After ingestion by macrophages, Legionella pneumophila enter spacious vacuoles that are quickly enveloped by endoplasmic reticulum (ER), then slowly transferred to lysosomes. Here we demonstrate that the macrophage autophagy machinery recognizes the pathogen phagosome as cargo for lysosome delivery. The autophagy conjugation enzyme Atg7 immediately translocated to phagosomes harbouring virulent Legionella. Subsequently, Atg8, a second autophagy enzyme, and monodansyl-cadaverine (MDC), a dye that accumulates in acidic autophagosomes, decorated the pathogen vacuoles. The autophagy machinery responded to 10–30 kDa species released into culture supernatants by Type IV secretion-competent Legionella, as judged by the macrophages’ processing of Atg8 and formation of vacuoles that sequentially acquired Atg7, Atg8 and MDC. When compared with autophagosomes stimulated by rapamycin, Legionella vacuoles acquired Atg7, Atg8 and MDC more slowly, and Atg8 processing was also delayed. Moreover, compared with autophagosomes of Legionella-permissive naip5 mutant A/J macrophages, those of resistant C57BL/6 J macrophages matured quickly, preventing efficient Legionella replication. Accordingly, we discuss a model in which macrophages elevate autophagy as a barrier to infection, a decision influenced by regulatory interactions between Naip proteins and caspases.
Autophagy is a conserved membrane traffic pathway by which eukaryotic cells capture cytoplasmic components for delivery to lysosomes. In response to nutrient starvation, autophagy breaks down macromolecules to generate substrates for essential biosynthetic pathways. In addition to cellular homeostasis, this degradative pathway contributes to numerous other physiological processes, including morphogenesis, cellular differentiation, tissue remodelling and antigen presentation (Nimmerjahn et al., 2003; Ogier-Denis and Codogno, 2003). Several pathological processes also involve autophagy, including carcinogenesis, myopathies and neurodegenerative diseases (Ogier-Denis and Codogno, 2003).
Autophagosome biogenesis is controlled by a unique protein conjugation system composed of several proteins now designated Atg (Klionsky et al., 2003). Atg7 (Apg7/Gsa7) is a broadly conserved ubiquitin E1-like activating enzyme that mediates the homotypic fusion of the isolating membranes to assemble the autophagosome (Mizushima et al., 1998; Tanida et al., 1999; Ohsumi, 2001; Tanida et al., 2001). When autophagy is stimulated, cytosolic Atg7 protein redistributes to the isolation membrane where it first catalyses the conjugation of Atg12 to Atg5, then of Atg8 (Apg8/MAP-LC3) to lipids, thereby promoting the formation and expansion of the nascent autophagosome (Tanida et al., 1999; Kabeya et al., 2000; Ohsumi, 2001). In mammalian cells, autophagosome formation requires class III phosphatidylinositol 3-phosphate kinases (PI3-kinases), because the pathway is blocked by the inhibitor 3-methyladenine (3MA; Seglen and Gordon, 1982). A negative regulator of autophagy is Tor kinase, which can be inhibited pharmacologically by rapamycin (Noda and Ohsumi, 1998). Autophagy is stimulated when pro-apoptotic caspases are inhibited (Martin and Baehrecke, 2004; Yu et al., 2004), suggesting that autophagy and apoptosis are co-ordinated cellular responses to stress.
Although macrophage delivery of the Legionella phagosome to the lysosomes via an ER intermediate is grossly similar to autophagy, a number of aspects are different (Swanson and Isberg, 1995Tilney et al., 2001; Ogier-Denis and Codogno, 2003). Autophagosomes decorated with ribosomes have not been observed; indeed, whether the ER contributes to autophagosome formation in mammalian cells remains a matter of debate (Hirsimaki, 1983; Dunn, 1990a; Ueno et al., 1991; Rich et al., 2003). Furthermore, the Legionella vacuole merges with the lysosomes after a delay of several hours (Sturgill-Koszycki and Swanson, 2000), a maturation period considerably longer than that of autophagosomes (Kopitz et al., 1990; Noda et al., 2002; Ogier-Denis and Codogno, 2003). Within the soil amoeba Dictyostelium discoidium, Legionella can replicate in several different autophagy mutant strains (Otto et al., 2004). Accordingly, autophagy itself does not appear to be a prerequisite for intracellular replication of Legionella in all cell types. Therefore, we tested the hypothesis that macrophages activate the autophagy pathway as a defence against Legionella infection by applying a combination of biochemical markers, pharmacological inhibitors, and bacterial and host mutants. The data support a model in which macrophages deliver Legionella vacuoles to lysosomes by autophagy, a cellular defence mechanism that pathogens must modulate to establish infection.
