6 h chloramphenicol treatment, 40% of the bacteria were enclosed in vacuoles. At 16 h the number had increased to 70%, whereas at 21 h greater than 90% of the bacteria had left their cytoplasmic location and now were re-enclosed within vacuoles (Fig. 3).

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Figure 3. Time course of escape of L. monocytogenes from phagosomes and subsequent re-entry into vacuoles.J774 cells were infected with Δacta bacteria and then treated with chloramphenicol, beginning at 3 h. Cells were fixed and processed for TEM at various times up to 24 h after infection. Time of addition of chloramphenicol is designated as t = 0 (denoted by the arrow). The number of bacteria enclosed within vacuoles or free in the cell cytoplasm was quantified by examination of thin sections in the TEM. From 1 to 3 h after infection, the number of intracellular bacteria found in vacuoles declined to very low levels. After chloramphenicol treatment, the number of bacteria in vacuoles increased dramatically, reaching 92% by 21 h. Data represent the mean ± SE of three separate experiments.

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Vacuole assembly around cytoplasmic bacteria

At 6 h after chloramphenicol treatment, bacteria within the cells were observed both in the cytoplasm, in vacuoles and also in different stages of what appeared to be internalization into vacuoles. These latter bacteria were surrounded by small vesicular structures and other membranes suggestive of various stages of vacuole formation. The initial step for this assembly process appeared to consist of an accumulation of small vesicles with variable morphology in the region around the bacterial surface (Fig. 4A). This was followed by an apparent fusion of the membranes to form larger membrane structures attaching to the bacterial surface (Fig. 4B). Eventually, larger, cisternal structures were detectable around the cytoplasmic bacteria, sometimes also engulfing other cytoplasmic components (Fig. 4C) until the bacteria were observed surrounded by a continuous double membrane (Fig. 4D).

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Figure 4. Stages of vacuole assembly around cytoplasmic L. monocytogenes. Electron micrographs of J774 cells infected with Δacta bacteria and then treated with chloramphenicol for 6 h. Bars, 0.5 µm. A. Cytoplasmic bacterium (B) with small membrane structures (arrows) in close proximity. B. Cytoplasmic bacterium (B) with a large membrane structure associated with its surface and apparently in the process of zippering around the bacterium (asterisk). Small vesicles (arrows) are in close proximity. C. An intracellular bacterium (B) that is almost enclosed by double membrane profiles. These profiles appear to be enclosing the bacterium and associated cell cytoplasm. Small channels (arrowheads) prevent the membranes from completely isolating the bacterium from the main body of the cell. D. An intracellular bacterium (B) enclosed within a double membrane vacuole (arrows).

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Inhibitors of autophagy reduce the entry of cytoplasmic bacteria into vacuoles

Inhibitors of PI3-kinase, such as 3-methyladenine and wortmannin, have been shown to inhibit the sequestration step of autophagy in a number of mammalian systems (Seglen and Gordon, 1982; Blommaart et al., 1997a). We therefore tested the effect of these inhibitors on the re-internalization of bacteria into vacuoles in J774 cells. Treatment of cells containing cytoplasmic bacteria with 3-methyladenine resulted in a marked reduction in the number of bacteria that entered vacuoles (Fig. 5A). Addition of 3-methyladenine to the culture media, applied at time t = 0 h along with chloramphenicol, resulted in a six- to eightfold decease in the number bacteria in vacuoles after 6 and 9 h of incubation. At the ultrastructural level, the cells and bacteria treated with 3-methyladenine had a similar morphology to untreated cells. Cytoplasmic bacteria were observed close to the cell nucleus, with small amounts of RER and vesicular membrane profiles nearby (Fig. 5B and C).

