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The evolutionarily conserved processes of endosome-lysosome maturation and macroautophagy are established mechanisms that limit survival of intracellular bacteria. Similarly, another emerging mechanism is LC3-associated phagocytosis (LAP). Here we report that an intracellular vacuolar pathogen, Legionella dumoffii, is specifically targeted by LAP over classical endocytic maturation and macroautophagy pathways. Upon infection, the majority of L. dumoffii resides in ER-like vacuoles and replicate within this niche, which involves inhibition of classical endosomal maturation. The establishment of the replicative niche requires the bacterial Dot/Icm type IV secretion system (T4SS). Intriguingly, the remaining subset of L. dumoffii transiently acquires LC3 to L. dumoffii-containing vacuoles in a Dot/Icm T4SS-dependent manner. The LC3-decorated vacuoles are bound by an apparently undamaged single membrane, and fail to associate with the molecules implicated in selective autophagy, such as ubiquitin or adaptors. The process requires toll-like receptor 2, Rubicon, diacylglycerol signaling and downstream NADPH oxidases, whereas ULK1 kinase is dispensable. Together, we have discovered an intracellular pathogen, the survival of which in infected cells is limited predominantly by LAP. The results suggest that L. dumoffii is a valuable model organism for examining the mechanistic details of LAP, particularly induced by bacterial infection.
Legionella species are opportunistic human pathogens that are normally found within phagocytic protozoa. Infection of humans occurs by incidental inhalation and replication of the organism within alveolar macrophages. This results in an atypical pneumonia termed Legionnaires’ disease. Legionella survive in human cells by manipulating host pathways conserved between simple eukaryotes and humans1. To subvert host cellular processes, the Dot/Icm type IV secretion system (T4SS) delivers bacterial proteins, termed effectors, into the cytosol of host cells2,3,4.
Although Legionella pneumophila accounts for the majority of diagnosed Legionnaires’ disease worldwide, at least 25 different Legionella species have been found in patients5,6. This includes the largely uncharacterized pathogen Legionella dumoffii7. Why L. pneumophila seems to dominantly cause the disease is unknown, though an O-antigen LPS modification is involved8,9. Dot/Icm T4SSs are required for intracellular replication of L. pneumophila10,11, L. longbeachae12 and L. oakridgensis13. The molecular strategies for intracellular survival of L. pneumophila have been well examined. Key hallmarks of intracellular L. pneumophila replication include association with mitochondria, remodeling of the vacuole into an endoplasmic reticulum (ER)-like compartment, decoration of L. pneumophila-containing vacuoles with ubiquitin and modulation of vacuolar phosphoinositides4,14. L. pneumophila also avoids trafficking and degradation within lysosomal compartments by subverting both canonical endocytic maturation and autophagy15,16,17,18.
Autophagy is a regulated and fundamental process in eukaryotic cells that acts to degrade and recycle cytoplasmic contents19,20. It involves at least 30 Atg proteins21 and many accessory proteins that govern substrate-targeting in selective autophagy22,23. The selective autophagic-process that removes bacteria is known as xenophagy, but herein we use autophagy for simplicity. Degradation occurs in terminal membrane-bound compartments termed autophagolysosomes. The process begins with formation of double-membrane autophagosomes that sequester components, including the hallmark autophagy marker LC3/Atg8, before fusing with lysosomes24. A number of pathogens that replicate inside membrane-bound compartments, including Chlamydia, Mycobacterium tuberculosis, and Salmonella enterica serovar Typhimurium (S. Typhimurium), gain the autophagy marker LC3 after invasion25,26,27.
Pathogens utilize diverse strategies and virulence factors to escape or subvert autophagy28,29. Pathogens such as Coxiella burnetii actually hijack autophagy and replicate in LC3-positive compartments30,31. Autophagy has been shown to be dispensable for L. pneumophila replication in the protozoan host Dictyostelium discoideum32. ATG9- knockout data in D. discoideum suggests autophagy might restrict L. pneumophila in host cells33. The potential for Atg machinery to limit L. pneumophila is also supported by Atg5-dependent inhibition of L. pneumophila replication in normally permissive macrophages following induction of metabolic starvation-triggered autophagy34. However, L. pneumophila is known to employ multiple strategies to interfere with autophagy. The effector RavZ irreversibly deconjugates LC3 and thus blocks autophagy17,35. The effector Spl was reported to inhibit autophagy by unknown mechanism36.
Key autophagy research has included examination of the contributing mechanisms and molecular triggers. At least six distinct processes or molecules have been found to target bacterial pathogens for decoration with LC3, including (1) innate immune Nod-like receptors37, (2) targeting of ubiquitin-modifications associated with the pathogen by various adaptor molecules that also bind to LC338,39,40,41, (3) sensing of damaged vacuolar membranes by galectin-8 and autophagy activation via the adaptor NDP5242, (4) Tecpr143, (5) a Drosophila peptidoglycan-recognition-protein44, and (6) activation of diacylglycerol (DAG) signaling45,46,47.
