Abstract
The cytoskeleton network of eukaryotic cells is essential for diverse cellular processes, including vesicle trafficking, cell motility and immunity, thus is a common target for bacterial virulence factors. A number of effectors from the bacterial pathogen Legionella pneumophila have been shown to modulate the function of host actin cytoskeleton to construct the Legionella-containing vacuole (LCV) permissive for its intracellular replication. In this study, we identified the Dot/Icm effector Lem8 (Lpg1290) as a protease that interferes with host motility. We show that the protease activity of Lem8 is catalyzed by a Cys-His-Asp motif known to be associated with diverse biochemical activities. Intriguingly, we found that Lem8 interacts with the host regulatory protein 14-3-3ζ, which activates its protease activity. Furthermore, Lem8 undergoes self-cleavage in a process that requires 14-3-3ζ. We identified the PH domain-containing protein Phldb2 involved in cell migration as a target of Lem8 and demonstrate that Lem8 plays a role in the inhibition of host cell migration. Our results reveal a novel mechanism of inhibiting host cell motility by L. pneumophila for its virulence.
Introduction
Legionella pneumophila is a Gram-negative intracellular bacterial pathogen ubiquitously found in freshwater habitats, where it replicates in a wide range of amoebae (Richards et al., 2013). It is believed that these natural hosts serve as the main replication niches for L. pneumophila in the environment and provide the primary evolutionary pressure for the acquisition and maintenance of virulence factors necessary for its intracellular lifecycle. Infection of humans by L. pneumophila occurs when susceptible individuals inhale aerosols generated from contaminated water, which introduces the bacterium to the lungs where it is phagocytosed by alveolar macrophages. Instead of being digested and cleared, internalized bacteria replicate within a membrane-bound compartment termed Legionella-containing vacuole (LCV), leading to the development of Legionnaires’ disease, a form of severe pneumonia (Cunha et al., 2016).
One feature associated with the LCV is its ability to evade fusion with the lysosomal network in the early phase (<8 h post-infection in mouse bone marrow-derived macrophages(BMDMs)) of its development and the quick acquisition of proteins of the endoplasmic reticulum (ER) origin (Kagan and Roy, 2002; Sturgill-Koszycki and Swanson, 2000; Swanson and Isberg, 1995). Biogenesis of the LCV requires the Dot/Icm type IV secretion system that injects more than 300 effector proteins into host cells (Qiu and Luo, 2017). These effectors function to modulate a wide cohort of host processes, including vesicle trafficking (Tan et al., 2011), protein synthesis (Shen et al., 2009), lipid metabolism (Gaspar and Machner, 2014), and autophagy (Choy et al., 2012) by diverse biochemical mechanisms. Coordinated activity of these effectors leads to the formation of the LCV which largely resembles the ER in its morphology and protein composition (Qiu and Luo, 2017).
The cytoskeleton of eukaryotic cells is composed of microfilaments derived from actin polymers, intermediate filaments and microtubules, which play distinct roles in maintaining cell shape, migration, endocytosis, intracellular transport and the association of cell with the extracellular matrix and cell-cell interactions (Jones et al., 2019). Due to its essential role in these cellular processes, components of the cytoskeleton, particularly the actin cytoskeleton is a common target for infectious agents. For example, Salmonella enterica Typhimurium utilizes a set of type III effectors, including SipC, SopE and SptP to reversibly regulate the rearrangement of host actin cytoskeleton to facilitate its entry into non-phagocytic cells (Kubori and Galan, 2003). Other bacterial pathogens such as Chlamydia, Orientia tsutsugamushi, and Listeria also exploit the actin cytoskeleton and microtubule networks to promote their movement in the cytoplasm of host cells and cell to cell spread (Cheng et al., 2018; Grieshaber et al., 2003; Kim et al., 2001).
Growing evidence indicates that manipulation of the actin cytoskeleton dynamics plays an important role in the intracellular lifecycle of L. pneumophila. It has been documented that chemical interference of the actin cytoskeleton structure impedes bacterial entry and replication (Charpentier et al., 2009). A number of Dot/Icm effectors have been shown to impose complex modulation of the host actin cytoskeleton. Among these, VipA promotes actin polymerization by functioning as a nucleator (Franco et al., 2012). LegK2 appears to inhibit actin nucleation by phosphorylating the Arp2/3 complex (Michard et al., 2015). The protein phosphatase WipA participate in this regulation by dephosphorylating several proteins involved in actin polymerization, including N-WASP, NCK1, ARP3, and ACK1, leading to dysregulation of actin polymerization (He et al., 2019). RavK is a metalloprotease that cleaves actin in host cells, abolishing its ability to form polymers (Liu et al., 2017). Ceg14 also appears to inhibit actin polymerization by a yet unrecognized mechanism (Guo et al., 2014). Interestingly, LegG1 has been demonstrated to promote microtubule polymerization and host cell migration by functioning as a guanine nucleotide exchange factor (GEF) for the Ran GTPase (Rothmeier et al., 2013; Simon et al., 2014). Counterintuitive to the role of LegG1, cells infected by L. pneumophila display defects in migration in a way that requires a functional Dot/Icm system (Simon et al., 2014), suggesting the existence of effectors that function to block cell migration.
Herein, we demonstrate that the L. pneumophila effector Lem8 (Lpg1290) (Burstein et al., 2009) is a cysteine protease that functions to inhibit host cell migration by targeting the microtubule-associated protein Phldb2 via a mechanism that requires the regulatory protein 14-3-3ζ.
Results
Lem8 is a Legionella effector with putative cysteine protease activities
One major challenge in the study of bacterial effectors is their unique primary sequences that share little similarity with proteins of known function. Bioinformatics analysis has been proven useful in the identification of putative cryptic functional motifs embedded in their structures. We used PSI-BLAST to analyze a library of the L. pneumophila Dot/Icm effectors (Zhu et al., 2011) and found that Lem8 harbors a putative Cys280-His391-Asp412 catalytic triad present in a variety of cysteine proteases (Fig. 1A). Further analysis by HHpred (Soding et al., 2005) revealed that Lem8 has high probability to have structural similarity with HopN1 and AvrPphB from Pseudomonas syringae (Rodriguez-Herva et al., 2012; Shao et al., 2002), as well as YopT from Yersinia enterocolitica (Shao et al., 2002) and PfhB1 from Pasteurella multocida (Shao et al., 2002) (Fig. S1).