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Figure 5. 3-Methyladenine inhibits the entry of cytoplasmic L. monocytogenes into vacuoles. J774 cells were infected with Δacta bacteria and then treated concomitantly with 3-methyladenine and chloramphenicol for 6 h. A. Quantification of the percentage of intracellular bacteria found within vacuoles in J774 cells in the presence (+) or absence (–) of 10 mM 3-methyladenine, an inhibitor of autophagy. Cells were fixed and examined by TEM at 6 h and 9 h after exposure to chloramphenicol. Data represent the mean ± SE of three separate experiments. A 6 h treatment with 10 mM 3-methyladenine markedly reduced the percentage of bacteria in vacuoles. B. Electron micrograph through infected J774 cells. Cytoplasmic bacteria (B) are in close proximity to the cell nucleus (N), profiles of rough endoplasmic reticulum (RER) and membrane organelles (arrow). C. Electron micrograph through infected J774 cells. In close proximity to the bacterium (B) are small, electron lucent vesicles (arrow) and small electron opaque vesicles (arrowhead). Also present is the cell nucleus (N) and elements of RER. Bars, 0.5 µm.

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Similarly, treatment of cells containing cytoplasmic bacteria with wortmannin resulted in a reduction in the number of bacteria that entered vacuoles. At 3 h after chloramphenicol, approximately 20% of the bacteria were in vacuoles. However, in cells treated with 100 nM wortmannin, less than 10% of the bacteria had entered vacuoles. At the 6 h time point, with this same concentration of wortmannin, bacterial entry into vacuoles was reduced by more than 50% (Fig. 6A). A lower concentration of wortmanin (20 nM) only partially inhibited entry of bacteria into vacuoles. Wortmannin treatment (100 nM) was also able to inhibit the entry of cytoplasmic bacteria into vacuoles in cells that were deprived of serum (Fig. 6B).

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Figure 6. High wortmannin concentrations inhibit the entry of cytoplasmic L. monocytogenes into vacuoles, whereas low concentrations result in an accumulation of vesicles around the bacteria. J774 cells were infected with Δacta bacteria and then treated concomitantly with wortmannin and chloramphenicol for 6 h. Wortmannin (100 or 20 nM) was replenished hourly. A and B. Quantification of the percentage of intracellular bacteria found within vacuoles in J774 cells in the presence or absence of wortmannin (WTM). Time points indicate the times after addition of chloramphenicol at which cells were fixed for TEM examination. Data represent the mean ± SE of three separate experiments. A 3 or 6 h treatment with 100 nM wortmannin reduced the percentage of bacteria found within vacuoles (A). The inhibitory effect was reduced if cells were treated with 20 nM wortmannin. The effect of a 6 h treatment with 100 nM wortmannin in serum-deprived cells (B). The percentage of bacteria found in vacuoles was reduced in the presence of 100 nM wortmannin. C and D. Electron micrographs through infected J774 cells treated with 20 nM wortmannin. The intracellular bacterium, B, appears to have normal morphology and is close to the cell nucleus, N (C). The cell cytoplasm around the bacterium is filled with small vesicular profiles. At higher magnification, the vesicular profiles surrounding the cytoplasmic bacterium, B, appear to have two forms, with contents that are either electron opaque (arrowhead) or electron lucent (arrows) (D). Bars, 0.5 µm.

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Ultrastructurally, the cells showed interesting morphological features when treated for 6 h with 20 mM wortmannin. The cytoplasmic bacteria had many small, electron-opaque and electron-lucent membrane structures clustered around them (Fig. 6C and D). These features are strikingly reminiscent of the small vesicular structures that possibly represent an early stage in the membrane assembly process around the cytoplasmic bacteria (Fig. 4A). Such an accumulation of small vesicular structures was not observed in cells treated with the higher (100 nM) concentration of wortmannin (data not shown).

The rough ER marker protein disulphide isomerase is localized to small vesicular structures around cytoplasmic bacteria

Whereas the precise origin of the membranes that form the AV is not yet established, it has been proposed that they may originate from ribosome-free regions of the ER (Dunn, 1994). Protein disulphide isomerase (PDI) is an abundant luminal protein of the rough ER and an established marker for this organelle (Noiva et al., 1991). Here we use anti-PDI antibodies to demonstrate an accumulation of PDI-positive vesicular structures around cytoplasmic bacteria during the early stages of autophagic vacuole formation (Fig. 7A–C), but not at later stages (Fig. 7D).