An alternative process for acquiring LC3 that is termed LC3-associated phagocytosis (LAP) has also been described for dead and entotic cells, zymosan (dead yeast) and a few bacteria48,49. A hallmark of this process is the formation of an LC3-positive phagosome with a single membrane50. Separately, single membrane phagosomes and association with LC3 have been described for the normally cytosolic pathogens Burkholderia pseudomallei and Mycobacterium marinum51,52,53. Compartments containing M. marinum54, Shigella flexneri55, Listeria monocytogenes46, and Yersinia pseudotuberculosis56,57 have been confirmed, at least in part, as LC3-positive single-membrane compartments. For the two latter organisms, the bacteria somehow replicate within this compartment. Restriction of intracellular bacterial replication by LAP has not been reported, though a survival defect was reported for B. pseudomallei in Beclin 1 siRNA treated cells58.
Here we profiled L. dumoffii-containing vacuoles (LdCVs) and found that the process of LAP conjugates LC3 to LdCVs, which results in bacterial degradation.
L. pneumophila avoids endocytic maturation and creates an ER-like niche. To assess whether L. dumoffii also interferes with the classical endocytic pathway, we examined acquisition of the tethering protein early endosomal antigen (EEA) 1, and the lysosomal marker LAMP-1. These markers have previously been used to examine Dot/Icm-dependent trafficking of L. pneumophila16,59. EEA1 association was not commonly observed on LdCVs in THP-1 cells (Fig. 1A). To examine whether or not this is a Dot/Icm-dependent inhibition we created an L. dumoffii mutant lacking the dotA gene. The L. dumoffii dotA strain failed to prevent EEA1 association (Fig. 1A), and was unable to replicate inside host cells (Figure S1A). Consistent with a block in endosomal maturation, LAMP1-association with LdCVs was rarely observed on LdCVs at one hour post-infection. However, we unexpectedly observed delayed acquisition of LAMP1 with ~10% of LdVCs positive at 3 to 4hours post-infection (Fig. 1B,C).
To test whether LdCVs become ER-associated, we examined phagosomal acquisition of two different markers: mtagRFP-KDEL (ER retention signal) and the ER-resident protein calnexin. On L. pneumophila-containing vacuoles (LpCVs), KDEL proteins transiently accumulate prior to maximal calnexin recruitment60. Similarly, mtagRFP–KDEL was highly enriched on LdCVs at 90min post-infection (Figure S1B). Calnexin was not detected on LdCVs at 3hours, but vacuoles containing replicating L. dumoffii were calnexin-positive (Figure S1C).
Taken together, L. dumoffii inhibits endosomal maturation and creates an ER-like niche, as previously described for L. pneumophila. However, the slow kinetics of maximal LAMP-1 acquisition was unexpected.
In addition to the classical endosome-lysosome pathway, another process that could deliver bacterial pathogens to LAMP-1-positive compartments is autophagy. Thus, we examined whether LdCVs acquire the autophagy marker LC3. In RAW 264.7 cells stably expressing GFP-LC3, GFP-LC3 localization was clearly observed on a minority of LdCVs (Fig. 2A). We then compared the LC3-recruitment phenotype of L. pneumophila and L. dumoffii in THP-1 cells (Fig. 2B). LpCVs rarely showed LC3 association, but LC3 was associated with up to ~20% of LdCVs. The peak of association was observed at three hours post-infection. LC3 was not observed on mature vacuoles containing replicating L. dumoffii (Fig. 2C). Importantly, we found that decoration with LC3 was Dot/Icm-dependent as our dotA deletion strain remained LC3-negative (Fig. 2B).
To examine whether LdCV LC3-acqusition involves host Atg machinery we assessed LC3 association to LdCVs in Atg7+/− and Atg7−/− mouse embryonic fibroblasts (MEFs). In the Atg7−/− MEFs LC3-was not observed (Fig. 2D). Thus, this finding suggests that autophagy components engage with L. dumoffii in a process that requires the virulence activities of the pathogen.
We next examined the molecular mechanism behind LC3-decoration of L. dumoffii. Recognition of substrates decorated with ubiquitin, a 76-amino acid polypeptide, is a common feature of selective autophagy61,62, and, to date, all Legionella species tested have been shown to reside in vacuoles that are decorated with ubiquitin63. Thus, we analyzed whether L. dumoffii also resides within a ubiquitin-positive compartment that could contribute towards host recognition by autophagy. To do this, we infected THP-1 cells and assessed ubiquitin-localization using the ubiquitin (clone FK2) antibody, which detects both mono- and poly-ubiquitin conjugates but not free ubiquitin, at various stages of infection. Although LpCVs were decorated with ubiquitin, we did not detect significant localization at any stage of infection with LdCVs (Fig. 3A,B). We also examined ubiquitin-localization in cells co-infected with both L. pneumophila (mCherry/Hoechst 33342 double stained in Fig. 3C) and L. dumoffii (Hoechst 33342 single stained). Whereas ubiquitin-decoration was consistently observed for LpCVs, LdCVs found in the same cell remained ubiquitin-negative. This result further confirmed that LdCVs are largely devoid of ubiquitin. We also confirmed that LC3-positive LdCVs are largely ubiquitin-negative (Fig. 3D,F). Salmonella enterica serovar Typhimurium (S. Typhimurium)-containing vacuoles (SCVs), which are established for ubiquitin-mediated recognition by autophagy64, were found to be mostly ubiquitin-positive (~90%) (Fig. 3E,F). These results suggest that formation of LC3-positive LdCVs occurs via ubiquitin-independent mechanisms.