Autophagy enzymes redistribute to Legionella phagosomes
To analyse whether the macrophage autophagy machinery could account for both the sequestration of the Legionella phagosome within ER and its subsequent delivery to the lysosomes, we examined the distribution of three molecular markers of autophagosomes in cells that had phagocytosed bacteria. Immediately after internalization, ∼50% of wild-type (WT) bacteria resided in spacious compartments surrounded by Atg7, a conjugating enzyme that initiates formation of the isolation vacuole (Fig. 1A and B; Mizushima et al., 1998; Tanida et al., 1999; Dorn et al., 2001; Ohsumi, 2001; Tanida et al., 2001; Klionsky et al., 2003). Accumulation of the autophagy-initiating enzyme was specific to phagosomes harbouring pathogenic bacteria, because Atg7 was rarely associated with vacuoles containing either PE dotA mutant (Fig. 1A and B) or E WT Legionella or avirulent Escherichia coli DH5α (Fig. 1B), microbes that macrophages efficiently deliver to the endosomal pathway (Berger et al., 1994; Roy et al., 1998; Joshi et al., 2001; Kagan and Roy, 2002). The appearance of Atg7 around vacuoles reflected a recruitment from cytosolic pools, because the amount of Atg7 protein detected by Western analysis did not change during the 30 min infection (A. Amer, unpubl.). As expected for autophagy, redistribution of Atg7 to the pathogen phagosomes required class III PI3-kinases, because Atg7-positive vacuoles were rare when macrophages were infected with Legionella in the presence of 10 mM 3MA (Seglen and Gordon, 1982). By 15 min after infection, the spacious vacuoles harbouring Legionella appeared more condensed (A. Amer, unpubl.), a pattern in good agreement with the previously described short-lived spacious phagosomes that contain Type IV secretion-competent Legionella and are rich in GM1 gangliosides and glycosylphosphatidylinositol-linked proteins (Watarai et al., 2001). The phagosomes progressively shed Atg7; by 3 h, the enzyme was rarely detected near vacuoles (Fig. 1B).
To examine by another approach whether macrophages handled Legionella phagosomes as cargo for the autophagy pathway, we examined the distribution of Atg8 (LC3), a second conjugating enzyme that is recruited to autophagosomes as they mature (Kabeya et al., 2000). When autophagy was stimulated by incubating macrophages in amino acid-free buffer for 1 h, numerous Atg8-positive vacuoles were detected (Fig. 2D). Beginning ∼4 h after infection of macrophages, Atg8 colocalized with > 40% of the vacuoles that contained PE WT Legionella, but not those that carried either PE dotA mutants (Fig. 1C and D) or avirulent E WT Legionella or E. coli (A. Amer, unpubl.). The transient association with Atg7 and the subsequent accumulation of Atg8 around vacuoles harbouring virulent Legionella is characteristic of autophagosome maturation (Kopitz et al., 1990; Noda et al., 2002; Ogier-Denis and Codogno, 2003; Yoshimori, 2004).
To analyse the interaction of the autophagy pathway with Legionella phagosomes by a third method, we utilized monodansyl-cadaverine (MDC), a fluorescent compound that accumulates in acidic autophagic vacuoles (Biederbick et al., 1995; Niemann et al., 2000; Munafo and Colombo, 2001). The results of several control assays verified that MDC behaved as a valid marker of macrophage autophagosomes. By 6 h after infection, MDC accumulated in ∼30% of the vacuoles that harboured virulent PE Legionella, but not avirulent dotA mutants (Fig. 3A and B). MDC persisted within mature replication vacuoles even 24 h after infection (Fig. 3A), a period when these organelles are acidic (Sturgill-Koszycki and Swanson, 2000). As expected for autophagy, formation of the MDC-positive vacuoles by infected cells was inhibited by 3MA (Fig. 3C). However, compared with autophagosomes induced by amino acid-free buffer, the pathogen vacuoles accumulated MDC more slowly (Fig. 3B and C). Therefore, macrophages appeared to handle phagosomes that contained virulent Legionella as cargo for autophagy, as judged by their sequential acquisition by a 3MA-sensitive process of the autophagy-initiating enzyme Atg7, the conjugating enzyme Atg8 and the fluorescent marker of mature acidic autophagosomes MDC. However, compared with typical autophagosomes, the pathogen-induced vacuoles matured more slowly.
