Justine Toinon

Monica Rolando

Magali Charvin

Didier Filopon

Lionel Schiavolin

Khadeeja Adam Sy

Hai-Chi Vu

Sarah Gallois-Montbrun

Antoine Alam

Christophe Rusniok

Bérangère Lombard

Damarys Loew

Carmen Buchrieser
email: carmen.buchrieser@pasteur.fr

Lionel Navarro
email: lionel.navarro@ens.psl.eu

A Legionella pneumophila effector impedes host gene silencing to promote virulence

RNA silencing is a gene silencing mechanism directed by siRNAs and miRNAs.

Human miRNAs act as central regulators of host-bacteria interactions. However, it is unknown whether human pathogenic bacteria could impede RNA silencing to promote virulence. Here, we show that the Legionella pneumophila type IV-secreted effector LegK1 suppresses siRNA-and miRNA-activities in human cells. This ability depends on its kinase activity and on a functional tryptophan-dependent Argonaute (Ago)binding platform. We further show that the capacity of LegK1 to activate NF-kB signaling contributes to silencing suppression, demonstrating a link between effectormediated NF-kB signaling activation and silencing suppression. LegK1 also promotes L. pneumophila growth in both amoeba and human macrophages, supporting a key role of this effector in virulence. In infected macrophages, the latter activity relies on the genetic targeting of human Ago4, highlighting a novel function of this host factor in antibacterial resistance.

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Introduction

Legionella pneumophila is a Gram-negative bacterium, which infects and replicates in freshwater amoebae and ciliated protozoa in the environment [START_REF] Rowbotham | Preliminary report on the pathogenicity of Legionella pneumophila for freshwater and soil amoebae[END_REF][START_REF] Newton | Molecular pathogenesis of infections caused by Legionella pneumophila[END_REF][START_REF] Boamah | From Many Hosts, One Accidental Pathogen: The Diverse Protozoan Hosts of Legionella[END_REF][START_REF] Mondino | Legionnaires' Disease: State of the Art Knowledge of Pathogenesis Mechanisms of Legionella[END_REF] . When contaminated water droplets are inhaled by humans, L. pneumophila incidentally infects lung alveolar macrophages, which can result in a severe form of pneumonia called Legionnaires' disease [START_REF] Mondino | Legionnaires' Disease: State of the Art Knowledge of Pathogenesis Mechanisms of Legionella[END_REF][START_REF] Escoll | From Amoeba to Macrophages: Exploring the Molecular Mechanisms of Legionella pneumophila Infection in Both Hosts[END_REF] . L. pneumophila has evolved a type IV secretion system, referred to as the Dot/Icm system, to promote pathogenesis [START_REF] Marra | Identification of a Legionella pneumophila locus required for intracellular multiplication in human macrophages[END_REF][START_REF] Berger | Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila[END_REF][START_REF] Segal | Host cell killing and bacterial conjugation require overlapping sets of genes within a 22-kb region of the Legionella pneumophila genome[END_REF] . This secretion system translocates over 330 effectors into host cells and is essential for the replication of L.

3 pneumophila in protozoan and in human macrophages [START_REF] Burstein | Genome-Scale Identification of Legionella pneumophila Effectors Using a Machine Learning Approach[END_REF][START_REF] Huang | The E Block motif is associated with Legionella pneumophila translocated substrates[END_REF][START_REF] Zhu | Comprehensive Identification of Protein Substrates of the Dot/Icm Type IV Transporter of Legionella pneumophila[END_REF][START_REF] Lifshitz | Computational modeling and experimental validation of the Legionella and Coxiella virulence-related type-IVB secretion signal[END_REF][START_REF] Gomez-Valero | More than 18,000 effectors in the Legionella genus genome provide multiple, independent combinations for replication in human cells[END_REF] . A significant proportion of L. pneumophila effectors resembles eukaryotic-like proteins or encode eukaryotic domains, and has likely been co-opted for subverting host functions [START_REF] Gomez-Valero | More than 18,000 effectors in the Legionella genus genome provide multiple, independent combinations for replication in human cells[END_REF][START_REF] Cazalet | Evidence in the Legionella pneumophila genome for exploitation of host cell functions and high genome plasticity[END_REF][START_REF] De Felipe | Evidence for Acquisition of Legionella Type IV Secretion Substrates via Interdomain Horizontal Gene Transfer[END_REF][START_REF] Lurie-Weinberger | The origins of eukaryotic-like proteins in Legionella pneumophila[END_REF] . Among them, the family of LegK proteins comprises proteins encoding eukaryotic-like kinase domains targeting specific host proteins [START_REF] De Felipe | Evidence for Acquisition of Legionella Type IV Secretion Substrates via Interdomain Horizontal Gene Transfer[END_REF] . For example, LegK1 is a serine/threonine protein kinase that phosphorylates the NF-kB inhibitor IkBa, as well as other IkB and NF-kB family members, resulting in a potent NF-kB signaling activation in human cells [START_REF] Ge | A Legionella type IV effector activates the NF-κB pathway by phosphorylating the IκB family of inhibitors[END_REF] . This effector contributes to the sustained Dot/Icm-dependent NF-kB signaling detected during L. pneumophila infection of human alveolar macrophages [START_REF] Losick | NF-kappaB translocation prevents host cell death after low-dose challenge by Legionella pneumophila[END_REF][START_REF] Abu-Zant | Anti-apoptotic signalling by the Dot/Icm secretion system of L. pneumophila[END_REF][START_REF] Bartfeld | Temporal resolution of two-tracked NF-κB activation by Legionella pneumophila[END_REF] . Furthermore, it triggers the induction of anti-apoptotic and host cell survival genes 18- 20 , which is thought to promote L. pneumophila growth in human macrophages.