We next examined involvement of signaling molecules and adaptor proteins that are commonly found to target pathogens for LC3-decoration. Once again we used S. Typhimurium as a positive control because it is reported to recruit the ‘danger’ signal galectin-8 and the LC3-binding adaptors p62, NDP52, and Tecpr-1 on its vacuoles38,39,42,43,65. Consistently, localization of GFP-galectin-8, GFP-NDP52, GFP-p62 and Tecpr1-GFP was readily observed on SCVs (Figs 3G and S2). In contrast, localization of these constructs was rarely (<2%) observed on LdCVs. Thus, it appears that decoration of L. dumoffii with LC3 is largely independent of ubiquitin, galectin-8, and the adapters NDP52, p62 and Tecpr-1.
It is emerging that Atg proteins play roles in processes beyond classical autophagy66. Indeed, two degradative pathways are associated with the core autophagy marker LC3/Atg8: autophagy and LC3-associated phagocytosis (LAP). Because LAP is not associated with ubiquitinated proteins47 and LdCVs lack ubiquitin and common autophagy adaptors, we decided to determine the pathway required for LC3-association. One of major differences between autophagy and LAP is the formation of a double-membrane-bound autophagosome during autophagy versus a single-membrane-bound LAP-phagosome. Thus, we examined the membranes of LC3-positive LdCVs using correlative light and electron microscopy (CLEM) at 2.5 to 3hours post-infection (Fig. 4). This technique allows confocal and electron- micrographs to be taken of the same cell57. We examined 15 separate LC3-positive LdCVs from seven independent dishes. The vast majority of LC3-positive bacteria resided in spacious vacuoles with a single membrane (Fig. 4A–C, LdCVs #1 and #2). Small intracellular vesicles were also observed within the lumen of these enlarged LC3-positive compartments (Fig. 4C, LdCV #1, white arrows). These may be artifacts or perhaps intraluminal vesicles67. LC3-negative vacuoles had attached vesicles (Fig. 4D, white arrows) and adjacent mitochondria (Fig. 4D, black arrow), which are both common hallmarks found in EM images of LpCVs68,69. Only a single double-membrane structure was found associated with an LC3-positive LdCV (Fig. 4A,B,E, #4). We also examined a rare LC3-positive L. pneumophila vacuole and observed a multi-membrane structure that is consistent with conventional autophagy (Fig. 4F). These results are consistent with the hypothesis in which a subset of LdCVs is targeted to LAP rather than conventional autophagy.
Because TLRs have been implicated in LAP-activation50, we examined the impact of TLR signaling on LC3-acquisition using murine bone-marrow derived macrophages (BMDMs) (Fig. 5). L. pneumophila is restricted within C57BL/6J (B6) mice by flagellin-induced Naip5 (Birc1e)/Nlrc4 (IPAF)-dependent Caspase-1-mediated cell death70,71,72,73. To avoid possible complications due to murine-specific restriction, we created a mutant L. dumoffii strain lacking the flagellin-encoding gene flaA. Our L. dumoffii flaA strain was capable of robust replication within BMDMs from B6 mice (Figure S3). In BMDMs obtained from TLR2−/− knockout mice, LC3-positive LdCVs were formed at a lower level than the TLR2+/− control (Fig. 5A–C). In comparison, TLR4 signaling was found to be dispensable for LC3-acquisition (Fig. 5C). Thus, these results support the link between TLR2 and LAP, which was previously established using the TLR2 agonist zymosan50.
A mechanistic variable known to distinguish LAP from conventional autophagy is the requirement for the ULK (Ulk1-Atg13-Fip200) complex: Ulk1 (Unc51-like kinase autophagy activating kinase (1) is not required for LAP50,74,75. Conversely, conventional autophagy is Ulk1-dependent. Under non-inducing conditions this protein is functionally inhibited by the mammalian-target of rapamycin (mTOR), but after relief from repression the ULK complex activates formation of the initial isolation membrane76. We found that TLR2, but not TLR4, expression in non-immune HEK293 cells allows LdCV enhanced LC3-recruitment (compare control siRNA columns in Fig. 5D). The result on the transfectable cell-line enabled us to analyze the role of Ulk1 using siRNA knockdown. HEK293 cells were co-transfected with TLR2 and siRNA-targeting Ulk1 or scrambled siRNA. Levels of LC3-association with L. dumoffii were found to be comparable despite the reduced level of Ulk1 (Figs 5D and S4A,B). To validate the effect of Ulk1 knockdown, in uninfected cells we compared the effect of the autophagy activator rapamycin on LC3-puncta formation following scrambled or Ulk1 siRNA treatment. As expected, the addition of rapamycin to cells treated with scrambled siRNA led to increased LC3 puncta, but in Ulk1-knockdown cells LC3-puncta were absent despite rapamycin treatment (Figure S4B bottom panels). Thus, unlike conventional autophagy, the acquisition of LC3 on LdCVs is independent of Ulk1. Treatment with rapamycin has also been described to enhance levels of LC3 associated with pathogens, including S. Typhimurium77. However, treatment with this drug did not enhance LC3-decoration of L. dumoffii. Rather levels were slightly decreased (Figure S4C).