The macrophage autophagy machinery responds to factors shed from virulent Legionella
Macrophages appeared to activate the autophagy machinery independently of phagocytosis of the pathogen. First, after a 5 min incubation with PE Legionella, only 21 ± 0.5% of the macrophages had ingested a bacterium, yet 54 ± 3% of the cells contained numerous Atg7-labelled donut-shaped compartments. Second, when actin polymerization was disrupted with cytochalasin D, phagocytosis of Legionella was decreased dramatically as expected (Elliott and Winn, 1986), yet 42 ± 1.7% of the macrophages still contained multiple Atg7-decorated vacuoles within 5 min of infection, a frequency significantly greater than the 5.5 ± 1.5% of the uninfected control cells that did so (Fig. 2B). Therefore, we tested the hypothesis that macrophages activate the autophagy machinery in response to soluble bacterial products.
Cell-free supernatant prepared from cultures of PE Legionella stimulated the autophagy machinery. After a treatment for 15 min, >50% of the macrophages contained multiple Atg7-labelled compartments (Fig. 2A and C). After a 5 h incubation with the sterile supernatant, multiple Atg8-labelled vesicles (Fig. 2D) were detected in 25 ± 2.5% macrophages, and by 6 h MDC-positive vacuoles were apparent (Fig. 3C). The maturation rate of the vacuoles was similar to that observed for phagosomes that contain virulent Legionella(Figs 1–3). In contrast, neither sterile bacteriological broth treated in the same manner nor supernatants obtained from cultures of non-infectious microbes, namely E Legionella, PE dotA mutants, or DH5αE. coli, induced formation of vacuoles labelled by Atg7, Atg8, or MDC (Fig. 2A; A. Amer, unpubl.). Stimulation of the autophagy machinery by Legionella soluble products was not accompanied by cytotoxicity, because macrophages remained viable after a 20 h incubation with supernatants that had been concentrated 20-fold (Experimental procedures; A. Amer, unpubl.).
To investigate by a biochemical approach whether soluble Legionella products stimulate autophagosome formation, we examined by Western assay processing of Atg8, a protein that the Atg7 enzyme conjugates to phosphatidylethanolamine during autophagosome biogenesis (Kabeya et al., 2000; Ichimura et al., 2000). Unlike control cells, macrophages that had been incubated with amino acid-free buffer, rapamycin, or Legionella sterile supernatants processed the Atg8 protein, as judged by the appearance of the membrane-associated lipidated species, whose electrophoretic mobility is greater than the cytosolic form (Fig. 2E). As expected, when autophagosome formation was inhibited by pretreating macrophages with 3MA, Legionella supernatants did not stimulate Atg8 lipidation (Fig. 2E). The conjugated Atg8 species was detected within 1 h of amino acid starvation or rapamycin treatment; in contrast, a 4 h incubation with Legionella culture supernatants was required for macrophages to process Atg8 (Fig. 2E; A. Amer, unpubl.). Thus, macrophages activate the autophagy pathway in response to soluble products released by Type IV secretion-competent PE Legionella by a mechanism that does not require phagocytosis of the bacteria.
To begin to characterize biochemically the Legionella autophagosome stimulating factor(s), which we shall refer to as ASF, three experiments were performed. ASF activity did not appear to reflect bacterial lysis, because the protein profile of concentrated culture supernatants was distinct from that of total bacterial lysates, as judged by SDS-PAGE (A. Amer, unpubl.). Treating PE culture supernatants with proteinase K dramatically reduced ASF activity (Fig. 2C). By fractionating the supernatants with filters that exclude either 5, 10, 30 or 100 kDa species, we determined that bacterial products of 10–30 kDa stimulated robust autophagosome formation, whereas fractions that contained molecules > 30 or <10 kDa had minimal effect (Fig. 2A and C). The active fraction of 10–30 kDa molecules was complex, as nine species were apparent after separation by SDS-PAGE and staining with Coomassie Blue. Thus, additional biochemical and molecular analysis can determine how macrophages activate the autophagy machinery in response to soluble 10–30 kDa proteinaceous molecules that are shed by Type IV secretion-competent PE phase Legionella.