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Figure 7. The rough ER marker protein disulphide isomerase (PDI) is localized to small vesicular structures around cytoplasmic bacteria. Anti-PDI labelling of ultrathin thawed cryosections through J774 cells infected with Δacta bacteria and treated with chloramphenicol for either 9 h (A–C) or 24 h (D). Bar, 0.5 µm. A. No co-localization of PDI with cytoplasmic bacteria, B. B. PDI-positive vesicles in close proximity to cytoplasmic bacteria (arrows); anti-PDI also labels the nuclear envelope (arrowheads). C. Large number of PDI-positive vesicular structures around bacterium, with some adjacent to the bacterial surface (arrow). D. The anti-PDI antibody labels the rough ER (arrows), but there is little or no labelling of the vacuole membrane (arrowheads) enclosing the bacterium, B.

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Depriving cells of serum increases the internalization rate of cytoplasmic bacteria

Nutrient or serum deprivation has previously been shown to act as a stimulus for autophagy, causing AVs to appear within 5–10 min of withdrawal, maturing into autolysosomes 10–20 min later (Mortimore et al., 1989; Dunn, 1990). When J774 cells were loaded with cytoplasmic bacteria and then chloramphenicol-treated in serum-free media, many bacteria were found enclosed within vacuoles with double membranes after only 3 h (Fig. 8A). Quantification of this phenomenon suggested that entry of bacteria into vacuoles was occurring at an increased rate. After 6 h exposure to chloramphenicol the percentage of bacteria in vacuoles was estimated and the results compared with those obtained from cells exposed to 10% serum. In the presence of 10% serum approximately 40% of the bacteria were in vacuoles (Fig. 8B). In contrast, when serum was omitted from the culture medium approx. 70% of the bacteria had entered vacuoles. This suggests that serum deprivation accelerates the rate at which cytoplasmic bacteria re-enter vacuoles.

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Figure 8. Depriving cells of serum increases the internalization rate of cytoplasmic L. monocytogenes. A. Cells grown in the absence of serum internalize cytoplasmic bacteria into double membrane vacuoles. The cells were fixed 3 h after exposure to chloramphenicol and examined by TEM. This micrograph shows a bacterium, B, enclosed by a double membrane (arrows). Other membrane profiles are present in the vacuole. Bar, 0.5 µm. B. Quantification of the percentage of intracellular bacteria found within vacuoles in J774 cells maintained in the presence or absence of serum. The cells were fixed 6 h after exposure to chloramphenicol and examined by TEM. Data represent the mean ± SE of three separate experiments.

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Reinternalized bacteria encounter the endocytic pathway

Autophagy has been shown to be a multistep process, the first step being the formation of a double-membrane AV. After fusion with endocytic organelles, the inner membrane is degraded followed by degradation of the sequestered cellular components in an autolysosome (Punnonen et al., 1993). Quantification of the number of bacteria within vacuoles at different times after chloramphenicol treatment revealed that at 6 h, bacteria were almost equally distributed within single and double-membrane vacuoles (27% versus 29%), with 44% of bacteria being free in the cytoplasm. At 18 h after chloramphenicol, the proportion of bacteria within single-membrane vacuoles had increased dramatically, to 70%, whereas only 3% were in double-membrane vacuoles. A further 27% were found free in the cytoplasm. This result suggests that the vacuole enclosing the bacteria initially forms as a double-membrane structure but matures into a single-membrane vacuole.

At later times (18–21 h) after chloramphenicol treatment, many of the internalized bacteria were observed within vacuoles with only a single enclosing membrane that also contained multivesicular structures (Fig. 9A), suggesting that these organelles were part of the endocytic pathway. To further examine the possibility that the internalized bacteria were meeting the endocytic pathway, J774 cells with intracellular bacteria that had been treated with chloramphenicol for 2 or 24 h were fixed and then double-labelled with antibodies to L. monocytogenes and to LAMP 1, a marker for late endocytic compartments (Fig 9B and C). Co-localization of LAMP 1 with the bacteria was quantified by light microscopy. Between 2 and 24 h, there was a threefold increase in the number of bacteria that co-localized with LAMP 1, although the number of bacteria per cell remained unchanged (Table 2). These results indicate that the majority of intracellular bacteria are delivered to LAMP 1-containing organelles by 24 h.