Taken all together, we concluded that LC3-recruitment to a subset of Dot/Icm T4SS+ LdCVs is mediated by LAP, a process independent of the mTOR-Ulk1 axis. Recently Rubicon was reported to be involved in LAP induction by fungal infection78. In BMDMs obtained from Rubicon−/− knockout mice, LC3-positive LdCVs were formed at a lower level than the Rubicon+/− control (Fig. 5E), providing further support for this notion. Further, unlike other pathogens that gain LC3 through multiple mechanisms, such as Salmonella, LAP appears to be the major mechanism targeting L. dumoffii for LC3-decoration.
Classical autophagy is often associated with membrane damage25,79, but whether membrane damage is important for LAP-activation is not clear. Galectin-3 has been used a marker for damaged phagosomes80. Thus, we examined the localization of galectin-3 on LdCVs as a measure of membrane integrity. As a control we again utilized S. Typhimurium, as galectin-3 has been found on SCVs42. Unlike SCVs, LdCVs were largely devoid of galectin-3 (Fig. 6A). To further examine the relationship between LAP-targeting of LdCVs and membrane damage, we next examined LC3-positive phagosomes for the presence of galectin-3. Whereas LC3-positive SCVs were commonly positive (~43%) for galectin-3, this marker was rare (<5%) on LC3-positive LdCVs (Fig. 6B,C). Taken together, our data demonstrated that LAP-activation on LdCVs is independent of membrane damage.
Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases produce reactive oxygen species (ROS) in response to phagocytosis or inflammatory mediators, and this ‘respiratory burst’ acts directly to kill invading organisms81. Additionally, a role for the NADPH oxidases in recruitment of LC3 to phagosomes (Fig. 7A), including phagosomes harboring S. Typhimurium and L. monocytogenes, was demonstrated using the NADPH oxidase inhibitor diphenyleneiodonium (DPI) and Nox2−/− mice45,46. Similarly, we found addition of 10μM DPI upon L. dumoffii infection drastically impaired LC3-acquisition (Fig. 7B). This suggests that NADPH oxidases are involved in activating LAP upon L. dumoffii infection.
In addition to other autophagic recognition mechanisms38,39,40,41,42,43,82, both S. Typhimurium and L. monocytogenes are decorated with diacylglycerol (DAG) during infection and this signal has been shown to be involved in LC3 association46,47,83. Manipulation of DAG levels using chemical inhibitors was found to perturb LC3-association with both organisms. Similarly, we found that specific chemical interventions that perturb DAG production (Fig. 7A), via inhibition of phospholipase D (PLD) by 1-butanol or phosphatidic acid phosphatase (PAP) by propranolol hydrochloride (Propr.), severely reduced association of LC3 with LdCVs (Fig. 7B). In contrast tert-butanol, an isomer of 1-butanol that has no inhibitory effect on PLD, had no effect on LC3 association with LdCVs. Normally, DAG kinase (DGK) reduces DAG levels by conversion of DAG to phosphatidic-acid. By inhibiting DGK with DGK inhibitor (R59022), which is expected to promote DAG accumulation, we reproducibly observed slightly but not significant enhanced levels of LC3-association with LdCVs (Fig. 7B). Furthermore, our data suggests both PAP and PLD function during L. dumoffii infection in a manner promoting LC3-association. The link between DAG and LC3 is thought to be PKCδ-binding via its C1-domain to vacuolar DAG, and subsequent activation of JNK and NADPH oxidases47. Indeed, inhibition of JNK activation using the drug SP600125 also reduced association of LC3 with LdCVs, though not as completely as inhibition of DAG production or NADPH oxidases (Fig. 7B). Taken together, our results support a role for both TLR2 and ROS generation via DAG-NADPH oxidases axis in triggering LAP.