Secretory traffic promotes association of Atg7 with the Legionella vacuole
Colocalization of pathogenic Legionella with autophagosomal vacuoles correlates with immediate evasion of the endocytic pathway
Legionella residence in ER-derived vacuoles is correlated with its immediate evasion of the endosomal pathway (Berger et al., 1994; Swanson and Isberg, 1995; Kagan and Roy, 2002; Molofsky and Swanson, 2004). Therefore, if the autophagy machinery assembles the ER-derived vacuole, drugs that inhibit autophagy are predicted to increase lysosomal degradation of Legionella. To test whether there is a reciprocal relationship between the autophagosome and phagolysosome pathways, we utilized two pharmacological inhibitors of autophagosome assembly. When accumulation of secretory vesicles and Atg7 by pathogen vacuoles was inhibited by BFA (Kagan and Roy, 2002), LAMP-1 was detected on ∼15% of the vacuoles harbouring Legionella (Fig. 4C), and ∼30% of the intracellular Legionella were degraded as early as 15 min after infection (Fig. 4D). Likewise, when autophagy was blocked by 3MA (Figs 1B and 3C), ∼30% of intracellular bacteria were visibly degraded and ∼40% were killed by 2 h after infection (Fig. 5A and B). The decrease in pathogen survival was not a consequence of 3MA toxicity, because the drug had no effect on either the viability of macrophages or Legionella (Experimental procedures; A. Amer, unpubl.), or the ability of macrophages to kill E Legionella, or the intracellular survival of dotA Legionella (Fig. 5B). Thus, both genetic and pharmacological studies of Legionella trafficking in macrophages suggest a reciprocal relationship between delivery of phagosomes to the autophagy pathway or directly to the lysosomes.
Slow autophagosome maturation correlates with survival of Legionella
Autophagy is a major catabolic pathway by which eukaryotic cells sequester organelles for lysosomal degradation, yet residence in autophagosomal vacuoles promoted Legionella survival in macrophage cultures (Figs 4 and 5). To account for this paradox, we postulated that to establish infection Legionella perturbs maturation of its autophagosome. Indeed, when autophagy was stimulated by treating macrophages with either rapamycin or amino acid-free buffer, the organelles matured considerably faster than vacuoles that contained the pathogen, as judged by comparing the kinetics of Atg7 and Atg8 colocalization with vacuoles and phagosomes (Fig. 6B and D; A. Amer, unpubl.). Thus, Legionella appears to encode factors that inhibit autophagosome maturation.
To investigate by a genetic approach whether the rate of autophagosome maturation affects infection by Legionella, we utilized macrophages obtained from a congenic mouse strain that is resistant to infection. Unlike permissive A/J mice, which harbour a naip5 mutation that reduces the amount of Naip proteins, neither C57BL/6 J mice nor their isolated macrophages support Legionella replication (Dietrich et al., 1995; Diez et al., 2000; Diez et al., 2003; Wright et al., 2003). When autophagy was induced by rapamycin or starvation, vacuoles formed by restrictive C57BL/6 J macrophages more quickly acquired and then shed both Atg7 and Atg8 than did the vacuoles of permissive A/J macrophages (Fig. 6A and B; A. Amer, unpubl.). Likewise, after infection with PE Legionella, autophagosomes formed by resistant C57BL/6 J macrophages matured more rapidly than those of permissive A/J macrophages, as judged by the kinetics of both Atg7 and Atg8 colocalization (Fig. 6C and D). As expected for efficient autophagosome maturation, ∼50% of the Legionella phagosomes formed by resistant C57BL/6 J macrophages accumulated the late endosomal and lysosomal protein LAMP-1 within 15 min of infection (Fig. 6E). In comparison, in permissive A/J macrophages, LAMP-1 is not detected on Legionella phagosomes until ∼8 h after infection (Sturgill-Koszycki and Swanson, 2000). Thus, macrophages with efficient autophagosome maturation were resistant to infection, whereas those with a sluggish autophagy pathway were susceptible to Legionella infection.
Here we report morphological, biochemical and genetic evidence that macrophages activate the autophagy machinery as an immediate response to infection by an intracellular pathogen. The autophagy pathway is activated by soluble factors released by infectious Legionella by a mechanism that does not require phagocytosis of the bacteria. However, vacuoles that harbour pathogens mature more slowly than autophagosomes stimulated by either rapamycin or amino acid-free buffer. Furthermore, the autophagy response of macrophages from mice that are permissive for Legionella infection is more lethargic than that of phagocytes from resistant mice. Accordingly, we postulate that macrophages activate autophagy as a barrier to infection; consequently, certain intracellular pathogens perturb autophagosome maturation to avoid or delay their delivery to toxic lysosomes.