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Figure 9. Re-internalized bacteria meet the endocytic pathway. J774 cells were infected with Δacta bacteria and then treated with chloramphenicol for 24 h. The cells were fixed and prepared for TEM or light microscopy. A. Electron micrograph through an infected J774 cell shows a bacterium, B, enclosed by a single membrane (arrows). Multivesicular structures (arrowhead) are also present in the vacuole, suggesting that this organelle is part of the endocytic pathway. Bar, 0.5 µm. B and C. Double-labelling of infected J774 cells with anti-LAMP 1 (B) and anti-L. monocytogenes (C). Labelling was detected by indirect immunofluoresence following sequential staining with specific primary antibodies and fluorescent-conjugated secondary antibodies. LAMP 1 labelling (B, arrows) co-localizes with intracellular bacteria (C, arrows). Bar, 10 µm.

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Table 2. . Co-localization of intracellular L. monocytogenes with LAMP 1.
Time after chloramphenicol (h)Bacteria per cellLAMP 1 co-localization (%)
  1. J774 cells were infected with Δacta bacteria and then treated with chloramphenicol for 2 or 24 h. The cells were fixed and double-labelled with anti-L. monocytogenes and anti-LAMP 1 antibodies. Co-localization of the two antibodies was quantified. Whereas the number of bacteria that co-localized with the late endosomal/lysosomal marker LAMP 1 increased with time, the number of bacteria per cell remained the same through the 24 h period indicating that bacterial growth was being inhibited. Data represent the mean ± SE of three separate experiments.

21025.1 ± 4.7
2410.279.2 ± 8.5

Discussion

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

Autophagy is the major inducible pathway for the general turnover of cytoplasmic components, and the only mechanism for the turnover of organelles including mitochondria and peroxisomes (Dunn, 1994; Kim and Klionsky 2000). The identification of autophagy by a combination of morphological and biochemical means is possible in mammalian cells because of the unique characteristics associated with this process. Autophagy is generally considered to be occurring if the following criteria are met: (i) if cytoplasmic components are taken into double-membrane vacuoles (Stromhaug et al., 1998); (ii) if the process is slowed or stopped using inhibitors of autophagy (Seglen and Gordon, 1982); (iii) if the process is stimulated by starvation (Mortimore et al., 1989; Susan and Dunn 2001), and (iv) if vacuoles containing engulfed cytoplasmic material fuse with the endocytic pathway (Tooze et al., 1990; Punnonen et al., 1993; Liou et al., 1997; Lucocq and Walker, 1997).

Our initial observations that cytoplasmic L. monocytogenes, when immobilized by treatment with chloramphenicol, were taken into vacuoles with double membranes, led us to consider the possibility that internalization of metabolically arrested L. monocytogenes might be a form of autophagy. As it has been established that cell-to-cell spread of wild-type Listeria also results in the formation of double-membrane structures around the bacteria (Tilney and Portnoy, 1989), we needed to carefully evaluate our observations. However, although these structures are morphologically similar to autophagosomes, they are actually vacuoles consisting of plasma membrane from two cells (Gedde et al., 2000). Whereas internalization of metabolically arrested Listeria into double-membrane vacuoles was also observed with wild-type bacteria, to ensure that we were not examining the unrelated process of cell-to-cell spread, we chose to focus our studies on the ΔactA mutant. This mutant form of L. monocytogenes grows and multiplies normally within host cells but is defective in actin recruitment and intra- and intercellular spread (Brundage et al., 1993).

We confirmed that little or no cell-to-cell spread of the ΔactA mutant was occurring by examining the percentage of cells infected over the period of time in which large numbers of bacteria were associating with cells. Exposure of J774 cells to L. monocytogenes results in rapid binding and internalization of the bacteria into the cells (Webster, 2002). The results reported here support earlier findings for the behaviour of the ΔactA mutant (Brundage et al., 1993). In addition, we have directly quantified the cellular distribution of wild-type and mutant bacteria. By labelling cytoplasmic bacteria with phalloidin, we confirmed that actin polymerization was not occurring around the ΔactA bacteria. The wild-type bacteria when in the cell cytoplasm clearly showed specific phalloidin association typical of the actin rockets that have been previously described (Tilney and Portnoy, 1989). The ΔactA bacteria had undetectable levels of phalloidin binding associated with them.