To elucidate whether this host response limits L. dumoffii inside host cells, or whether L. dumoffii utilizes Atg-proteins for its intracellular survival strategy we first examined whether compartments positive for both LC3 and LAMP-1 exist. Classically, when LC3-positive autophagosomes fuse with lysosomes, autophagolysosome will be decorated with both LC3 and LAMP-1 before degradation of phosphatidylethanolamine (PE)-conjugated LC3 found within the lumen of the membrane-bound compartment. In RAW cells stably expressing GFP-LC3, LC3-positive LdCVs were also positive for LAMP1 by indirect immunofluorescence (Fig. 8A). In THP-1 cells, using dual staining, we found that at 3hours post-infection ~50% of LC3-positive LdCVs were also positive for LAMP-1 (Fig. 8B,C). Thus, delayed acquisition of LAMP-1 of LdCVs is associated with LAP. As a control we also examined LC3-positive LdCVs for the presence of ubiquitin. LdCVs were rarely positive (~10%) for both LC3 and ubiquitin, consistently with our previous finding that only ~2% of total LdCVs are ubiquitin-positive (Fig. 3B).
To examine the fate of bacteria within LC3-positive LdCVs we then performed live imaging experiments. We did not observe LC3-positive LdCVs being expelled from the cells, as is the case for uropathogenic E. coli67, but rather the mCherry expressing bacteria within LC3-positive compartments disappeared over time (Figure S5). Given that a number of pathogens interfere with host processes that acidify lysosomes to create a niche in which to replicate, we also confirmed that LdCVs become acidified by live imaging of RAW cells stably expressing GFP-LC3 infected with Hoescht-stained L. dumoffii strains in the presence of LysoTracker® Red (Figure S6). As a positive control we tracked dotA mutant L. dumoffii. dotA mutants were found within LC3-negative but acidic (lysotracker-positive) compartment shortly after infection, which is consistent with trafficking through the canonical endocytic pathway (Figure S6A, top panels). Conversely, acidified L. dumoffii vacuoles were not observed at one hour post-infection (Figure S6A, bottom panels). However, at 3hours, LC3-positive but not acidified vacuoles were mainly observed and at 4–5hours a positive lysotracker signal was associated with ~10% of L. dumoffii and LC3-positive LdCVs were rare (Figure S6B). Thus, acidification occurs post acquisition of LC3. This data further supports degradation of pathogenic L. dumoffii that are recognized by Atg-components.
Because LAP of L. dumoffii results in bacterial degradation, we hypothesized that this innate immune response acts to restrict L. dumoffii. We examined this hypothesis using a bacterial viability-based assay. In wild-type MEF cells, ~25% more L. dumoffii remained viable in comparison with in Atg7 knockout MEF cells at five hours post infection (Fig. 8D). Likewise, in BMDMs derived from TLR2−/− knockout mice, ~80% more L. dumoffii remained viable in comparison with in BMDMs derived from TLR2+/− mice (Fig. 8E). Taken together, these results demonstrated that LAP can limit intracellular survival of a virulent bacterial pathogen that resides within a membrane-bound compartment within host cells.
Some L. pneumophila strains including Lp01 we used in this study encode the effector protein RavZ, which irreversibly deconjugates LC3 from phospholipid membrane17 and thus interferes LAP78. To explore the exact role of RavZ in LAP avoidance of LpCVs, we examined ubiquitin and LC3 recruitment to LpCVs containing a RavZ detetion strain in THP-1 cells at three hours post infection, In this condition we observed LC3 recruitment to LdCVs but rarely to LpCVs (Fig. 9A,B) like LpCVs containing the wild-type strain (Fig. 3B). This clearly indicates that the LAP avoidance of L. pneumophila is not solely due to the effector RavZ function.
On the other hand, L. dumoffii NY23 used in this study does not encode RavZ ortholog. When L. pneumophila RavZ or its catalytic mutant derivative (RavZC258A) was ectopically expressed in L. dumoffii, the resulting LdCVs failed to acquire LC3 in a RavZ activity-dependent manner (Fig. 9C), suggesting that the catalytic activity of RavZ is sufficient to block LAP as previously reported78.
To lay a foundation for studies examining species-specific Legionella effector functions during intracellular infection, we set out to phenotypically compare L. pneumophila and the largely unstudied pathogen L. dumoffii. L. dumoffii was found to share some of L. pneumophila’s intracellular survival strategies, namely inhibition of the canonical endocytic pathway and creation of an ER-like intracellular niche. However, we identified two points of difference between the organisms: L. dumoffii’s decoration with LC3 and lack of vacuole ubiquitination. Extending this insight, we show that LAP is the main process for LC3-decoration of L. dumoffii in host cells. LC3-decoration of LdCVs was also found to be Dot/Icm T4SS-dependent (Fig. 2). Thus, we propose that the majority of pathogenic (T4SS+) L. dumoffii survive in host cells by creating an ER-like intracellular niche, but a portion of L. dumoffii are recognized by the host and decorated with LC3 in a ubiquitin-independent manner consistent with LAP (Fig. 10). Our data are consistent with the previous report that found ~20% of intracellular L. dumoffii were LAMP-1 positive at 4hours post-infection in J774 mouse macrophages84.