Lem8 is a protein of 528 residues coded for by the gene lpg1290 in L. pneumophila strain Philadelphia 1, it was first identified as a substrate of the Dot/Icm transporter by a machine learning approach (Burstein et al., 2009). The translocation of Lem8 by the Dot/Icm system into host cells during L. pneumophila infection was later validated by two independent reporter systems (Huang et al., 2011; Zhu et al., 2011). Consistent with these results, we observed Dot/Icm-dependent translocation of Lem8 into host cells using the β-lactamase- and CCF2-based reporter assay. Approximately 60% of the cells infected with a Dot/Icm competent strain expressing the β-lactamase-fusion emited blue fluorescence signals. No translocation was detected when the same fusion was expressed in the dotA- mutant defective in the Dot/Icm system (Berger and Isberg, 1993) (Fig. 1B).
The expression of many Dot/Icm substrates peaks in the post-exponential phase, probably due to the demand for high quantity of effectors to thwart host defense in the initial phase of LCV construction (Luo and Isberg, 2004; Segal, 2013). Thus, we evaluated the expression pattern of lem8 throughout the entire growth cycle of L. pneumophila in broth. Intriguingly, unlike most of effectors, the expression of lem8 is detected at high levels in the lag phase of its growth cycle in bacteriological medium. A decrease in protein abundance is detected 9 h after the subcultures have started and is maintained constant throughout the remaining 15 h experimental duration (Fig. 1C). These results suggest that Lem8 may play a role in the entire intracellular lifecycle of of
L. pneumophila
Next, we attempted to determine whether the putative cysteine protease motif is important for the effects of Lem8 on eukaryotic cells. We first tested whether Lem8 is toxic to yeast and if so, whether the Cys280-His391-Asp412 motif is required for such toxicity. Expression of Lem8 from the galactose-inducibe promoter caused cell growth arrest (Fig. 1D). Mutations in Cys280, His391 or Asp412 did not affect the stability of the protein in yeast, but abolished such toxicity (Fig. 1D). Thus, the putative cysteine protease activity conferred by the predicted Cys280-His391-Asp412 catalytic triad very likely is important for the effects of Lem8 on eukaryotic cells.
Genomic analysis reveals that in addition to L. pneumophila, lem8 or its homolog is present only in L. waltersii, one of the 40 Legionella species whose genomed had been fully sequenced (Burstein et al., 2016). Such a low prevalence suggests that Lem8 plays a role in the survival of the bacteria in specific inhabits, or its role in other Legionella species is substituted by genes of little sequence similarity that may have arisen by convergent evolution. We probed the role of lem8 in L. pneumophila virulence by examining intracellular replication of the Δlem8 mutant in the protozoan host Dictyostelium discoideum and in BMDMs. In both host cells, intracellular growth of the Δlem8 mutant was indistinguishable to that of the wild-type strain (Fig. S2), which is akin to most mutants lacking one single Dot/Icm substrate gene (Qiu and Luo, 2017).
Lem8 directly interacts with the regulatory protein 14-3-3ζ
To identify the host target of Lem8, we performed a yeast two-hybrid screening against a mouse cDNA Library (Clontech) using Lem8C280S fused to the DNA binding domain of the transcriptional factor GAL4 as bait. Plasmid DNA of the library was introduced into the yeast strain PJ69-4A (James et al., 1996) expressing the bait fusion and colonies appeared on selective medium were isolated and the inserts of the rescued plasmids capable of conferring the interactions were sequenced. We found that 50 out of the 96 independent clones analyzed harbored portions of the gene coding for 14-3-3ζ, a member of a chaperone family important for the activity of a wide variety of proteins in eukaryotic cells (Pennington et al., 2018). Robust interactions occurred in the yeast two- hybrid system when full-length 14-3-3ζ was fused to the AD domain of Gal4 (Fig. 2A).
We further explored the interactions between 14-3-3ζ and Lem8 by reciprocal immunoprecipitation (IP) assays. Flag-tagged 14-3-3ζ was coexpressed with GFP- tagged Lem8 or GFP in HEK293 cells. IP using the Flag antibody specifically precipitated GFP-Lem8, whereas GFP was not detectable in similar experiments. Reciprocally, IP with GFP antibodies specifically pulled down Flag-tagged 14-3-3ζ (Fig. 2B). These results suggest that Lem8 forms a complex with 14-3-3ζ in mammalian cells.
To determine whether Lem8 directly binds to14-3-3ζ, we purified recombinant proteins and used GST pulldown assasy to analyze their interactions. We found that mixing His6-Lem8 and GST-14-3-3ζ in reactions led to the formation of stable protein complexes that can be retained by GST beads (Fig. 2C).
Members of the 14-3-3 family commonly recognize phospho-serine and/or phospho- threonine sites of client proteins for binding (Muslin et al., 1996). Yet, we did not detect phosphorylation on Lem8 purified from mammalian cells or E coli using a pan phospho- serine/threonine antibody. As a control, this antibody detected phosphorylation on CTNNB1, a known phosphorylated target of 14-3-3ζ (Tian et al., 2004). As expected, no signal was detected for ExoS, a non-phosphorylated 14-3-3 interacting effector from P. aeruginosa (Henriksson et al., 2002) (Fig. S3).
To determine the region in Lem8 involved in binding 14-3-3ζ, we constructed a series of Lem8 deletion mutants and examined their interactions with 14-3-3ζ by immunoprecipitation. Whereas removing as few as 25 residues from the amino terminal end of Lem8 abolished its ability to bind 14-3-3ζ, a Lem8 mutant lacking the last 50 residues can still robustly interact with 14-3-3ζ, and deleting an additional 50 residues from this end abolished the binding (Fig. 2D). Thus, either 14-3-3ζ recognizes a large region of Lem8 or deletion from either end of this protein caused significant disruptions in its structure and abolished its ability to interact with 14-3-3ζ.