The interpretation that macrophages handle Legionella phagosomes as cargo for the autophagy pathway is supported by a large body of morphological and genetic data presented here and elsewhere. When immature, both autophagosomes and vacuoles that contain Legionella are limited by a double membrane that forms by homotypic fusion of numerous small vesicles derived from the secretory pathway, a process described in the autophagy field as nucleation-assembly elongation (Noda et al., 2002). Dependent on the sequential activities of the Sar1/COPII and ARF/COPI systems, vesicle production from the ER promotes biogenesis of both Legionella replication vacuoles and yeast autophagosomes (Aridor et al., 1995; Scales et al., 1997; Kagan and Roy, 2002; Hamasaki et al., 2003; Derre and Isberg, 2004). As they mature, both organelles colocalize first the autophagy enzyme Atg7, next Atg8, and then the fluorescent dye MDC (Mizushima et al., 1998; Tanida et al., 2001). At any given time, only a subset of the vacuoles colocalized with a particular autophagy marker, most likely because the associations are transient, phagosome formation is not synchronous, and the fluorescence signal is limiting. Finally, in their terminal stage of development, both autophagosomes and Legionella vacuoles acquire lysosomal features, including LAMP-1, the acid hydrolase cathepsin D, and an acidic pH (Hirsimaki, 1983; Dunn, 1990b; Sturgill-Koszycki and Swanson, 2000).
Analysis of Legionella trafficking indicates that membranes derived from the secretory pathway contribute to autophagosome biogenesis by macrophages. The ER resident proteins Bip and calnexin, the recombinant ER marker KDEL-YFP, the small GTPases ARF1 and Rab1, the v-SNARE Sec22, and ribosomes all colocalize with the Legionella vacuole, and its membrane acquires a thickness characteristic of ER (Horwitz, 1983b; Swanson and Isberg, 1995; Tilney et al., 2001; Kagan and Roy, 2002; Nagai et al., 2002; Molofsky and Swanson, 2004). Furthermore, when vesicular traffic from the ER is inhibited by BFA, association of the autophagy enzyme Atg7 with Legionella vacuoles is reduced (Fig. 4B). Therefore, studies of Legionella–macrophage interactions provide another example where ER is the source of autophagosome isolation membranes (Hirsimaki, 1983; Dunn, 1990a; Ueno et al., 1991; Rich et al., 2003). In particular, we predict that Atg7 binds to the secretory vesicles that attach to the cytoplasmic face of the L. pneumophila phagosome to mediate their homotypic fusion, thereby generating an isolation membrane around the pathogen phagosome (Fig. 7), a model compatible with the ultra-structural studies of Tilney and colleagues (Tilney et al., 2001).
Microbial traffic along the autophagy pathway is not unique to Legionella (Kirkegaard et al., 2004). Both the picoranviruses poliovirus and herpes simplex virus type 1 encode proteins that induce the formation of double membrane vesicles that resemble autophagosomes (Talloczy et al., 2002; Kirkegaard et al., 2004). In HeLa cells, Brucella abortus is engulfed by autophagosome-like vacuoles that contain the ER components sec61β and calnexin and also accumulate MDC (Pizarro-Cerda et al., 1998a; 1998b). In endothelial cells, Porphyromonas gingivalis reside in vacuoles that colocalize with Atg7 and Bip (Dorn et al., 2001). Leishmania spp. and Coxiella burnetii, pathogens that multiply in lysosomes, may be delivered to their replicative niche by autophagy (Schaible et al., 1999; Beron et al., 2002). When caspase 1-deficient macrophages are infected with Salmonella that express the type III secretion system substrate SipB, numerous autophagosomes accumulate (Hernandez et al., 2003). At least in tissue culture models of infection, the intracellular pathogens Legionella, P. gingivalis, C. burnetii and B. abortus benefit from autophagy, because these bacteria survive poorly in cells treated with 3MA (Pizarro-Cerda et al., 1998b; Dorn et al., 2001; Beron et al., 2002). On the other hand, the autophagy machinery can effectively eliminate cytoplasmic Group A Streptococcus and inhibit the survival of Mycobacterium tuberculosis (Gutierrez et al., 2004; Nakagawa et al., 2004). Likewise, when intracellular Listeria monocytogenes are killed by antibiotic treatment, the cytoplasmic bacteria are engulfed and eliminated within double-membrane vacuoles that contain the ER protein disulphide isomerase (Rich et al., 2003). Moreover, efficient autophagy appears to prevent Legionella replication in C57BL/6 J macrophages (Fig. 6). Therefore, autophagy can be a defence mechanism against intracellular microbes that some pathogens acquired the means to overcome.