Having excluded the possibility that bacteria found enclosed in vacuoles with double membranes were a result of cell-to-cell spread, we examined the effect of 3-methyladenine and wortmannin, two established inhibitors of autophagy, on the internalization of cytoplasmic bacteria. Treatment with either inhibitor led to a marked reduction in the percentage of internalized bacteria, thus linking the internalization process with autophagy. These autophagy inhibitors have been shown to inhibit the sequestration of cytoplasmic components and organelles (Blommaart et al., 1997a). Interestingly, numerous small vesicular structures were observed around the cytoplasmic bacteria when cells were treated with a low concentration of wortmannin, similar to what was observed in cultures without inhibitor after about 3 h of chloramphenicol treatment. It is known that the effects of wortmannin are reversible (Kovacs et al., 2000; Petiot et al., 2000) Thus it is possible that the lower concentration of wortmannin results in a partial inhibition of AV formation and that the small vesicular structures represent an early stage of AV formation. Combined, all of these observations suggest that AVs may assemble by fusion of small cytoplasmic vesicles around the target destined for autophagic sequestration, rather from any preformed organelle. Interestingly, previous studies have described lipid-rich precursor structures in the cytoplasm of cells undergoing autophagy and suggested that double-membrane AVs may form by elongation and closure of this ‘isolation membrane’, thus sequestering cytoplasmic components (Mizushima et al., 2001).

The dramatic difference between the two main hypotheses being proposed to explain the origin of AVs may be attributable to different cell systems being studied, or to the stages of AV maturity being examined by different investigators. The observation that purified AVs contain no markers typical for rough ER, endosomes or lysosomes, led to the proposal that AVs are comprised of unique, pre-existing organelles called phagophores (Stromhaug et al., 1998). On the other hand, several studies localized ER proteins to nascent AVs (Reunanen and Hirsimaki, 1983; Dunn, 1990; Furuno et al., 1990; Ueno et al., 1991), which led to the proposal that AVs form by invagination of cisternae from the ER (Dunn, 1994). Our own observations of the ER marker PDI in association with the early stages of AV formation supports the concept that AV membranes may originate from elements of the rough ER. However, the absence of PDI labelling at later stages suggests that the presence of ER proteins in AV membranes may be a transient phenomenon. The model system described here, using metabolically arrested bacteria as targets for autophagy, may provide a novel approach for directly examining the cellular events of AV assembly.

It is well-established that the autophagic pathway eventually meets the endocytic pathway (Tooze et al., 1990; Liou et al., 1997; Lucocq and Walker, 1997). In order to determine whether the vacuoles containing internalized bacteria fused with the endocytic pathway we examined intracellular bacteria to see if there was an increase in the co-localization with LAMP 1, an established marker of late endocytic organelles. After a short incubation with choramphenicol, few intracellular bacteria were co-localized with the LAMP 1 marker. However, after 24 h incubation the majority of intracellular bacteria had LAMP 1 associated with them. In addition, the majority of bacteria were within vacuoles with only a single enclosing membrane (later stage AV), many of which also contained multivesicular structures. These data strongly suggest that the bacteria were delivered to the endocytic pathway during this 24 h time period.

The internalization of L. monocytogenes from the cell cytoplasm is a process with characteristics that strongly link it to autophagy. The reason that metabolic inhibition of the bacteria by treatment of the cells with chloramphenicol results in the bacteria becoming targets for autophagy is unknown at present, but understanding the mechanism may shed some light on the general process by which a cell distinguishes cargo destined for the autophagic pathway. The selectivity of the sequestration event has been the subject of some debate as there is evidence for both selective and non-selective uptake of cellular components into AVs (Lardeux and Mortimore, 1987; Kopitz et al., 1990). For example, peroxisomes are selectively removed from the cell by autophagy after cessation of drug treatment (Luiken et al., 1992; Kondo and Makita, 1997) as are mitochondria during apoptosis (Xue et al., 2001). Perhaps metabolically inhibited cytoplasmic bacteria are targeted for degradation by the cell in a similar manner to non-functional or damaged cellular organelles.