Lem8 undergoes 14-3-3ζ-dependent auto-cleavage
Since Lem8 harbors the predicted Cys280-His391-Asp412 catalytic triad associated with proteases from diverse bacterial pathogens, we next investigated whether Lem8 cleaves 14-3-3ζ. Incubation of recombinant His6-Lem8 with His6-14-3-3ζ at room temperature for 2 h did not lead to detectable cleavage of 14-3-3ζ. Unexpectedly, a protein with a molecular weight slightly smaller than that of Lem8 was detected in this reaction (Fig. 3A). The production of this smaller protein did not occur in reactions that contained the Lem8C280S mutatant or when the cysteine protease-specific inhibitor E64 was included in the reactions (Fig. 3A), suggesting that this band represents a fragment of Lem8 produced by self-cleavage. Intriguingly, the self-cleavage did not occur in samples containing only Lem8, suggesting that the self-cleavage activity of Lem8 requires 14-3- 3ζ as a co-factor.
Dictyostelium discoideum, the protozoan host of L. pneumophila codes for one 14- 3-3 protein with 66% identity and 78% similarity to that of human 14-3-3ζ (Eichinger et al., 2005), we investigated whether the D. discoideum 14-3-3 (14-3-3Dd) can activate Lem8 by incubating His6-Lem8 with GST-14-3-3Dd or human 14-3-3ζ (14-3-3ζHs). In each case, we observed the production of a protein with a size clearly smaller than Lem8 as early as 2 h after the reaction has started. (Fig. 3B). Thus, Lem8 can be activated by 14-3-3 from both humans and a protist.
To determine the self-cleavage site of Lem8, we incubated His6-Lem8 with His6-14- 3-3ζ at room temperature for 16 h. Proteins resolved by SDS-PAGE were stained with Coomassie brilliant blue and bands corresponding to full-length and cleaved Lem8 were excised, digested with trypsin and sequenced by mass spectrometry, repsectively (Fig. 3C). Analysis of the tryptic fragments identified a semi-tryptic fragment - A468PQPTPQRQ476- present in the cleaved protein but not in the full-length protein, suggesting that the cleavage occurs between Gln476 and Arg477 (Fig. 3C). To narrow down the potential self-cleavage site, we compared the abundance of identified tryptic peptides from the full-length and cleaved Lem8 and found that the abundance of - A468PQPTPQR475- was similar between two sets of samples, whereas peptide -A478QSLSAETER487- was present only in full-length samples but not in the cleaved ones (Fig. S4A), suggesting that the cleavage site was between R475 and R487. Consistent with this notion, the signal of a semi-tryptic fragment -L464CEKAPQPTPQRQ476- was identified in the cleaved protein but not in the full-length protein, suggesting that the cleavage occurs between Gln476 and Arg477 (Fig. 3C).
To determine whether Lem8 undergoes auto-cleavage via the recognition of the protein sequence around Gln476, we introduced mutations to replace residues Pro473, Gln474, Arg475 and Gln476 with alanine and incubated this Lem8 mutant (called 4A) with 14-3-3ζ. Unexpectedly, although at a lower rate, self-cleavage still occurred in this mutant (Fig. 3D). We further examined the self-cleavage of Lem8 by fusing GFP to the carboxyl end of Lem8, Lem8C280S and Lem8 △ C50, respectively. These fusion proteins were expressed in HEK293T cells and the cleavage was probed by immunoblotting with GFP- specific antibodies. We found that a fraction of Lem8-GFP and Lem8△C52-GFP has lost the GFP portion of the fusions, an event that did not occur in Lem8C280S-GFP (Fig. S4B). Thus, although the amino acids adjacent to Gln476 play a role in its self-cleavage, other factors such as the overall structure of Lem8 may contribute to the recognition of the cleaving site.
Lem8 targets Phldb2 for cleavage
It has been reported that some bacterial cysteine proteases cleave both themselves and their substrates in the host by recognizing sites with similar sequences. For instance, AvrpphB and Avrrpt2, two type III effectors from P. syringae cleave themselves as well as their host targets PBS1 and RIN4, respectively (Chisholm et al., 2005; Shao et al., 2003). Importantly, in each case, the sequences of the recognition sites for both self- cleavage and cellular target cleavage are very similar. In fact, this feature has been exploited to predict the potential host substrates of these effectors by bioinformatic analyses. Therefore, we performed BLAST searches and obtained 10 candidate proteins that contain sequence elements resembling the self-cleavage site of Lem8, including Phldb2, Rasgrp2, Pak6, Exoc8, Ankrd13B, Chkb, Ppp6R1, Kiaa1033, Gnal and Gpr61.
The predicted recognition sites in these proteins locate in the middle or at sites close to either their amino or carboxyl ends (Fig. 4A). Further experiments revealed that one of the candidates, Pleckstrin homology-like domain family B member 2 (Phldb2) can be cleaved by Lem8 in a process that requires an intact Cys280-His391-Asp412 catalytic triad. The predicted Lem8 recognition site locates between the 1106th residue and the 1119th residue in this protein of 1253 amino acids (Fig. 4A). In HEK293T cells, expression of Lem8 led to a considerable reduction of endogenous Phldb2 (Fig. 4B). To confirm this finding, we added an HA and a Flag tag to the amino and carboxyl end of Phldb2 respectively, and co-expressed the double tagged protein in HEK293T cells with Lem8 or each of the mutants with mutations in one of the three sites (C280S, H391A and D412A) predicted to be critical for catalysis. Detection of tagged Phldb2 by immunoblotting with the Flag-specific antibody indicated that the protein levels in cells expressing Lem8 were reduced comparing to samples in which the catalytically inactive mutants were expressed. Athough to a lesser extent, reduction in Phldb2 was also observed in experiments in which the tagged protein was detected with the HA antibody (Fig. 4C).
Phldb2 is a phosphatidylinositol-3,4,5-triphosphate (PIP3) binding protein and is associated with the plasma membrane (Paranavitane et al., 2003), we next examined how Lem8-mediated cleavage impacts its cellular localization. In HEK293T cells, when GFP-Phldb2 was ectopically expressed, the GFP signals mainly were associated with the plasma membrane, and this pattern of distribution remains unchanged in cells co- expressing enzymatically inactive Lem8 mutants (Fig. 4D). In constrast, in cells co- expressing wild-type Lem8, the GFP signals redistributed to occupy the entire cytoplasm, including the nuclei of transfected cells, a pattern similar to that of GFP itself (Fig. 4D). These observations suggest that GFP tag had been cleaved from the GFP-Phldb2 fusion to assume its typical localization in these cells. We also analyzed how Lem8 impacts the subcellular localization of endogenous Phldb2. In cells expressing mCherry-Lem8C280S, Phldb2 is mainly associated with the plasma membrane. In contrast, in cells expressing mCherry-Lem8, the association of Phldb2 with the plasma membrane almost became undetectable (Fig. S5A).