MicroRNAs (miRNAs) are small non-coding RNAs (sRNAs) that post-transcriptionally regulate genes through the targeting of sequence complementary mRNA targets. In mammals, the miRNA biogenesis pathway involves the cleavage of primary miRNA transcripts by the Drosha-DGCR8 complex, which further releases hairpin-based miRNA precursors that are subsequently processed by the RNAse III enzyme Dicer [START_REF] Bartel | Metazoan MicroRNAs[END_REF] .

The resulting miRNA duplexes then bind to an Argonaute (Ago) protein, and one strand, the guide, remains associated to this silencing effector to form a miRNA-Induced Silencing Complex (miRISC) [START_REF] Duchaine | Mechanistic Insights into MicroRNA-Mediated Gene Silencing[END_REF][START_REF] Iwakawa | Life of RISC: Formation, action, and degradation of RNA-induced silencing complex[END_REF] . The miRISC further directs silencing of mRNA targets through endonucleolytic cleavage (slicer activity), translational repression and/or mRNA degradation [START_REF] Bartel | Metazoan MicroRNAs[END_REF][START_REF] Duchaine | Mechanistic Insights into MicroRNA-Mediated Gene Silencing[END_REF][START_REF] Jonas | Towards a molecular understanding of microRNAmediated gene silencing[END_REF] . Four structurally conserved Ago proteins (Ago1-4) are encoded by the human genome [START_REF] Sasaki | Identification of eight members of the Argonaute family in the human genome[END_REF][START_REF] Müller | Argonaute Proteins: From Structure to Function in Development and Pathological Cell Fate Determination[END_REF] . Human Ago2 is the most characterized Ago member that possesses a slicer activity directed by a catalytic tetrad, DEDH (Asp-Glu-Asp-His), embedded in its PIWI domain [START_REF] Liu | Argonaute2 Is the Catalytic Engine of Mammalian RNAi[END_REF][START_REF] Meister | Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs[END_REF][START_REF] Schirle | The Crystal Structure of Human Argonaute2[END_REF][START_REF] Sheu-Gruttadauria | Phase transitions in the assembly and function of human miRISC[END_REF] . This factor is the central component of the miRISC [START_REF] Li | AGO2 and its partners: a silencing complex, a chromatin modulator, and new features[END_REF] . Mechanistically, the phosphorylation of Ago2 at serine 387 triggers its binding to trinucleotide repeat containing 6 (TNRC6) proteins, which are tryptophan (W)-rich cofactors that associate with the PIWI domain of Ago2 through W-repeats (W-motifs) found in their N-terminal Ago-binding domains (ABDs) [START_REF] Schirle | The Crystal Structure of Human Argonaute2[END_REF][START_REF] Till | A conserved motif in Argonaute-interacting proteins mediates functional interactions through the Argonaute PIWI domain[END_REF][START_REF] Horman | Akt-mediated phosphorylation of Argonaute 2 downregulates cleavage and up-regulates translational repression of microRNA targets[END_REF][START_REF] Bridge | Argonaute Utilization for miRNA Silencing Is Determined by Phosphorylation-Dependent Recruitment of LIM-Domain-Containing Proteins[END_REF] . These molecular events are essential for High Molecular Weight (HMW)-miRISC assembly by recruiting downstream effector proteins [START_REF] Duchaine | Mechanistic Insights into MicroRNA-Mediated Gene Silencing[END_REF][START_REF] Iwakawa | Life of RISC: Formation, action, and degradation of RNA-induced silencing complex[END_REF] , including those involved in decapping (Decapping protein 1/2, DCP1/2), RNA unwinding (DEAD-Box RNA helicase 6, DDX6), and deadenylation (carbon catabolite repressor 4/ negative regulator of transcription, CCR4/NOT) [START_REF] Chen | A DDX6-CNOT1 complex and W-binding pockets in CNOT9 reveal direct links between miRNA target recognition and silencing[END_REF][START_REF] Rouya | Human DDX6 effects miRNA-mediated gene silencing via direct binding to CNOT1[END_REF] . In addition, TNRC6 proteins use W-motifs embedded in their C-terminal silencing domains to directly bind CCR4-NOT and PAN2-PAN3 (poly(A)-specific ribonuclease 2) deadenylase complexes, thereby contributing to HMW-miRISC activity [START_REF] Chen | A DDX6-CNOT1 complex and W-binding pockets in CNOT9 reveal direct links between miRNA target recognition and silencing[END_REF][START_REF] Braun | GW182 proteins directly recruit cytoplasmic deadenylase complexes to miRNA targets[END_REF][START_REF] Chekulaeva | miRNA repression involves GW182-mediated recruitment of CCR4-NOT through conserved W-containing motifs[END_REF][START_REF] Fabian | miRNA-mediated deadenylation is orchestrated by GW182 through two conserved motifs that interact with CCR4-NOT[END_REF][START_REF] Christie | Structure of the PAN3 pseudokinase reveals the basis for interactions with the PAN2 deadenylase and the GW182 proteins[END_REF] . This series of molecular events leads to the translational repression, destabilization and degradation of mRNA targets, and in turn regulates diverse biological processes, including development (e.g. neurological development), differentiation, stress signaling and antiviral responses [START_REF] Alisch | Argonaute2 is essential for mammalian gastrulation and proper mesoderm formation[END_REF][START_REF] Morita | One Argonaute family member, Eif2c2 (Ago2), is essential for development and appears not to be involved in DNA methylation[END_REF][START_REF] Li | Induction and suppression of antiviral RNA interference by influenza A virus in mammalian cells[END_REF][START_REF] Qiu | Human Virus-Derived Small RNAs Can Confer Antiviral Immunity in Mammals[END_REF][START_REF] Ngondo | Argonaute 2 Is Required for Extra-embryonic Endoderm Differentiation of Mouse Embryonic Stem Cells[END_REF][START_REF] Lessel | Germline AGO2 mutations impair RNA interference and human neurological development[END_REF] . Human Ago1 and Ago4 are slicer-deficient and thus trigger gene silencing through endonucleolytic-independent mechanisms [START_REF] Liu | Argonaute2 Is the Catalytic Engine of Mammalian RNAi[END_REF][START_REF] Hauptmann | Turning catalytically inactive human Argonaute proteins into active slicer enzymes[END_REF] . In contrast, human Ago3 retains a canonical DEDH catalytic tetrad and was recently shown to trigger RNA cleavage when loaded with specific guide miRNAs, in particular 14-nt 3' end-shortened miRNA variants [START_REF] Park | Human Argonaute2 and Argonaute3 are catalytically activated by different lengths of guide RNA[END_REF][START_REF] Nakanishi | Anatomy of four human Argonaute proteins[END_REF] . Although very little is known about the biological functions of these three Ago proteins, gene deletions of Ago1 and Ago3 have been retrieved in patients suffering from neurological disorders [START_REF] Tokita | Five children with deletions of 1p34.3 encompassing AGO1 and AGO3[END_REF][START_REF] Sakaguchi | Further evidence of a causal association between AGO1, a critical regulator of microRNA formation, and intellectual disability/autism spectrum disorder[END_REF] . Furthermore, several biological functions have been ascribed to mammalian Ago4. This factor has been shown to (i) regulate meiotic entry and meiotic sex chromosome inactivation in mice germ cells [START_REF] Modzelewski | AGO4 Regulates Entry into Meiosis and Influences Silencing of Sex Chromosomes in the Male Mouse Germline[END_REF] , (ii) translationally inhibit the second cistron of the CACNA1A locus in a miRNA-dependent manner, thereby inhibiting the growth of neurons and Purkinje cells in mice [START_REF] Miyazaki | An miRNA-mediated therapy for SCA6 blocks IRES-driven translation of the CACNA1A second cistron[END_REF] , (iii) interact with the de novo methyltransferase DNMT3A and in turn direct cytosine methylation of some mature miRNAs, which inhibits their functions and is associated with poor prognosis in glioma [START_REF] Cheray | Cytosine methylation of mature microRNAs inhibits their functions and is associated with poor prognosis in glioblastoma multiforme[END_REF] , (iv) mediate RNA-dependent DNA methylation in human cells [START_REF] Chalertpet | Argonaute 4 as an Effector Protein in RNA-Directed DNA Methylation in Human Cells[END_REF] , and (v) promote antiviral resistance in mice [START_REF] Adiliaghdam | A Requirement for Argonaute 4 in Mammalian Antiviral Defense[END_REF] . Therefore, despite early assumptions that mammalian Ago proteins function redundantly, they can also exhibit very specialized functions.