In conclusion, the present studies have revealed the autophagic pathway as another possible host defence mechanism against invading pathogens. Whereas the cytosol has been considered to be permissive for the growth of bacteria, recent evidence suggests the existence of innate cytosolic host surveillance mechanisms (O’Riordan and Portnoy 2002; O’Riordan et al., 2002). Virulence factors produced by the bacteria can induce or repress the expression of specific host genes (Cohen et al., 2000; Goebel and Kuhn 2000). However, if cytoplasmic bacteria are prevented from adapting to the intracellular niche (by antibiotic treatment, for example), this may enable the host cell to access the autophagic pathway as a route for their removal from the cytoplasm and subsequent delivery to the endocytic pathway for degradation.

Experimental procedures

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

Reagents

Chemical fixatives, most resins and materials used for processing cells for electron microscopy were obtained from Electron Microscopy Sciences (Ft Washington, PA). Eponate 12, a substitute for Epon was purchased from Ted Pella (Redwood, CA). Tissue culture supplies were obtained from Cellgro (through VWR Scientific) and all other chemicals were obtained from Sigma Chemical Co (St Louis, MO).

Cells and bacteria

The macrophage-like cell line J774A.1 (TIB 67) was obtained from ATCC (Manassa, VA). The cells were maintained in Dulbecco's Minimal Essential Medium (DMEM) containing 10% fetal calf serum (FCS) and antibiotics (penicillin and streptomycin) at 37°C in an atmosphere of 5% CO2 and 95% humidity. For experiments, the cells were seeded into 60 mm Petri dishes (with or without coverslips) and cultured for 3–5 days at 37°C before use.

The virulent wild-type Listeria monocytogenes strain 10403S and its avirulent ΔactA mutant (DP-Ll942 (Brundage et al., 1993)) were generous gifts from Dr D. Portnoy (University of Berkeley, CA). Stock cultures of bacterial suspensions were stored under liquid nitrogen in 30% glycerol. For use, the bacteria were grown on brain heart infusion (BHI) agar plates (Sigma). Colonies from agar plates were cultured in BHI broth and incubated overnight at room temperature. Bacterial suspensions (2 × 109 cfu ml−1) from these overnight cultures were used to infect the J774 cells.

Infection of J774 cells with L. monocytogenes

The cells were washed to remove antibiotics and 2 ml of DMEM containing 20 µl (for EM studies) or 0.5 µl (for light microscopy studies) of overnight bacterial culture was added. For light microscopy, the J774 cells were grown on sterile 12 mm glass coverslips at the bottom of the culture dish. Cells were exposed to bacteria for 1 h, the bacteria washed away, and then incubated for a further 5 min at 37°C to allow completion of internalization. The cells were washed and exposed to gentamicin (1 mM) for 10 min to kill extracellular bacteria, and then immediately fixed and processed for microscopy or incubated further (see below). This short treatment with a high concentration of gentamicin is rapidly bactericidal, but has no effect on intracellular L. monocytogenes (Brundage et al., 1993).

Chloramphenicol treatment

Cells containing internalized bacteria were incubated for a total of 3 h to allow the bacteria to escape from the phagosome and enter the cell cytoplasm. Chloramphenicol (20 µg ml−1 final concentration) was then added to the culture medium and the cells incubated for a further 3–24 h at 37°C, after which they were fixed and processed for either light or electron microscopy. In some experiments, chloramphenicol treatment was carried out in the absence of serum. Time of addition of chloramphenicol was designated as t = 0 h.