We next examined whether the cleavage of Phldb2 by Lem8 occurs in a cell-free reaction. HA-Phldb2-Flag expressed in HEK293T cells isolated by immunoprecipitation was incubated with Lem8 or its inactive mutants with or without 14-3-3ζ. Cleavage of Phldb2 occurred only in reactions containing wild-type Lem8 and 14-3-3ζ (Fig. 4E). Taken together, these results establish Phldb2 as a target of Lem8.
Our results using the double tagged Phldb2 suggest that Lem8 likely cleaves Phldb2 not only at the predicted site located in the carboxyl end of the protein, but also targets its amino terminal portion (Fig. 4C-E). To test this hypothesis, we constructed two Phldb2 mutants by replacing residues Arg1111 and Gln1112 (Phldb2AA1) or Gln1112 and Arg1113 (Phldb2AA2) within the predicted recognition sequence with alanine. Each of these mutants was co-expressed with Lem8 in HEK293T cells by transfection. Comparing to samples expressing enzymatically inactive Lem8, the amounts of protein detected by the amino terminal Flag epitope and the carboxyl end HA tag both decreased in cells co- expressing wild-type Lem8 (Fig. S5B). We validate this notion by making constructs in which GFP was fused to the amino terminal end of Phldb2 and three of its truncation mutants, Phldb2△N50, Phldb2△N100 and Phldb2△N200, respectively. Each of these fusion proteins was co-expressed with Lem8 or Lem8C280S in HEK293T cells and the protein level of these fusions was probed by immunoblotting with GFP-specific antibodies. In each case, a fraction of the protein has lost the GFP portion of the fusions when co- expressed with Lem8 but not with Lem8C280S (Fig. S5C). Intriguingly, the cleavage also occurred in fusion proteins in which the GFP is fused to the carboxyl terminus of Phldb2 or Phldb2△C153 (Fig. S5C). These results are consistent with the notion that Lem8 targets Phldb2 at multiple sites.
We also attempted to detect Lem8-mediated cleavage of endogenous Phldb2 in cells infected with L. pneumophila. Although Lem8 translocated into infected cells by a Dot/Icm-competent strain expressing Lem8 from a multicopy plasmid is readily detetable, we were unable to detect Phldb2 cleavage in these samples (Fig. S5D). The most likely reason for the inability to detect Lem8 activity against Phldb2 in infected cells is the low abundance or instability of the cleaved protein or a combination of both.
14-3-3ζ binds Lem8 by recognizing a coiled-coil motif in its amino terminal region
Using the online MARCOIL sequence analysis software (Gabler et al., 2020), we identified a putative coiled coil motif located in the amino region of Lem8. Coiled coil is a common structural element in proteins, particularly those of eukaryotic origin; it is formed by 2-7 supercoiled alpha-helices (Liu et al., 2006), and often is involved in protein-protein interactions, thus playing an important role in the formation of protein complexes (Burkhard et al., 2001). To determine the role of this region in the activity of Lem8, we introduced mutations to replace Leu58 and Glu59, the two sites predicted to be essential for the coild coil structure in Lem8, with glycine (called Lem8GG) (Fig. 5A). When tested in yeast, these mutations have completely abolished the toxicity of Lem8 without affecting its expression or stability (Fig. 5B ). These mutations may affect the cysteine protease acitivity of Lem8, its interaction with the regulatory protein 14-3-3ζ or its ability to recognize substrates.
We examined the ability of Lem8GG to cleave Phldb2 by coexpressing them in HEK293T cells. Whereas wild-type Lem8 consistently cleaves this substrate, Lem8GG has lost such activity despite a similar expression level (Fig. 5C). To test the self-cleavage of Lem8GG, we expressed Lem8-GFP or Lem8GG-GFP in HEK293T cells and probed the fusion proteins by immunoblotting with GFP-specific antibodies. Comparing to Lem8-GFP, the protein levels of Lem8GG-GFP and in Lem8C280S-GFP were similarly higher, indicating that the loss of the GFP portion of the fusion occurred in Lem8-GFP but not in Lem8GG- GFP (Fig. 5D). Finally, we examined the impact of these mutations on the interaction between Lem8 and 14-3-3ζ. Albeit Lem8GG expressed similarly to the wild-type, it has largely lost the ability to bind 14-3-3ζ in immunoprecipitation assays (Fig. 5E). Together with the observation that Lem8 mutants lacking as few as 25 residues from its amino terminal end are unable to bind 14-3-3ζ, these results suggest that the regulatory protein most likely bind Lem8 by recognizing the coiled coil motif located in its amino end region.
Auto-cleaved Lem8 maintains the cysteine protease activity
It has been well-established that some proteins, particularly enzymes are made as precursors or zymogens that need either auto-processing or cleavage by other enzymes to exhibit their biological functions. One such example is caspases involved in cell death regulation and other important cellular functions. These enzymes are synthesized as zymogens before being activated by proteolytic cleavage in response to stimulation (Shalini et al., 2015). In some cases, auto-processing leads to changes or even loss of their enzymatic activity (Kapust et al., 2001; Zhang et al., 2018). To investigate whether Lem8 that has undergone self-cleavage still possesses the cysteine protease activity, we tested the cleavage of Phldb2 by Lem8△C52, its self-processed form. Similar to full-length Lem8, Lem8△C52 was able to reduced the protein levels of Phldb2. In contrast, other truncation mutants, including Lem8△N25, Lem8△N50 and Lem8△C100 have lost the capacity to cleave Phldb2 (Fig. 6A). In addition, Lem8△C52, but not Lem8△N25 or Lem8△C100, cleaved the GFP tag from from the GFP-Phldb2 fusion and released the GFP signals from the plasma membrane (Fig. 6B). Intriguigingly, although their ability to cleave Phldb2 appears similar, under our experimental conditions, the protein level of Lem8△C52 is considerably lower than that of Lem8 (Fig. 6A), suggesting that the self-processed form has higher activity.
We next examined whether the protease activity of Lem8△C52 still requires 14-3-3ζ binding. Results from immunoprecipitation and pulldown assays with purified protiens clearly showed that Lem8 △ C52 robustly binds 14-3-3ζ (Fig. 6C-D). Furthermore, incubation of Lem8△C52 with Phldb2 isolated from cells did not lead to its cleavage, but the inclusion of 14-3-3ζ allowed the cleavage to occur (Fig. 6E), indicating that Lem8△C52 still requires 14-3-3ζ for its protease activity.