Human miRNAs have been extensively studied in the context of host-bacteria interactions [START_REF] Aguilar | MicroRNAs at the Host-Bacteria Interface: Host Defense or Bacterial Offense[END_REF] . They regulate various cellular processes during bacterial infection, including innate immune responses, host cell cycle, cell death and survival pathways, autophagy, and cytoskeleton organization [START_REF] Aguilar | MicroRNAs at the Host-Bacteria Interface: Host Defense or Bacterial Offense[END_REF] . For example, L. pneumophila triggers the differential expression of 85 human miRNAs during infection of macrophages [START_REF] Herkt | A MicroRNA Network Controls Legionella pneumophila Replication in Human Macrophages via LGALS8 and MX1[END_REF] . Among them, miR146a was found up-regulated and required to restrict the growth of L. pneumophila in human macrophages [START_REF] Herkt | A MicroRNA Network Controls Legionella pneumophila Replication in Human Macrophages via LGALS8 and MX1[END_REF] . Furthermore, a trio of down-regulated miRNAs, namely miR-125b, miR-221 and miR-579, was shown to target repressors of L. pneumophila replication in macrophages [START_REF] Herkt | A MicroRNA Network Controls Legionella pneumophila Replication in Human Macrophages via LGALS8 and MX1[END_REF] . Interestingly, L. pneumophila infection is not only modulating the expression of human miRNAs, but L. pneumophila is itself translocating active small RNAs (sRNAs) via extracellular vesicles (EVs) into the host cell [START_REF] Sahr | Translocated Legionella pneumophila small RNAs mimic eukaryotic microRNAs targeting the host immune response[END_REF] . Indeed, 15 sRNAs were identified that mimic human miRNAs and two of these, namely RsmY and tRNA-Phe, down-regulate selected sensor and regulator proteins of the host innate immune response in a miRNA-like manner [START_REF] Sahr | Translocated Legionella pneumophila small RNAs mimic eukaryotic microRNAs targeting the host immune response[END_REF] . Intriguingly, a recent study also shows that human airway cells make use of EVs to deliver let-7b-5p into Pseudomonas aeruginosa and reprogram bacterial gene expression, thereby reducing biofilm formation and antibiotic resistance [START_REF] Koeppen | Let-7b-5p in vesicles secreted by human airway cells reduces biofilm formation and increases antibiotic sensitivity of P. aeruginosa[END_REF] . Altogether, these studies, among many others, highlight a central role played by sRNAs in the reprogramming of both host and bacterial gene expression during infections.