A clue to how Legionella tolerates the catabolic autophagy pathway is the slow maturation of its vacuole (summarized in Fig. 7; Noda et al., 2002; Ogier-Denis and Codogno, 2003). Whereas Atg7-labelled vacuoles are difficult to detect in cells treated with rapamycin or amino acid free buffer, the enzyme remained associated with some Legionella vacuoles as long as 60 min (Figs 1B and 6A; A. Amer, unpubl.). Second, macrophages accumulated Atg8-labelled vesicles within 1 h of rapamycin treatment or amino acid deprivation (Fig. 6B; A. Amer, unpubl.), but the pathogen compartments did not colocalize with Atg8 for 4 h (Figs 1D and 6D). Third, vesicular MDC was detected 2 h after macrophage autophagy was stimulated by starvation or rapamycin (Fig. 3C; A. Amer, unpubl.), but the dye was not visible within Legionella vacuoles for 4–6 h (Fig. 3B). By similar criteria, the autophagic vacuoles stimulated by soluble factors released by Legionella also matured slowly (Figs 2D and 3C). Finally, A/J-derived macrophages, which are permissive for Legionella infection, have a slower rate of autophagosome maturation than cells from C57BL/6 J mice (Fig. 6). Their lethargic maturation likely explains why Legionella autophagosomal vacuoles accumulate ribosomes; normally, the ER-derived isolation vacuole rapidly converts into an autophagolysosome, masking its origin as a biosynthetic membrane. Presumably, when confronted by the autophagy response of its host phagocyte, Legionella must retard maturation of its vacuole to secure time to differentiate to an acid-tolerant form (Sturgill-Koszycki and Swanson, 2000; Molofsky and Swanson, 2004). Herpes Simplex Virus applies a different strategy to evade degradation in autophagolysosomes: the neurovirulence viral protein ICP34.5 antagonizes eukaryotic initiation factor-2-α (eIF2α) kinase-dependent autophagy (Talloczy et al., 2002; Kirkegaard et al., 2004).
Analysis of Legionella infection of macrophages strengthens the emerging concept of a regulatory link between autophagy and programmed cell death, two cellular responses to stress (Gozuacik and Kimchi, 2004; Martin and Baehrecke, 2004; Yu et al., 2004; Xue et al., 1999). In mice, susceptibility to Legionella infection is conferred by a mutation in naip5, a gene originally identified as neuronal apoptosis inhibitor protein (Diez et al., 2003; Wright et al., 2003; Dietrich et al., 1995). The Naip5 protein is a member of a large family of endogenous caspase inhibitors, based both on its BIR, NOD and LRR domains (Liston et al., 2003) and the ability of BIR domains from human Naip to bind to and inhibit caspase 3 and caspase 7 (Maier et al., 2002). The data presented here are consistent with a number of previous observations that together suggest that Naip proteins promote efficient autophagosome maturation, perhaps by inhibiting caspase-3. First, macrophages from both A/J and C57Bl mice increase expression of Naip proteins after ingesting Legionella, Salmonella typhimurium, or latex beads (Diez et al., 2000). However, compared with permissive naip5 mutant macrophages from A/J mice, C57BL/6 J-derived macrophages express more Naip protein, and their autophagosomes more rapidly merge with lysosomes (Fig. 6C and D). Second, when human monocytes are infected by Legionella, the cells activate caspase-3 and engulf the pathogen within ER, but the vacuoles do not fuse with lysosomes for at least 2 h, perhaps because Rabaptin-5 is degraded (Molmeret et al., 2004). In contrast, when caspase-3 activity is inhibited pharmacologically, the phagocytes instead rapidly deliver Legionella to lysosomes, preventing their replication (Molmeret et al., 2004). Third, the pan-caspase inhibitor zVAD triggers toxic levels of autophagy in human monocytic cells or mouse peritoneal macrophages; a similar cell response is observed when caspase 8 protein is reduced by RNA interference (Yu et al., 2004). The existing data support the idea that autophagy functions as an adaptive response to infection that is efficient when caspase activity is low, whereas programmed cell death is an emergency response triggered when caspase activity is high. How Naip proteins regulate both autophagy and apoptosis can be examined using Legionella infection of macrophages as an experimental system.