Lem8 inhibits migration in mammalian cells
Phldb2 is a PIP3 binding protein involved in microtubule stabilization (Lansbergen et al., 2006; Paranavitane et al., 2003), thus playing a pivotal role in cell motility. Depletion of Phldb2 significantly reduces the migration of MDA-231 cells in the haptotactic migration assay (Astro et al., 2014). As Lem8 cleaves Phldb2, we hypothesized that Lem8 may affect cell migration. To test this, we first established HEK293T-derived cell lines that stably express GFP, GFP-Lem8 or GFP-Lem8C280S. Immunoblotting confirmed that Lem8 and Lem8C280S robustly expressed in the respective cell lines. Furthermore, in the cell line expressing Lem8, the level of Phldb2 was drastically reduced comparing to that in the line expressing GFP or Lem8C280S (Fig. 7A). We then used the wound-healing scratch assay (De Ieso and Pei, 2018) to examine the impact of ectopic Lem8 expression on cell motility. Confluent monolayer of each cell lines was scratched using a pipette tip and the migration of cells into the gap was monitored over a period of 24 h. Results from this experiment showed that the percentage of wound closure at 24 h after wounding was around 50% in samples using cells expressing GFP or GFP-Lem8C280S. In the same experimental duration, cells expressing GFP-Lem8 only filled the gap by 26%, which was significantly slower that of the controls (Fig. 7B). Thus, ectopic expression of Lem8 inhibits mammalian cell migration.
An earlier study has shown that in the under-agarose migration assay, L. pneumophila inhibits the chemotaxis of mouse macrophages towards cytokines CCL5 and TNF-α in a Dot/Icm-dependent manner (Simon et al., 2014). Yet, the Dot/Icm substrates responsible for this inhibition remain elusive. We further studied whether Lem8 contributes to the inhibition of infected cell migration. To test this, we performed the scratch assay with cells infected with wild-type L. pneumophila or several strains relevant to lem8. The percentage of wound closure by cells infected with wild-type L. pneumophila or Δlem8(pLem8) was significantly lower than that with cells infected with the Δlem8 mutant. Consistent with its lack of the protease activity, Lem8C280S was unable to complement the defects displayed by the Δlem8 mutant (Fig. 7C). Thus, the inhibition of cell migration by L. pneumophila during infection is caused at least in part by the activity of Lem8.
Discussion
Intracellular bacteria manipulate cellular processes to create a niche that supports their survival and replication in host cells by virulence factors that target proteins important for the regulation of these processes. These virulence factors often attack host regulatory proteins by diverse posttranslational modifications (PTMs) such as phosphorylation (Krachler et al., 2011), ubiquitination (Zhou and Zhu, 2015), AMPylation (Yarbrough et al., 2009), acetylation (Mukherjee et al., 2006) and ADP-ribosylation (Cohen and Chang, 2018). Proteolytic processing is a type of PTM that can lead to the activation, inactivation or destruction of target proteins, causing alterations in cellular structure or signaling that benefit the pathogen. For instance, the type III effector EspL from enteropathogenic Escherichia coli functions as a cysteine protease that antagonizes host inflammatory response by degrading several proteins involved in necroptotic signalling (Pearson et al., 2017). Our results herein establish Lem8 as a cysteine protease that directly targets the microtubule associated protein Phldb2, therefore contributing to the inhibition of host cell migration by L. pneumophila. Lem8 joins a growing list of Legionella effectors with protease activity, including the serine protease Lpg1137 that inhibits autophagy by cleaving syntaxin 17 (Arasaki et al., 2017) and the metalloprotease RavK that attacks actin to disrupt the actin cytoskeleton of host cells (Liu et al., 2017).
One interesting feature associated with Lem8 is the requirement of 14-3-3ζ for its activity. In line with the notion that amoebae are the primary host of L. pneumophila, the sole 14-3-3 protein from D. discoideum similarly activates Lem8. In mammals, members of the 14-3-3 family, including 14-3-3ζ often bind their client proteins by recognizing phosphorylated pockets with relatively conserved sequences such as RSX[pS/pT]XP (mode I) and RXXX[pS/pT]XP (mode II) (pS, phospho-serine, pT, phospho-threonine, X, any residue) (Morrison, 2009). Intriguingly, neither of these two motifs is present in Lem8. Consistently, using a pan phospho-serine/threonine antibody capable of detecting phosphorylation of vimentin, another 14-3-3ζ binding protein in mammalian cells, we cannot detect phosphorylation on Lem8 purified from mammalian cells or E. coli (Fig. S3). The binding of 14-3-3 proteins to unmodified clients is not unprecedented. All isoforms of 14-3-3 bind non-phosphorylated ExoS of P. aeruginosa by recongnizing the DALDL element (Henriksson et al., 2002), which bears sequence similarity to the unphosphorylated target WLDLE, an artificial R18 peptide inhibitor derived from a phage display library (Petosa et al., 1998). Elements with a sequence similar to these established recogniztion sites are not present in Lem8 nor is there one resembling those in other nonphosphorylated binding targets of 14-3-3, including GPIb-α (Gu and Du, 1998), p75NTR-associated cell death executor (NADE) (Kimura et al., 2001) and CLIC4 (Suginta et al., 2001).
Two lines of evidence suggest that 14-3-3ζ recognizes a coiled coil motif in the amino terminal portion of Lem8. First, deletion of as few as 25 residues from the amino terminus end of Lem8 abolished its interaction with 14-3-3ζ (Fig. 2D). Second, the integrity of a predicted coiled coil motif in the amino terminal portion of Lem8 is required for its binding to the regulatory protein (Fig. 5). Coiled coil motifs have long been known to be important for protein-protein interaction but its involvement in binding 14-3-3 has not yet been established. The binding of 14-3-3 to TRIM25 had been suggested to be mediated by recognizing a coiled coil domain, but the mechanism of such binding or whether phosphorylation is required remains unclear (Gupta et al., 2019). Future study, particularly structural analysis of the Lem8-14-3-3ζ complex may allow a definite identification of the region in Lem8 recognized by 14-3-3ζ, which will surely shed light on the additional features of the sequences recognizable by these imporant regulatory proteins.