Although the role of human miRNAs in host-bacteria interactions is now wellestablished, there is currently no evidence indicating that human pathogenic bacteria can interfere with RNA silencing as part of their virulence functions. This has, however, been previously characterized in a phytopathogenic Pseudomonas syringae strain, which delivers several type III-secreted effectors into the host cell to suppress different steps of the Arabidopsis miRNA pathway 61 . It has also been extensively characterized in mammalian viruses that produce Viral Suppressors of RNA silencing (VSRs) to replicate in host cells [START_REF] Li | Induction and suppression of antiviral RNA interference by influenza A virus in mammalian cells[END_REF][START_REF] Qiu | Human Virus-Derived Small RNAs Can Confer Antiviral Immunity in Mammals[END_REF][START_REF] Aqil | The HIV-1 Nef Protein Binds Argonaute-2 and Functions as a Viral Suppressor of RNA Interference[END_REF][START_REF] Li | RNA Interference Functions as an Antiviral Immunity Mechanism in Mammals[END_REF][START_REF] Maillard | Antiviral RNA Interference in Mammalian Cells[END_REF] . In this study, we found that the L. pneumophila type IVsecreted effector LegK1 acts as a bona fide Bacterial Suppressor of RNA silencing (BSR). We show that LegK1 efficiently suppresses siRNA-and miRNA-activities in human cells through both its kinase activity and a functional Ago-binding platform. We further demonstrate that the ability of LegK1 to activate NF-kB signaling contributes to silencing suppression. In addition, we found that LegK1 promotes L. pneumophila growth in both human macrophages and amoeba, supporting a virulence function of this effector in biologically relevant host cells. Importantly, the genetic targeting of human Ago4 by LegK1 was found required to promote L. pneumophila growth in macrophages. Therefore, besides identifying the first BSR from a pathogenic bacterium, this study unveils an unexpected link between effector-mediated NF-kB signaling activation and silencing suppression, as well as a novel function of human Ago4 in host-bacteria interactions.

Results

The L. pneumophila effector LegK1 suppresses siRNA-directed silencing in human cells

The known Ago-binding function of W-motifs, present in some endogenous silencing factors as well as in some VSRs [START_REF] Aqil | The HIV-1 Nef Protein Binds Argonaute-2 and Functions as a Viral Suppressor of RNA Interference[END_REF][START_REF] El-Shami | Reiterated WG/GW motifs form functionally and evolutionarily conserved ARGONAUTE-binding platforms in RNAi-related components[END_REF][START_REF] Azevedo | Argonaute quenching and global changes in Dicer homeostasis caused by a pathogen-encoded GW repeat protein[END_REF][START_REF] Giner | Viral Protein Inhibits RISC Activity by Argonaute Binding through Conserved WG/GW Motifs[END_REF][START_REF] Garcia | Ago hook and RNA helicase motifs underpin dual roles for SDE3 in antiviral defense and silencing of nonconserved intergenic regions[END_REF][START_REF] Karran | Tomato ringspot virus coat protein binds to ARGONAUTE 1 and suppresses the translation repression of a reporter gene[END_REF] , prompted us to examine whether effectors from human pathogenic bacteria have also evolved such Ago-binding platforms to interfere with RNA silencing. To test this hypothesis, we retrieved the protein sequences from 25 genomes of pathogenic bacteria and used them to the Wsearch algorithm to search for putative W-motifs [START_REF] Zielezinski | Integrative data analysis indicates an intrinsic disordered domain character of Argonaute-binding motifs[END_REF] (Figure S1a). The score of each W-motif was determined using the animal Bidirectional Position-Specific Scoring Matrix (BPSSM), generated from experimentally-verified Ago-binding proteins and their orthologous sequences [START_REF] Zielezinski | Integrative data analysis indicates an intrinsic disordered domain character of Argonaute-binding motifs[END_REF] . Among the candidate proteins exhibiting the highest Wscore, we selected a subset of secreted virulence factors and tested their possible effects on the siRNA-based CXCR4 reporter 71 (Figure S1b). This reporter system relies on the co-transfection of two plasmids along with exogenous siRNAs targeting the 3'UTR of CXCR4. The first plasmid expresses the firefly luciferase fused to the 3'UTR of CXCR4, while the second one expresses the renilla luciferase, which served as a transfection control. We first showed that the silencing of the luciferase reporter was compromised in the ago2 -/-and ago1 -/-/ago2 -/-CRISPR/Cas9-based mutant HeLa cell lines, as revealed by an increased luminescence intensity compared to control cells (Figure S1c,d). In contrast, the silencing of the luciferase remained effective in ago1 -/-and dicer -/-cell lines (Figure S1c,d). Collectively, these results indicate that the CXCR4 reporter system is dependent on Ago2, but not on Ago1. They also indicate that Dicer is not required for this process, which is consistent with the exogenous delivery of mature anti-CXCR4 siRNAs. Significantly, when monitoring luminescence intensity in HeLa cells expressing individually these 8 candidate bacterial W-effectors (Figure S1e-g), we found that LegK1 from Legionella pneumophila (strain Paris) was the sole effector capable of inhibiting the silencing of the CXCR4 reporter, as found in ago2 -/-cells or in cells expressing the VSR Hepatitis B virus HBx 72 (Figure S1h,i).