The relationship between Dot/Icm-dependent LdCV maturation and LAP prompts us to question whether there is a link between phagosome maturation and LAP initiation. Regarding the balance of LAP-targeting over autophagy, our data indicates that vacuolar integrity is a likely contributing factor. Based on the lack of galectin-3 (or any other galectins we tested) association with LdCVs (Fig. 6), LAP-activation appears independent of membrane damage. Thus, vacuolar integrity of LdCVs may skews recognition away from membrane-damage responsive autophagy25,79. Alternatively, it remains possible that our membrane damage marker (galectin-3) is somehow removed from LdCVs. LAP’s predominance may simply be due to the absence of phagosomal ubiquitin. In this scenario, TLR- and DAG-signaling and delayed phagosome maturation would provide positive LAP-activation signals and ubiquitin-negative intact phagosomes would avoid activation of classical autophagy.
Why LdCVs are subjected to LAP but LpCVs are not is an intriguing question. The L. pneumophila strain Lp01 used in this study produces the effector protein RavZ, which has an activity to irreversibly deconjugate LC3 and thus inhibit autophagy-related pathways including macroautophagy and LAP17,78. We examined the behavior of a RavZ deletion derivative and found that RavZ is not required for the LAP avoidance by L. pneumophila (Fig. 9A,B). This suggests that either L. pneumophila has additional effectors to inhibit LAP or L. dumoffii has an effector to promote LAP. The effector Spl36 is one of such candidates which may inhibit LAP, however, L. dumoffii appears to encode the Spl ortholog. On the other hand, L. dumoffii ectopically producing RavZ failed to engage in LAP (Fig. 9C), indicating that L. dumoffii lacks sophistication to have a single effector protein like RavZ which efficiently blocks autophagy-related pathways, unlike some strains of L. pneumophila. Clarification of molecular mechanisms underlying L. dumoffii engagement in LAP awaits future study.
A remaining question is the physiological importance of LAP for bacterial restriction in the context of other immune defense mechanisms. Recently, LAP has been proposed to facilitate enhanced antigen presentation by delaying phagosome maturation85. It is tempting to speculate that in higher eukaryotes, LAP-mediated enhanced antigen presentation by macrophages infected with L. dumoffii engages innate or adaptive responses that limit disease progression, whereas the LAP-avoiding pathogen like L. pneumophila avoids this enhanced host response and causes more disease.
Rubicon is a Class III PI(3) kinase-associated protein86, which recruits PI(3) kinase to phagosomes and supports prolonged localization of PI3P78. Then Rubicon interacts with the NOX2 complex recruited in a PI3P-mediated manner and stabilizes the NOX2 complex, resulting induction of ROS production78. In contrast, LpCVs were reported to acquire PI3P right after infection in a model natural host Dictyostelium discoideum87, but soon PI4P becomes a dominant phosphoinositide species, which possibly involves the concerted actions of host PI(4) kinases, and host and bacterial phosphoinositide phosphatases88,89,90,91. Currently phosphoinositide dynamics on LdCVs are not known, and future studies focusing on PI3P pool on LAP-targeted versus LAP-escaping LdCVs will shed light on the requirement in LAP induced by fungal and bacterial infection, and the difference in fates of LdCVs and LpCVs. Furthermore LAP-targeting of LdCVs may be used as a model system examining the finer mechanistic details of LAP, particularly induced by bacterial infection.
Unless otherwise noted, all chemicals were purchased from Sigma. Rapamycin was purchased from Santa Cruz (sc-3504). Restriction and molecular cloning enzymes were purchased from New England Biolabs or Toyobo Co. Ltd. Primary antibodies used include rabbit polyclonal antibodies to L. pneumophila (Biodesign #B65051G) and Legionella dumoffii (Denka Seiken). Primary antibodies used for immunofluorescence experiments are listed in figure legends. Secondary conjugate antibodies Alexa Fluor® 488 (Life Technologies #A11029), Alexa Fluor® 488 (Life Technologies #A11034), Rhodamine Red-X (Life Technologies #R6393), and Rhodamine Red-X (Life Technologies #R6394) and Alexa Fluor® 568 (Life Technologies #A11077) were purchased from Thermo Fischer Scientific.
Bacterial strains used in this study are described in Table S1. Legionella strains were grown on charcoal-yeast extract plates or in aces-buffered yeast extract broth as already described92. When required, drugs were included in the bacteriological media at the following concentrations: for strains of Legionella species, streptomycin 100μg/ml, chloramphenicol 3μg/ml and kanamycin 10μg/ml; for strains of E. coli, ampicillin 100μg/ml, kanamycin 25μg/ml and chloramphenicol 20μg/ml; for Salmonella enterica serovar Typhimurium (S. Typhimurium), chloramphenicol 20μg/ml.