Analysis of Legionella trafficking in macrophages has also revealed a reciprocal relationship between delivery of phagosomes directly to the endosomal pathway or their capture by autophagy. When autophagosome formation is inhibited by 3MA or by BFA, or production of ER-derived vesicles is decreased by dominant-interfering SNARE proteins, macrophages efficiently deliver Legionella phagosomes to the endosomal pathway, preventing bacterial replication (Figs 4–6; Watarai et al., 2001; Kagan and Roy, 2002; Kagan et al., 2004). Accordingly, as one means to evade immediate destruction in lysosomes, PE Legionella may release its autophagosome-stimulating factor, ASF (Fig. 3). Although more analysis is required to understand the impact of ASF on Legionella pathogenesis, it is the first activity shown to be released by a Dot/Icm Type IV secretion system-dependent mechanism and also to alter macrophage membrane traffic.
Rather than function as a virulence factor, Legionella ASF can be considered a candidate Pathogen-Associated Molecular Pattern molecule because ASF triggers autophagy, an alternate route to toxic lysosomes. Consistent with the view that autophagy is a barrier to infection, when compared with C57BL/6 J mice, A/J mice are more susceptible to several other intracellular pathogens including Trypanosoma cruzi, Plasmodium chabaudi and Listeria monocytogenes (Stevenson and Tam, 1993; Gonçalves da Costa et al., 2002; Czuprynski et al., 2003). Future studies can examine directly the role of autophagy in other intracellular infections. Furthermore, genetic studies of Legionella growth in D. discoidium indicate that autophagy per se is not required for bacterial replication (Otto et al., 2004); instead, the pathogen may replicate within acidic lysosomal vacuoles not only in A/J macrophages (Sturgill-Koszycki and Swanson, 2000) but also in soil amoebae. Compared with mammalian macrophages, D. discoidium appear to have a more robust phagolysosome pathway, based on the smaller fraction of virulent Legionella that survive phagocytosis, the death of dot/icm mutants, and the prolonged lag period before Legionella replication can be detected (Otto et al., 2004). It is also noteworthy that, unlike mammalian macrophages, D. discoidium lacks caspases, and its single paracaspase gene is not required for programmed cell death (Roisin-Bouffay et al., 2004). Therefore, D. discoidium is a valuable experimental model to analyse some aspects of Legionella biology, but it may not mimic the macrophage response to pathogens.
Bone marrow-derived macrophages from either A/J or C57BL/6 J mice (The Jackson Laboratory) were cultured as described previously (Swanson and Isberg, 1995). Unlike cells from humans and A/J mice, C57BL/6 J macrophages are resistant to Legionella infection. The susceptibility of A/J mice to Legionella infection is conferred by a mutation in naip5 that reduces the amount of Naip proteins (Dietrich et al., 1995; Diez et al., 2003; Wright et al., 2003).
Autophagy was stimulated by incubating macrophages for the periods indicated either with amino acid-free buffer as described (Swanson and Isberg, 1995) or with 0.2 µg rapamycin (LC Laboratories) per ml RPMI/FBS (Noda and Ohsumi, 1998). To infect macrophages synchronously, PE Legionella were added at a multiplicity of infection (MOI) <5 to ice-cold macrophages, then the preparations were centrifuged 10 min at 400 g at 4°C, transferred to a 37°C water bath for 5 min, then washed three times with warm RPMI/FBS to remove extracellular bacteria.
For localization of markers on Legionella phagosomes, 3.5 × 105 macrophages cultured on 12 mm glass coverslips in 24 well tissue culture plates were infected synchronously as described above. At the times designated, the preparations were fixed and permeabilized as described (Swanson and Isberg, 1995). Antibodies were diluted into PBS with 5% goat serum (PBS/GS) as follows: rabbit anti-Legionella (a gift from Dr Ralph Isberg, Tufts University School of Medicine), 1:100; rat antilysosomal-associated membrane protein 1 (LAMP1; 1D4B; Developmental Hybridoma Bank), 100%; rabbit anti-Atg7 (Dorn et al., 2001), 1:100; rabbit anti-Atg8/LC3 (a gift from Dr T. Yoshimori, National Institute of Genetics, Japan), 1:200; rat anti-Bip (SantaCruz), 1:100; all fluorescent secondary antibodies (Molecular Probes) were diluted 1:3000. Non-specific antibody binding was reduced by preincubating preparations overnight with PBS/GS. Cells were incubated with antibodies for 1 h at 37°C, then washed three times in PBS/GS. Bacteria were stained with either anti-Legionella antibody or 0.1 µM of the nucleic acid dye 4′,6′-diamino-2-phenylindole (DAPI; Molecular Probes) in PBS. After staining with specific antibodies, mounted coverslips were examined as previously described (Berger et al., 1994; Sturgill-Koszycki and Swanson, 2000). Colocalization with Atg7 was confirmed with confocal microscopy. To score autophagosome formation, a macrophage was defined as positive if it contained > 5 donut- or C-shaped Atg7-labelled structures. Autophagosomes were labelled with MDC as previously described (Biederbick et al., 1995).