The self-cleavage of Lem8 has allowed us to identify its recognition sequence and several candidate cellular targets. One unexpected observation is that mutations in the identified recogniztion element reduced but did not abolished self-cleavage (Fig. 3D). Thus, the primary sequence may not the only factor that dictates the specificity of Lem8 in substrate recognition. Other factors such as the overall structure of substrates may contribute to the determination of the cleavage site. The low level of promiscuity in cleavage site selection may allow Lem8 to more effectively to bring down the protein level of its cellular targets, which may explain the requirement of 14-3-3ζ for its activity. If a host co-factor is not needed for its activity, Lem8 may cleave itself or even other proteins in L. pneumophila cells. For Lem8, self-cleavage in the absence of 14-3-3ζ in bacterial cells will be disastrous because the cleaved product will lose the portion of the protein that harbors translocation signals recognized by the Dot/Icm system (Luo and Isberg, 2004; Nagai et al., 2005). Likewise, the requirement of CaM by the Dot/Icm effector SidJ to inhibit the activity of members of the SidE family is to ensure that such inhibition does not occur in bacterial cells (Bhogaraju et al., 2019; Black et al., 2019; Gan et al., 2019; Sulpizio et al., 2019). The promiscuity in cleavage site recognition by Lem8 is also supported by the observation that this protease appears to cleave Phldb2 at multiple sites (Figs. 4 and S5).
Interference with host cell motility appears to be a common strategy used by bacterial pathogens. For example, Salmonella enterica Typhimurium inhibits the migration of infected macrophages and dendritic cells in a process that requires its type III effector SseI, which binds to IQGAP1, an important regulator of cell migration (McLaughlin et al., 2009). Similarly, the phosphatidylinositol phosphatase IpgD from Shigella flexneri contributes to the inhibition of chemokine-induced migration of human T cells (Konradt et al., 2011). The observation that cells infected with the wild-type L. pneumophila or strain Δlem8(pLem8) migrated significantly slower than those infected with the Δlem8 mutant or its complementation strain expressing the Lem8C280A mutant suggests a role of Lem8 in cell mobility inhibition (Simon et al., 2014).
Akin to most L. pneumophila Dot/Icm effectors, Lem8 is not required for proficient bacterial intracellular growth in commonly used laboratory hosts such as D. discoideum. Lem8 may be required for the survival of the bacteria in some specific inhabits or other Dot/Icm effectors may substitute its role by distinct mechanisms, thus contributing to such inhibition. Future studies aiming at the identification and characterization of Dot/Icm effectors involved in attacking host cells motility will continue to provide insights into the mechanisms of not only bacterial virulence but also the regulation of eukaryotic cell migration.
Materials and Methods
Bacterial stains, plasmids and cell culture
E. coli strain DH5α was used for plasmid construction and strain BL21(DE3) or XL1blue was used for recombinant protein production and purification. All E. coli strains were grown on LB agar plates or in LB broth at 37°C. For maintenance of plasmids in E. coli, antibiotics were added in media at the following concentrations: ampicillin,100 μg/mL; kanamycin, 30 μg/mL. All L. pneumophila strains were derived from the Philadelphia 1 strain Lp02 and the dotA- mutant strain Lp03 (Berger and Isberg, 1993) and were listed in Table S1. L. pneumophila was cultured in N-(2-acetamido)-2-aminoethanesulfonic acid buffered yeast extract medium (AYE) or on charcoal buffered yeast extract plates (CYE). When necessary, thymidine was added into AYE at a final concentration of 0.2 g/mL. pZL507 and its derivatives which allow expression of His6-tagged proteins (Xu et al., 2010) in L. pneumophila were maintained by thymidine autotrophic. Deletion of the Lem8 coding gene lpg1290 from the genome of L. pneumophila was performed as described previously (Liu and Luo, 2007).
Plasmids used in this study are listed in Table S1. Genes were amplified by polymerase chain reactions (PCR) using Platinum™ SuperFi II Green PCR mix (Invitrogen, cat# 12369050). The PCR product was digested with restriction enzymes (New England Biolabs, NEB), followed by ligated to linearized plasmid using T4 DNA ligase (NEB). For site-directed mutagenesis, plasmid was reacted with primer pairs designed to introduce the desired mutations using Quikchange kit (Agilent, cat# 600670). After digestion with the restriction enzyme DpnI (NEB, cat# R0176), the products were transformed into E. coli strain DH5α. All substitution mutants were verified by double strand DNA sequencing. The sequences of primers used for molecular cloning are listed in Table S1.
HEK293T and Hela cells purchased from ATCC were cultured in Dulbecco’s modified minimal Eagle’s medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS). Bone marrow cells were isolated from 6- to 10-week-old female A/J mice (GemPharmatech, Co., Ltd.) and were differentiated into BMDMs using L929-cell conditioned medium as described previously (Conover et al., 2003). PCR-based test (Sigma, cat# MP0025) was used to validate the absence of potential mycoplasma contamination in all mammalian cell lines. pAPH-HA, a derivative of pVR1012 (Wang et al., 2018) suitable for expressing proteins with an amino HA tag and a carboxyl Flag tag.
Yeast manipulation
Unless otherwise indicated, yeast strains used in this study were derived from W303 (Thomas and Rothstein, 1989); yeast was grown at 30°C in yeast extract, peptone, dextrose medium (YPD) medium or in appropriate amino acid dropout synthetic media supplemented with 2% of glucose or galactose as the sole carbon source.
For assessment of inducible protein toxicity, Lem8 or its derivatives were cloned into pYES2/NTA (Invitrogen) in which their expression is driven by the galactose-inducible promoter. Yeast transformation was performed using the lithium acetate method (Gietz et al., 1995). After growing in selective liquid medium with 2% raffinose, yeast cultures were serially diluted (five-fold) and 10 μL of each dilution was spotted onto selective plates containing glucose or galactose. Plates were incubated at 30°C for 3 days before image acquisition.
To screen Lem8-interacting protein(s), Gal4-based two-hybrid screening against the mouse cDNA library (Clontech) was performed as described before (Mitsuzawa et al., 2005). Briefly, Lem8C280S was inserted into pGBKT7 (Banga et al., 2007) to give pGBKLem8, which was transformed into the yeast strain PJ-64A (James et al., 1996) and the resulting strain was used for yeast two-hybrid screening. The mouse cDNA library was amplified in accordance with the manufacturer’s instructions and the plasmid DNA was transformed into strain PJ-64A (pGBKLem8). Transformants were plated onto a selective synthetic medium lacking adenine, tryptophan, leucine, and histidine, ccolonies appeared on the selective medium were verified for interactions by reintroducing into strain PJ-64A (pGBKLem8) and inserts of those that maintained the interaction phenotype were sequenced to identify the interacting proteins.