Altogether, these data indicate that LegK1 suppresses the silencing of an Ago2dependent siRNA-based reporter system.

LegK1 suppresses siRNA-and miRNA-directed gene silencing through both its kinase activity and a predicted tryptophan-dependent Ago-binding platform

LegK1 is an experimentally validated Dot/Icm type IV-secreted effector possessing a eukaryotic-like serine/threonine kinase activity [START_REF] Ge | A Legionella type IV effector activates the NF-κB pathway by phosphorylating the IκB family of inhibitors[END_REF] . Here, we additionally found that LegK1 contains a putative Ago-binding platform composed of four predicted W-motifs (Figure S1j). The tryptophan residues located at positions 41, 283 and 293 of the LegK1 protein sequence, namely W41, W283 and W293, displayed the highest Wscore, and were thus selected for further analyses (Figures 1a andS1j). We first substituted each of these three tryptophans by phenylalanine (W>F; LegK1-3WF). In parallel, we generated a K121A substitution in the LegK1 ATP-binding site (LegK1-KA), which has been previously shown to abolish the kinase activity of this effector [START_REF] Ge | A Legionella type IV effector activates the NF-κB pathway by phosphorylating the IκB family of inhibitors[END_REF] . Both LegK1-3WF and LegK1-KA proteins accumulated to the same levels as wild-type (WT) LegK1 proteins in HEK293T cells (Figure 1b), indicating that these mutated proteins are stable when expressed in human cells. We further analyzed their ability to suppress RNA silencing. To this end, we used a siRNA-based GFP reporter system, which relies on the co-transfection of a plasmid expressing the enhanced GFP (eGFP), together with anti-GFP siRNAs targeting a single site located in the 5' region of the eGFP coding sequence. This siRNA-based reporter system has been previously used to characterize VSRs from different human pathogenic viruses [START_REF] Fabozzi | Ebolavirus proteins suppress the effects of small interfering RNA by direct interaction with the mammalian RNA interference pathway[END_REF][START_REF] Li | Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing[END_REF] . We found that this reporter was robustly silenced by the anti-GFP siRNAs, as revealed by low accumulation of eGFP proteins when expressed in HeLa and HEK293T cells (Figure 1c-e). In contrast, the eGFP protein accumulation was moderately increased in the ago2 -/-HeLa cell line expressing the anti-GFP siRNA duplexes, an effect which was found enhanced in the ago1 -/-/ago2 -/-mutant cells (Figure 1c). These results indicate that both Ago1 and Ago2 proteins direct the silencing of this reporter system. Of note, the fact that the reporter was not fully derepressed in ago1 -/-/ago2 -/-HeLa cells suggests that Ago3 and/or Ago4 must additionally contribute to its silencing. When the same analysis was conducted in HEK293T cells expressing LegK1, we found an efficient suppression of eGFP silencing, a process which was not observed in cells expressing the empty vector or the type III-secreted YopM effector from Yersinia pestis, which is deprived of silencing suppression activity (Figure 1d,e). It is noteworthy that the silencing suppression effect mediated by LegK1 was even more pronounced than the one triggered by T6B, a TNRC6B-derived peptide containing multiple W-motifs, which was shown to disrupt the function of multiple Ago proteins [START_REF] Hauptmann | Biochemical isolation of Argonaute protein complexes by Ago-APP[END_REF] (Figure 1e). Taken together, these data indicate that LegK1 efficiently suppresses the silencing of an siRNA-based reporter controlled by multiple Ago proteins. Importantly, we found that the LegK1-3WF and LegK1-KA mutant versions no longer triggered silencing suppression of this siRNA-based GFP reporter (Figure 1e). Therefore, both the kinase activity and the putative Ago-binding platform of LegK1 are essential for the silencing suppression of this reporter.