Bacterial plasmids used in this study are listed in Table S2. pMMB207mCherry was created by cloning mCherry into pAM239. To induce constitutive expression of mCherry an internal region of the lacI repressor was disrupted. To create the dotA, flaA L. dumoffii and ravZ L. pneumophila mutants, the plasmids pSR47SLddotA, pSR47SLdflaA and pSR47SLdravZ were created using primer pairs list in Table S3. Clones were sequenced using the primers pSR47Sac1 and pSR47SXba1. The ravZ C258A mutation was introduced using QuickChange II Site-Directed Mutagenesis Kit (Agilent Technologies) according to the manufacture’s instruction. The lentiviral vector for the stable expression of GFP-LC3 was generated as follows. The genes encoding GFP and LC3 were amplified using the primer pairs listed in Table S3 with pEGFP-LC393 as a template. The PCR product was subsequently amplified with attB adaptor primers and cloned into pDONR201 (Invitrogen) with Gateway BP Clonase II Enzyme Mix (Invitrogen). The sequence encoding GFP-LC3 was then transferred to the lentiviral expression vector pLEXEF.pur94, using Gateway LR Clonase II Enzyme Mix (Invitrogen), to obtain pLEXEF.pur.GFP-LC3.
Strain Ld00 is a spontaneous streptomycin-resistant mutant of L. dumoffii NY23. Chromosomal deletion mutants of L. dumoffii were made using the pSR47S-based plasmids described above and following the method previously described for L. pneumophila95. Legionella strains constitutively expressing mCherry were created by introduction of pMMB207::mCherry by electroporation using the conditions described for L. pneumophila2. S. Typhimurium expressing pMMB207::mCherry was also created by electroporation and selection on chloramphenicol resistance.
HEK293 cells stably expressing the FcγRII receptor were obtained from the laboratory of Craig Roy (Yale University, USA)96. HeLa cells lines stably expressing the FcγRII were created and kindly provided by Dr. Kohei Arasaki (Tokyo University of Pharmacy and Life Sciences, Japan). Wild-type MEF cells were provided by Dr. Miwa Sasai (Osaka University, Japan). Immortalized Atg7-deficient MEFs were kindly provided by Dr. Tatsuya Saitoh (Osaka University, Japan) on behalf of Dr. Masaaki Komatsu (Niigata University, Japan). TLR297, TLR498, Rubicon99 KO mice were previously described. Murine BMDMs were obtained from mice as previously described100, and used fresh or stored as described101. All mammalian cell lines were maintained in 5% CO2 at 37°C. Mouse embryonic fibroblasts (MEF), HeLa, HEK293 and FcγRII-expressing stable variants96 were maintained in Dulbecco’s modified Eagle’s growth medium (Gibco, Life Technologies) supplemented with 10% fetal calf serum. THP-1 cells were routinely passaged as non-adherent cells. RAW264.7 and THP-1 cells were cultured in RPMI 1640 medium (Gibco, Life Technologies) supplemented with 10% fetal calf serum. For knockdown experiments, HEK293 cells were seeded into 24-well tissue culture plates to achieve 40% confluency 24hours later when plasmid DNA and siRNA was added to wells. Two-days later, media was changed to DMEM plus 5% FBS for the final overnight incubation. Infection experiments in complete media were performed at 72hours post-transfection. For infection experiments with THP-1 cells, cells were differentiated in tissue culture wells at densities of 2×105 cells per well of the 24-well dishes and 5×104 cells for the 96-well dishes. Three days prior to use, THP-1 cells were incubated in media containing phorbol 12-myristate 13-acetate (PMA, Sigma). After 24–48hours media was replaced with fresh media lacking PMA. The next day, infection experiments were performed. To create the RAW 264.7 cell line stably expressing GFP-LC3, the ViraPower Lentiviral Expression System (Invitrogen) was used to produce lentiviruses, and the resulting viral supernatant was used to transduce the lentiviral construct into RAW 264.7 cells. Subsequently, stable GFP-LC3-expressing cells were established by selection with puromycin (Clontech). RAW cells stably expressing GFP-LC3 were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum.
HEK293 and HeLa cell transfections were performed using Lipofectamine 2000 (Invitrogen) or Lipofectamine LTX+ (Invitrogen) according to the manufacturer’s instructions. For co-transfection assays in HEK293 cells, plasmid DNA (200 ng) was mixed with siRNA (50nM) and Lipofectamine 2000 before co-transfection into cells. Scrambled siRNA and Ulk1 siRNA for RNAi experiments were ordered from Sigma Genosys. The sequence of the Ulk1 siRNA used was previously reported102. Lipofectamine LTX+ (Invitrogen) was used to transfect MEF cells using 1.75μl of LTX, 0.5μl ‘plus’ reagent and 0.5μg DNA per well of the 24-well dishes.
Infections with L. pneumophila or L. dumoffii were performed with bacteria growth in 2-ml AYE both cultures for 20hours. The final OD600 of L. dumoffii cultures used for infection was ~2.8 (range of 2.7 to 3.2), whereas L. pneumophila cultures were used at ~4.0 (range of 3.8 to 4.4). HEK293 FcγRII and HeLa FcγRII cells were infected with L. dumoffii at a multiplicity of infection (moi) of one as previously described for L. pneumophila90. For non-FcγRII-mediated uptake, an moi of 100 to 300 was used. For infection of RAW264.7 cells stably expressing GFP-LC3, THP-1 and MEF cells an moi of 10 to 30 was used. For dual infection experiments of L. pneumophila mCherry and L. dumoffii, 10 times more L. pneumophila was used than L. dumoffii because we observed better uptake of L. dumoffii than L. pneumophila. To propagate Salmonella for infections, overnight cultures were diluted to OD600 of 0.17 and grown for ~3hours until the OD600 reached 1.8–2.5, and finally bacteria were added to cells at an moi of 100. At 20min post-infection, 100μg/ml gentamycin was added and maintained until cells were washed and fixed. In all experiments cells were fixed with 4% PFA for 15min at 37°C. After staining coverslips were stained with Hoechst at 1μg/ml and mounted using the ProLong gold antifade reagent (Invitrogen). For growth curves in mammalian cells an MOI of 0.5 was used and performed as previously described90. For experiments using C57BL/6 BMDM and MEF cells, infections were performed using L. dumoffiiflaA.