To validate the interactions between 14-3-3ζ and Lem8, its full-length gene was inserted into pGADGH (Banga et al., 2007) and the plasmids were introduced yeast strain PJ-64A (pGBKLem8). Yeast strains harboring the indicated plasmid combinations were streaked on Leu- and Trp- synthetic medium to select for plasmids and the transformants were transferred to Leu-, Trp-, Ade-, and His- medium to examine protein-protein interactions measured by cell growth.
Antibodies and immunoblotting
Polyclonal antibody against Lem8 were generated according to the protocol described before (Guide for the Care and Use of Laboratory Animals, 1996; J. Derrell Clark, 1997). Briefly, 1 mg of emulsified His6-Lem8 with complete Freund’s adjuvant was injected intracutaneously into a rabbit 4 times at 10-day intervals. Sera of the immunized rabbit containing Lem8-specific antibodies were used for affinity purification of IgG with an established protocol (Harlow, 1999).
Samples from cells or bacteria lysates were prepared by adding 5×SDS loading buffer and heated at 95°C for 10 min. The soluble fraction of the lysates was resolved by SDS-PAGE, proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Pall Life Sciences). The membranes were blocking with 5% nonfat milk for 30 min, followed by incubated with primary antibodies at the indicated dilutions: α-Phldb2 (Sigma, cat# HPA035147, 1:1000), α-HA (Sigma, cat# H3663, 1:3000), α-Flag (Sigma, Cat# F1804, 1: 3000), α-GFP (Proteintech, cat# 50430-2-AP, 1:5000), α-GST (Proteintech, cat# 66001-2, 1:10000), α-His (Sigma, cat# H1029, 1: 3,000), α-ICDH (1: 10,000) (Xu et al., 2010), α-Lem8 (1: 5,000), α-PGK (Abcam, cat# ab113687, 1:2,500) and α-Tubulin (Bioworld, cat# AP0064, 1:10,000). After washed 3 times, the membranes were incubated with appropriate HRP-labeled secondary antibodies and the signals were taken and analyzed by Tanon 5200 Chemiluminescent Imaging System.
Transfection and immunoprecipitation
When grown to approximately 80% confluence, HEK293T cells were transfected using Lipofectamine 3000 (Invitrogen, cat# L3000150) according to the manufacturer’s protocol. Twenty-four hours after transfection, cells were lysed using a lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% Triton X-100, PH 7.5) for 10 min on ice, followed by centrifugation at 12,000g at 4°C for 10 min. Beads coated with Flag- (Sigma, cat# F2426), HA- (Sigma, cat# E6779) or GFP-specific antibodies (Sigma, cat# G6539) were washed twice with lysis buffer and then mixed with the prepared cell supernatant. The mixture was incubated on a rotatory shaker at 4°C overnight. The resin was washed with the lysis buffer for five times, followed by boiling in the Laemmli buffer at 95°C for 10 min to release the bound Flag- or HA-tagged proteins. For proteins used in biochemical reactions, the Flag- or HA-tagged proteins were eluted with Flag peptide (Sigma, cat# F4799) or HA peptide (Sigma, cat# I2149), respectively.
Protein expression and purification
Lem8 and its mutants were amplified by PCR and cloned into pQE30 to express His6-tagged proteins. The plasmids were transformed into in E. coli strain XL1blue and grown in LB broth. When the cell density reached an OD600 of 0.8, isopropyl-β-D- thiogalactopyranoside (IPTG) was added into the cultures at a final concentration of 0.2 mM to induce the expression of target proteins for 14 h at 16°C. Cells collected by centrifugation were re-suspended in a lysis buffer (1×PBS, 2 mM DTT and 1 mM PMSF), and were lysed with a cell homogenizer (JN-mini, JNBIO, Guangzhou, China). The lysates were centrifugated at 20,000g for 30 min at 4°C twice to remove cell debris. The supernatant was incubated with Ni2+-NTA beads (QIAGEN) at 4°C for 1 h, followed by washed with 50x bed volumes of 20 mM imidazole to remove unbound proteins. The His6-tagged proteins were eluted with 250 mM imidazole in PBS buffer. Purified proteins were dialyzed in a storage buffer (30mM NaCl, 20 mM Tris, 10% glycerol, pH 7.5) overnight at 4°C and then stored at -80°C.
14-3-3ζ and its homologous genes were cloned into pGEX6p-1 to express GST- tagged proteins. The plasmids were transformed into E. coli strain BL21(DE3). Protein expression induction and purification was carried out similarly with Glutathione Sepharose 4B (GE Healthcare) beads. The resin was collected and washed for with wash buffer (lysis buffer plus 200 mM NaCl). The GST-tagged proteins were eluted with 10 mM glutathione and stored at -80°C after dialysis.
In vitro cleavage assays
For auto-cleavage assays, 5 μg His6-Lem8 or its mutants was incubated with or without 2.5 μg 14-3-3ζ in 50 μl reaction buffer (50 mM Tris, 150 mM NaCl, PH 7.5) at room temperature for the indicated time points. For Phldb2 cleavage, Flag-Phldb2 purified from HEK293T cells were added into reactions with or without Lem8 and 14-3- 3ζ at room temperature for the indicated time. In each case, samples were analyzed by SDS-PAGE followed by immunoblotting or Coomassie brilliant blue staining.
GST pulldown assay
GST-14-3-3ζ or GST bound to Glutathione Sepharose 4B was incubated with His6- Lem8 in a binding buffer (50 mM Tris, 137 mM NaCl, 13.7 mM KCl) for 2 h at 4 °C. After washing three times with the binding buffer, beads were boiled in the Laemmli buffer at 95°C for 10 min and the samples were resolved by SDS-PAGE. Proteins were detected by Coomassie brilliant blue staining.
Bacterial infection, immunostaining and image analysis
For infection experiments, L. pneumophila strains were grown in AYE broth to the post-exponential growth phase (OD600=3.3-3.8). When necessary, complementation strains were induced by 0.1 mM IPTG for another 4 h at 37°C before infection.