10

To investigate whether LegK1 additionally suppress miRNA functions, we made use of a previously characterized GFP-based construct that contains two artificial miRNA precursors embedded in an intron sequence located upstream of the GFP coding sequence [START_REF] Carré | a Biosensor to Detect Alterations in miRNA Biogenesis and in Small RNA Silencing Guided by Perfect Target Complementarity[END_REF] . This construct produces two mature anti-GFP miRNAs, which are perfectly complementary to the GFP mRNA sequence [START_REF] Carré | a Biosensor to Detect Alterations in miRNA Biogenesis and in Small RNA Silencing Guided by Perfect Target Complementarity[END_REF] . These artificial miRNAs were previously shown to trigger the self-silencing of the GFP reporter in a Drosha-and DGCR8-dependent manner [START_REF] Carré | a Biosensor to Detect Alterations in miRNA Biogenesis and in Small RNA Silencing Guided by Perfect Target Complementarity[END_REF] . Using this reporter system, we show that LegK1 triggers an increased GFP protein level compared to cells expressing the control effector YopM (Figure 1f,g). This was, however, not the case in response to T6B, suggesting that the Ago-binding activity of this peptide does not sufficiently alter the function(s) of the Ago protein(s) that silence(s) this reporter (Figure 1g). Therefore, unlike T6B, LegK1 can suppress miRNA-directed silencing of this GFP-based reporter.

In contrast, the GFP protein levels remained low in HEK293T cells expressing LegK1-KA and LegK1-3WF mutants (Figure 1g), indicating that these mutants no longer suppress miRNA activity. Altogether, these data reveal that LegK1 can efficiently suppress siRNA-and miRNA-activities through both its kinase activity and a predicted W-dependent Ago-binding platform.

LegK1 interacts with RISC factors, including Ago2 and its PIWI domain

The presence of predicted W-motifs in the protein sequence of LegK1 and their relevance in silencing suppression, suggested that this effector could interact with human Ago proteins. To test this possibility, we expressed the Flag-tagged LegK1-WT, LegK1-KA and LegK1-3WF versions in HEK293T cells and immunoprecipitated their cellular partners. Using Western blot analysis, we demonstrated that Ago1, Ago2 and Ago4 proteins were recovered from Flag-LegK1 immunoprecipitates, which was not observed after immunoprecipitation of Flag-eGFP from control cells (Figure 2a,b). This result indicates that LegK1 interacts with protein complexes containing Ago1, Ago2 and Ago4 proteins. Interestingly, we also recovered in the Flag-LegK1 immunoprecipitates the TNRC6A, PABPC1 and DDX6 proteins, which are wellcharacterized HMW-miRISC factors [START_REF] Chen | A DDX6-CNOT1 complex and W-binding pockets in CNOT9 reveal direct links between miRNA target recognition and silencing[END_REF][START_REF] Rouya | Human DDX6 effects miRNA-mediated gene silencing via direct binding to CNOT1[END_REF][START_REF] Fabian | Mammalian miRNA RISC recruits CAF1 and PABP to affect PABP-dependent deadenylation[END_REF][START_REF] Mathys | Structural and biochemical insights to the role of the CCR4-NOT complex and DDX6 ATPase in microRNA repression[END_REF][START_REF] Kamenska | The DDX6-4E-T interaction mediates translational repression and P-body assembly[END_REF] , suggesting that LegK1 interacts with mature Ago-RISCs engaged in RNA target repression. Importantly, the above interactions were maintained with the catalytic mutant LegK1-KA, while they were significantly reduced with the LegK1-3WF mutant (Figure 2a,b). These data suggest that the Ago-binding platform of LegK1 is required to interact with Ago-RISCs, while its kinase activity is dispensable for this process. Furthermore, we found that RNAse A treatment did not alter the ability of Flag-LegK1 to interact with GFP-Ago2 (Figure 2c). LegK1 thus interacts with human Ago2 in an RNA-independent manner. We next investigated whether LegK1 directly interacts with human Ago2. For this end, we conducted an in vitro interaction assay using GST-Ago2 and His6-SUMO-LegK1 2:386 -deprived of its predicted transmembrane domain-, which were both expressed and purified from E. coli (Figure 2d,e). We found that the GST-tagged Ago2 protein efficiently bound to His6-SUMO-LegK1 2:386 , while it did not interact with the TAP negative control (Figure 2e). Because the PIWI domain of human Ago2 is known to possess W-binding interfaces 32,80-82 , we additionally expressed and purified a CBPtagged PIWI domain of human Ago2 (Figure 2d,f). Using an in vitro pull-down assay, we found that the CBP-PIWI 517:859 -His6 recombinant protein interacted with His6-SUMO-LegK1 2:386 , however this interaction was not further improved in the presence of RNA extracts from HEK293T cells (Figure 2f). Therefore, we show that LegK1 directly interacts with human Ago2 likely through interaction surfaces embedded in its 12 PIWI domain. Our data also suggest that one or several W-motifs of LegK1 orchestrate the interaction between this bacterial effector and human Ago proteins.

The two W-motifs embedded in the kinase domain of LegK1 exhibit binding capacity to human Ago proteins

To further examine the contribution of the selected W-motifs of LegK1 in Ago-binding, we first attempted to express and purify the LegK1-3WF mutant version from E. coli.

However, this mutant formed insoluble inclusion bodies, preventing further in vitro pulldown experiments (Figure S2). To circumvent this issue, we next chemically synthesized biotinylated peptides containing each candidate W-motif (W41, W283 and W293), surrounded by native amino acid residues, referred to here as W1, W2 and W3 peptides (Figure 3a). In parallel, we synthetized identical peptides where each tryptophan was substituted by phenylalanine, named F1, F2 and F3 mutant peptides.