To ascertain whether MDC behaves as a specific marker for autophagosomes in our experimental model, several control assays were performed. First, compared to untreated cells, macrophages in which autophagy was stimulated for 2 h by either amino acid-free buffer or rapamycin stained brightly with MDC while the untreated macrophages showed a fine reticular background staining with the MDC (Fig. 2C; Amer et al., 2005). To distinguish the characteristic vesicular distribution of MDC in induced autophagosomes, each experiment was performed and scored in comparison with positive and negative controls. Formation of vacuoles that accumulate MDC required class III PI3-kinases, since the organelles were rare when macrophages were starved in the presence of 3MA. When uninfected macrophages whose lysosomes had been labeled by endocytosis of Texas Red-ovalbumin were incubated with MDC, the dye was rarely detected in the abundant lysosomal network (Amer et al., 2005). Nor was appreciable MDC retained by macrophages that were fed E. coli or E phase Legionella, microbes that are efficiently digested in lysosomes, or dotA mutant Legionella, bacteria that persist in non-lysosomal LAMP-1-positive compartments. Thus, MDC did not readily accumulate in acidic lysosomes during our treatment regimen but instead behaved as a valid marker of macrophage autophagosomes.
The supernatants of E or PE phase Legionella cultures were collected by centrifugation at 7000 g for 15 min, sterilized with 0.22 µm filters, then concentrated 20× by centrifugation in 5 kDa Ultrafree-4 centrifugal filters (Vivascience). Supernatant preparations were diluted to 2× final concentration with RPMI/FBS. Sterile AYE broth concentrated similarly served as a control. Size fractionation was achieved by centrifugation of culture supernatants in Amicon Ultra Centrifugal Filter Devices (Millipore) with 5, 10, 30, 100 kDa molecular weight limits. Proteins were digested by treating PE phase supernatants with 1 µg proteinase K per 2.5 µg of protein for 12 h at 37°C.
A/J mouse macrophages treated to the conditions indicated were lysed with Mammalian Protein Extraction Reagent (PIERCE). Samples were separated by SDS-PAGE, transferred to nitrocellulose membranes (Bio-Rad), incubated with Atg8-specific antibody, then developed with WesternBreeze Chromogenic Western Blot Immunodetection Kit (Invitrogen).
3-methyladenine, cytochalasin D and BFA treatment
To inhibit autophagy, macrophages were incubated for 60 min before and during the infection with 10 mM 3-methyladenine (Sigma), as 2 and 5 mM 3MA had no detectable effect (Seglen and Gordon, 1982). At 90 min after infection, the cells were washed three times with RPMI/FBS then incubated for the additional period indicated. To inhibit phagocytosis by depolymerizing actin filaments, macrophages were incubated with 1 µg cytochalasin D per ml RPMI/FBS for 45 min before infection. Dimethyl sulphoxide (0.1%), which was used to dissolve the cytochalasin D, did not alter the growth of the bacteria in macrophage cultures nor affect macrophage viability (A. Amer, unpubl.). To inhibit vesicular traffic from the ER, BFA (Klausner et al., 1992) was added to the macrophage cultures 45 min before infection with Legionella and maintained throughout the incubation period. A 1 h incubation of uninfected macrophages with 3MA, BFA, or cytochalasin D at 5× the working concentration had no effect on macrophage viability as judged by Alamar Blue reduction (Byrne and Swanson, 1998).
Much gratitude is given to Dr William Dunn (College of Medicine, University of Florida) and Dr Tamotsu Yoshimori (National Institute of Genetics, Japan) for generously providing antibodies used in this work. We thank Brenda Byrne for technical assistance, Dr Atsuki Nara (National Institute of Genetics, Japan) for insightful discussions and John-Demian Sauer for helpful comments on the manuscript. Our research is supported by the National Institute of Allergy and Infectious Diseases of the National Institute of Health, Grant 2 R01 AI040694-06AI.