To determine intracellular bacterial growth, D. discoideum or BMDMs of A/J mice were infected with relevant L. pneumophila at a multiplicity of infection (MOI) of 0.05. 2 h after adding the bacteria, the cells were washed using warm PBS to remove the extracellular bacteria. D. discoideum and BMDMs were maintained in 22°C and 37°C, respectively. At the indicated time points, cells were lysed with 0.2% saponin and appropriately diluted lysates were plated on CYE plates. After 4-day incubation at 37°C, the counts of bacterial colonies were calculated to evaluate the growth.
To determine the impact of the infection on cell migration, HEK293T cells transfected to express FcγRII receptor (Qiu et al., 2016) were infected with the indicated bacterial strains. 2 h after infection, cells were washed using warm PBS and were used for the wound healing assay.
To determine the cellular localization of Lem8 in infected cells, BMDMs were infected with relevant L. pneumophila strains at an MOI of 10 for 2 h. The samples were immunostained as described earlier (Haenssler et al., 2015). Briefly, we washed the samples 3 times with PBS to remove extracellular bacteria, and fixed the cells with 4% paraformaldehyde at room temperature for 10 min. After three times washes, cells were permeabilized using 0.1% Triton X-100 and then were blocked with 4% goat serum for 1 h. Samples were incubated with rat anti-Legionella antibodies (1:10,000) and rabbit anti- Lem8 antibodies (1:100) at 4°C overnight. followed by incubated with appropriate fluorescence-labeled secondary antibodies at room temperature for 1 h. After stained by Hoechst 33342 (Invitrogen, cat# H3570, 1:5000), samples were inspected using an Olympus IX-83 fluorescence microscope.
To detect the cleavage of endogenous Phldb2 by Lem8, Hela cells transfected to express mCherry-Lem8 or mCherry-Lem8C280S were stained with Phldb2-specific antibodies (1:100) as described above. The images were taken using a Zeiss LSM 880 confocal microscope. The determine the impact on ectopically expressed Phldb2, mCherry-Lem8 or mutants each was co-transfected with GFP-Phldb2 into HEK293T cells seededonto glass coverslips (Nest, cat# 801001). Fixed samples were stained with Hoechst, cell images were acquired by a confocal microscope.
Production of lentiviral particles and transduction
For production of lentiviral particles carrying lem8 or its mutants, the gfp-lem8 fusion was inserted into pCDH-CMV-MCS-EF1a-Puro (System Biosciences, cat# CD510B-1). The plasmids were co-transfected with pMD2.G (gift from Dr. Didier Trono, Addgene#12259) and psPAX2 (gift from Dr. Didier Trono, Addgene #12260) into HEK293T cells grown to about 70% confluence. Supernatant was collected after 48 hours incubation, followed by filtration with 0.45-μm syringe filters. After measuring the titers using qPCR with the Lentivirus Titer Kit (abm, cat# LV900), the packed lentiviral particles were used to infect newly prepared HEK293T cells at an MOI of 10. After incubation for 2 days, cells were sorted by BD Influx™ cell sorter to establish cell lines stably expressing the gene of interest.
Mass spectrometry analysis of Lem8 self-cleavage site
Recombinant His6-Lem8 was incubated with His6-14-3-3 for 8 h and the samples were separated by SDS-PAGE. After Coomassie brilliant blue staining, bands corresponding full-length His6-Lem8 or cleaved were excised and subjected to in-gel digestion with trypsin. Peptides were loaded into a nano-LC system (EASY-nLC 1200, Thermo Scientific) coupled to an LTQ-Orbitrap mass spectrometer (Orbitrap Velos, Thermo Scientific). Peptides were separated in a capillary column (75 µm x 15 cm) packed with C18 resin (Michrom BioResources Inc., 4 µm, 100 Å) with the following gradient: solvent B (100 ACN, 0.1% FA) was started at 7% for 3 min and gradually raised to 35% in 40 min, then rapidly increased to 90% in 2 min and maintained for 10 min before column equilibration with 100% solvent A (97% H2O, 3% ACN, 0.1% FA). The flow rate was set at 300 nL/min and eluting peptides were directly analyzed in the mass spectrometer. Full-MS spectra were collected in the range of 350 to 1500 m/z and the top 10 most intense parent ions were submitted to fragmentation in a data-dependent mode using collision-induced dissociation (CID) with the max injection time of 10 milliseconds. MS/MS spectra were searched against the Legionella pneumophila (strain Philadelphia 1) database downloaded from UniProt using Mascot (Matrix Science Inc.). The signals of Lem8 tryptic peptides were compared between full-length and cleaved samples to narrow down the potential cleavage site(s) within specific peptides, cleaved Lem8 semi-tryptic peptides were inspected manually.
Wound healing assay
Wound healing assays were performed as previously described (Liang et al., 2007). Briefly, HEK293T cells were seeded into 6-well plates and incubated until the confluency reached about 90%. The cell monolayer was scraped in a straight line using a p200 pipet tip to create a “wound”, followed by washing with growth medium to remove the debris. Reduced-serum medium (1% serum) was added and the cells were placed back in a 37°C incubator. 2 h and 24 h after making the scratch, images of the cell monolayer wound were taken using an Olympus IX-83 fluorescence microscope. For each image,distances between one side of the wound and the other were quantitated by Image J (http://rsb.info.nih.gov/ij/). The wound healing rate was calculated by the following formula: % wound healing = (0 h distance – 24 h distance)/24 h distance × 100.
Data quantitation, statistical analyses
All data were represented as mean ± standard deviation (SD). Student’s t-test was applied to analyze the statistical difference between two groups each with at least three independent samples.
Author contributions
LS, ZQL, YL and YT conceived the projects, LS, YL, YT, JL, DH, and YZ performed the experiments. KY and XL performed the mass spectrometric analysis. SL, YT, YL, XL, and ZQL analyzed data. SL drafted the first version of the manuscript, and ZQL revised the manuscript with input from all authors.
Acknowledgements
The authors thank Dr. Shaohua Wang for plasmids, the study was funded in part by Jilin Science and Technology Agency grant 20200403117SF (LS), 20200901010SF (DL), National Natural Science Foundation of China grant 21974002 (XL), Beijing Municipal Natural Science Foundation grant 5202012 (XL), and the National Institutes of Health grant R01AI127465 (ZQL).
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