A pull-down experiment of each individual peptide was performed to elute bound proteins. Using this approach, we found that the W1 peptide did not bind to any of the Ago proteins, indicating that W41 is not a functional Ago-binding motif (Figure 3b). In contrast, the W2 and W3 peptides exhibited a clear binding to human Ago1, Ago2, Ago3 and Ago4, but not to the negative control GAPDH (Figure 3b,c). Therefore, the W2 and W3 peptides can interact with the four Ago proteins. Interestingly, the binding to these Ago proteins was lost in the presence of the F2 peptide, providing evidence that the W283 residue is a functional Ago-binding motif (Figure 3b). The F3 mutant peptides remained, however, competent in interacting with the four Ago proteins (Figure 3b). To assess whether the W293 residue is functional, we further synthesized a peptide carrying a substitution of the tryptophan residue into alanine (Figure 3a), a point mutation that has been the most extensively used on W-rich proteins to alter Ago-binding [START_REF] Till | A conserved motif in Argonaute-interacting proteins mediates functional interactions through the Argonaute PIWI domain[END_REF][START_REF] El-Shami | Reiterated WG/GW motifs form functionally and evolutionarily conserved ARGONAUTE-binding platforms in RNAi-related components[END_REF][START_REF] Lian | The C-terminal half of human Ago2 binds to multiple GW-rich regions of GW182 and requires GW182 to mediate silencing[END_REF][START_REF] Takimoto | Mammalian GW182 contains multiple Argonaute-binding sites and functions in microRNA-mediated translational repression[END_REF][START_REF] Eulalio | The GW182 protein family in animal cells: New insights into domains required for miRNA-mediated gene silencing[END_REF] . Indeed, the resulting A3 peptide no longer interacted with Ago proteins (Figure 3c), supporting a key role for the W293 motif in the interaction with human Ago proteins. Collectively, these data indicate that both the W283 and W293 motifs, which are embedded in the kinase domain of LegK1, behave as canonical Agobinding motifs.

The W283 and W293 motifs of LegK1 and their surrounding residues exhibit sequence conservation among Legionella species

To get some insights into the sequence conservation of the W-motifs of LegK1, we retrieved and aligned the protein sequences of putative LegK1 homologs from the Legionella protein sequences available on the National Center for Biotechnology Information (NCBI). More specifically, we selected for this analysis LegK1 homologous protein sequences exhibiting a protein identity of at least 40% to the L. pneumophila LegK1 strain Paris. According to these criteria, LegK1 was found present in 15 other Legionella species 4,13 (Figure 3d). Although the W41 motif was found conserved among all the Legionella species analysed, its surrounding residues were highly variable (Figure 3d). Furthermore, they did not fulfill the typical features of residues neighbouring functional W-motifs, which are composed of small, polar and nonhydrophobic residues [START_REF] Zielezinski | Integrative data analysis indicates an intrinsic disordered domain character of Argonaute-binding motifs[END_REF] . This observation is thus consistent with our experimental data indicating that this Ago-binding motif is not functional (Figure 3b). In contrast, the W293 motif of the strain L. pneumophila Paris, and its surrounding residues, were conserved among all Legionella species analysed (Figure 3d). The equivalent W293 motifs present in the different LegK1 homologous protein sequences analysed here thus probably maintain their Ago-binding capacity. When we analysed the sequence conservation of the W283 motif, and its surrounding residues, we found that these amino acids were also conserved among a cluster including L. pneumophila, L. norrlandica and L. waltersii (Figure 3d, blue clade). However, we noticed sequence variations in the remaining Legionella species analysed, which were not only found in the surrounding residues, but also in the tryptophan itself (tryptophan to tyrosine residues (W>Y); Figure 3d). Given that a tyrosine is one of the least probable residues that forms a functional W-motif [START_REF] Zielezinski | Integrative data analysis indicates an intrinsic disordered domain character of Argonaute-binding motifs[END_REF] , this observation suggested that the equivalent W283 motifs from these Legionella LegK1 protein sequences had lost, or alternatively never gained, the ability to bind Ago proteins. Consistent with this hypothesis, a biotinylated peptide corresponding to the region of the L. quateirensis LegK1 sequence carrying the W>Y natural variation, named Y2, did not bind to any of the human Ago proteins (Figure 3e,f). Therefore, the equivalent W283 motifs from LegK1 homologs should be functional in L. norrlandica and L. waltersii, but not in the remaining Legionella species.

Concomitant mutations in the two functional W-motifs of LegK1 abolish the ability of this effector to phosphorylate I𝜅Ba and to suppress RNA silencing

The fact that the two functional W-motifs are embedded in the kinase domain of LegK1 prompted us to further investigate the relationship between the Ago-binding platform and the kinase activity of this effector. To this end, we examined the possible impact of single and concomitant W-motif mutations on the ability of LegK1 to phosphorylate I𝜅Ba, a known substrate of this effector protein [START_REF] Ge | A Legionella type IV effector activates the NF-κB pathway by phosphorylating the IκB family of inhibitors[END_REF] . More specifically, we expressed the WT or the mutant versions of LegK1 in HEK293T cells, and further incubated the immunoprecipitates from each cell lysate with purified recombinant CBP-I𝜅Ba-His6.