Inhibitor studies

Inhibitors were added to cell cultures at the same time as the chloramphenicol. 3-methyladenine was dissolved in hot water to 200 mM and added directly into the cultures at a final concentration of 10 mM, while stock solutions of wortmannin in DMSO (5 mM) were diluted first in culture media to achieve a final concentration of 20 or 100 nM. Wortmannin was readded to the cultures hourly.

Light microscopy

For light microscopy, cells grown on glass coverslips were fixed in 4% formaldehyde in 200 mM Hepes, pH 7.4, and then either Giemsa-stained or immunolabelled. Giemsa staining followed protocols recommended by the supplier. For immunocytochemistry, the cells were treated with 0.01% saponin in PBS containing 0.1% gelatin and then sequentially with specific primary and fluorescent-conjugated secondary antibodies. A L. monocytogenes-specific polyclonal antiserum (Difco Laboratories, Detroit, MI) was used for identification of bacteria, in combination with rat anti-mouse LAMP 1 (clone 1D4B, Developmental Studies Hybridoma Bank, Iowa City, IA). Some cells were double-labelled with a phalloidin-rhodamine conjugate (Sigma). After labelling, the coverslips were mounted on glass slides and examined by epifluorescence microscopy using an Axiophot II light microscope (Carl Zeiss, Thornwood, NY) or by confocal laser scanning microscopy using a Zeiss LSM 410 scanning laser two photon confocal microscope.

Electron microscopy

Cells grown on plastic substrates were fixed by immersion in 2.5% glutaraldehyde in 200 mM sodium cacodylate buffer, pH 7.2. After 5 min, the cells were scraped off the plastic with a small Teflon scraper and pelleted by centrifugation. The pellets were left in fixative for 2 h, and then washed in cacodylate buffer. The pellets were processed as previously described (Webster, 2002), embedded in resin, sectioned and examined using a CM120 BioTwin transmission electron microscope (FEI-Veeco, Hillsboro, OR), operating at 80 kV.

Immunoelectron microscopy

For immunocytochemistry at the EM level, cell monolayers were fixed for 1 h in 4% phosphate-buffered paraformaldehyde. After fixation, the cells were prepared for ultracryomicrotomy using previously described methods (Webster, 1999). Briefly, the cryosections were retrieved using sucrose droplets, mounted onto coated specimen grids, labelled with a rabbit polyclonal antibody to PDI (Dr S. Fuller, University of Oxford, UK) and then with protein A-gold (PAG; obtained from Department of Cell Biology, University of Utrecht), and finally contrasted by incubating and drying in the presence of aqueous methyl cellulose and uranyl acetate (final concentration of 0.3% uranyl acetate). Control experiments consisted of treating sections with PAG alone, with non-relevant antibodies and PAG. Antibody binding was visualized by TEM.

Quantification

Cell counts of immunolabelled and Giemsa-stained coverslips were performed using Stereo Investigator (MicroBrightField, VT), a quantification software package offering unbiased, on-screen estimators, working from a PC connected to an Axioplan II light microscope (Carl Zeiss, Thornwood, NY). Quantification of the numbers of bacteria enclosed within vacuoles was performed on resin-embedded thin sections. For each sample, sections were taken from five different parts of the embedded cell pellet and examined in the TEM using sequential random scan sampling methods (Watt et al., 2002). Cytoplasmic bacteria were scored positive or negative for being enclosed in a vacuole. Approximately 200–300 cells were sequentially sampled for each experiment. In some cases, it was additionally documented whether the vacuole containing bacteria had a single versus a double membrane.

Acknowledgements

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

This research was funded in part by the Hope for Hearing Foundation of Los Angeles in the form of an internship granted to C.B. C.B., who was a student at Marlborough School, Los Angeles while working on this project would like to thank her mother, Dr Annette Newmann, and Dr Arlene Forsheit, her science teacher, for their help and support. We thank Siva Wu for excellent technical assistance, Dan Portnoy for bacteria, Steve Fuller for the anti-PDI antibody and Norma Andrews for some great ideas. The monoclonal antibody 1D4B (developed by Thomas August) was obtained from the Developmental Studies Hybridoma Bank, Iowa City, IA 52242.

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  6. Experimental procedures
  7. Acknowledgements
  8. References
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