Knockdown efficiency was validated by quantitative RT-PCR using ThunderbirdTM SYBR® qPCR mix on cDNA synthesized by the SuperScriptTM III (Invitrogen#18080–051) kit from total RNA extracted from cells at 72h post-knockdown using the RNeasy kit (Qiagen). The primer pairs used to assess transcript levels are listed in Table S2.
Epifluorescence micrographs were taken using a TE2000 (Nikon) inverted microscope and this microscope was used for all counting experiments. Confocal micrographs were taken using a LSM510 microscope (Zeiss) with a 100x/1.4 numerical aperture objective, or a Fluoview FV10i (Olympus) microscope with a 60×/1.35 numerical aperture objective. Correlative light electron microscopy (CLEM) experiments were performed as previously described103. Basically, RAW264.7 cells stably expressing EYGP-LC3 were cultured on glass bottom dishes (with grids) and then infected with mCherry-expressing Legionella dumoffii for 2.5–3hours. After fixing, confocal images were taken before processing the samples for electron microscopy.
To perform the L. dumoffii survial assay in Atg7+ and Atg7- MEF cells, MEFs were seeded at 5×104 per well in 24-well format - to reach ~80% confluency at the time of infection. Fresh 2-day heavy patch L. dumoffiiflaA expressing mCherry were grown overnight in AYE plus chloramphenicol broth until OD600 reached 2.8–3.0. Bacteria were added to wells at 2×105 per well. Plates were spun at 1000rpm for 5minutes and then incubated for 5hours under standard cell culture conditions. At 5hours post-infection, wells were gently washed two times with PBS to remove extracellular bacteria. Cells were then lysed in 1ml of sterile water per well. To promote host cells lysis, cells were freezed and thawed before vigorously resuspending the contents of each well and plating for bacterial viability on CYE media. For each cell type, sixteen independent wells were assessed from four separate plates and each well was plated in duplicate. Data are shown compared to control cells (wild-type) average bacterial survival, which was normalized to 100%. Significance was determined by the students’ T-Test.
Differentiated BMDMs were seeded in 24-well plates at 1.5×105 cells per well. The following day, L. dumoffiiflaA expressing mCherry were added at 5×105 per well and spun for 5min at 1000rpm. At 5hours post-infection, wells were treated as described for MEF cells with a final well volume of 1ml. One hundred μl of 1/100 dilutions were plated to enumerate bacterial survival. In triplicate experiments, six wells were assessed for each cell type. Data are shown compared to control cells (wild-type) average bacterial survival, which was normalized to 100%. Significance was determined by the students’ T-Test.
To statistically assess significance, calculations were performed using the paired Student’s t-test (homoscedastic two-tailed, paired) using Excel software (Microsoft). In all graphs error bars represent standard error of the mean (SEM). Where appropriate, p values or ns for not significant were denoted.
All animal experiments were performed in accordance with the institutional guidelines and were approved by the Animal Care and Use Committee of the Research Institute for Microbial Diseases, Osaka University, Japan (Biken-AP-H26-10-0, IFReC-AP-H27-07-0).
How to cite this article: Hubber, A. et al. Bacterial secretion system skews the fate of Legionella-containing vacuoles towards LC3-associated phagocytosis. Sci. Rep. 7, 44795; doi: 10.1038/srep44795 (2017).
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We thank members of the Nagai laboratory for helpful advice. Hiroko Omori’s role in CLEM experiments is much appreciated. We are grateful to F. Higa (Ryukyu Univ.) who provided us L. dumoffii strains. We also gratefully acknowledge those who provided materials including K. Arasaki, M. Sasai and T. Saitoh for cells, as indicated in materials and methods. This work was supported in part by MEXT/JSPS KAKENHI Grants 15H01322, 16H05189, 16K14724 (to T. Ku) and 15H04728 (to H.N.).
The authors declare no competing financial interests.
Author Contributions A.H., T. Ku and H.N. designed research; A.H. and T. Ku performed research; A.H., T. Ku and H.N. analyzed data; C.C., T. Ka and T.Y. assisted mouse experiments and discussed the data; T.M. assisted CLEM experiments and discussed the data; M.O. assisted experiments on autophagic pathways and the discussed data; A.H., T. Ku and H.N. wrote the paper.