Western blot analyses from these cell-free extracts, using an antibody recognizing phosphorylation at Ser-32 of I𝜅Ba, revealed strong LegK1-induced phosphorylation of CBP-I𝜅Ba-His6 (Figures 4a andS3). While the single LegK1-W283F and LegK1-W293F mutants triggered comparable phosphorylation levels at Ser-32 of I𝜅Ba, the LegK1-W293A mutant was partially compromised in this process (Figure 4a). Importantly, the phosphorylation at Ser-32 of I𝜅Ba was fully abolished in the presence of LegK1-W283F-W293A, despite the stable accumulation of this double mutant in HEK293T cells (Figure 4a). These effects were similar to the ones detected with the LegK1-3WF and the LegK1-KA mutants (Figure S3). Therefore, the concomitant mutations in the two functional W-motifs of LegK1 are sufficient to abolish its catalytic activity.

We next assessed the possible impact of these W-motif mutations on the silencing suppression activity of LegK1 using the siRNA-based GFP reporter in HEK293T cells.

We found that the LegK1-W293A mutant, and particularly the LegK1-W283F-W293A double mutant, were altered in their ability to suppress siRNA-guided silencing, as revealed by an impaired GFP derepression (Figure 4b). These effects were comparable to the ones observed in HEK293T cells expressing YopM, LegK1-3WF or LegK1-KA mutants (Figure 1e). We conclude that the W293 motif has a major role in LegK1-triggered silencing suppression and that both the W283 and W293 motifs must cooperatively orchestrate this process. Altogether, these data indicate that the kinase, Ago-binding and RNA silencing suppression activities of LegK1 are tightly interconnected.

LegK1-triggered NF-kB signaling activation contributes to RNA silencing suppression

Given that the kinase and silencing suppression activities of LegK1 were found to be interconnected, we reasoned that LegK1 could directly phosphorylate human Ago proteins to alter their functions. To test this hypothesis, we investigated whether LegK1 could phosphorylate human Ago2. Nevertheless, we did not observe any increase in the phosphorylation status of the human Ago2 protein in the presence of LegK1 (Figure S3, Supplementary text), indicating that LegK1 unlikely phosphorylate this host factor. Instead, these results suggest that LegK1 could suppress RNA silencing by phosphorylating other RISC component(s) and/or proteins that indirectly regulate RNA silencing activity. Because LegK1 has been shown to directly phosphorylate IkBa, resulting in a potent NF-kB signaling activation 17 (Ge et al., 2009), and because its catalytic activity was found here to be essential for RNA silencing suppression (Figure 1e, g), we further investigated whether LegK1-triggered NF-kB signaling activation could contribute to silencing suppression. For this purpose, we tested if the Caffeic Acid Phenethyl Ester (CAPE), a well-established NF-kB signaling inhibitor [START_REF] Natarajan | Caffeic acid phenethyl ester is a potent and specific inhibitor of activation of nuclear transcription factor NF-kappa B[END_REF][START_REF] Lee | Caffeic acid phenethyl ester-mediated Nrf2 activation and IkappaB kinase inhibition are involved in NFkappaB inhibitory effect: structural analysis for NFkappaB inhibition[END_REF] , could alter the ability of LegK1 to suppress silencing of the siRNA-based GFP reporter in HEK293T cells. Interestingly, while LegK1 triggered a significant derepression of siRNA-directed silencing of the GFP reporter compared to the control vector, we found that this effect was significantly reduced upon CAPE treatment, which concomitantly prevented LegK1-induced phosphorylation at Ser-32 of I𝜅Ba (Figure 4c). Of note, the low GFP protein accumulation detected in this condition was not due to a destabilization of the GFP in response to CAPE, because a normal GFP protein level was observed upon CAPE treatment of HEK293T cells expressing LegK1 and control siRNAs (Figure 4c). The CAPE-dependent suppression of NF-kB signaling can thus restore the ability of siRNAs to silence the GFP reporter in the presence of LegK1.

Collectively, these results demonstrate that the silencing suppression activity of LegK1 is in part achieved through activation of NF-kB signaling directed by its kinase activity.

LegK1 promotes the growth of L. pneumophila in both Acanthamoeba castellanii and human macrophages

To determine whether LegK1 contributes to the virulence of L. pneumophila, we first generated an isogenic legK1 deletion mutant, referred to here as the Lpp DlegK1 strain. Whole-genome sequencing of Lpp DlegK1 confirmed a unique deletion in the legK1 gene, which did not affect the fitness of this bacterium in vitro (Figure S4a,b).

We next infected Acanthamoeba castellanii, a natural host of L. pneumophila, with the L. pneumophila WT and DlegK1 strains and further monitored bacterial numbers during the course of the infection. Interestingly, the growth of the Lpp DlegK1 strain was reduced in A. castellanii compared to that of L. pneumophila WT (Figure 5a).

Because L. pneumophila can accidentally infect humans where it replicates in alveolar macrophages, we conducted the same assay in macrophages derived from THP-1 cells. Similarly, a reduced intracellular replication of the DlegK1 strain was observed compared to the one achieved by the WT strain (Figure 5b). These growth defects were partially complemented in both A. castellanii and human macrophages, respectively, when LegK1 was expressed in trans in the Lpp DlegK1 strain (Figure 5c,d). These data indicate that the reduced growth of Lpp DlegK1 in both A. castellanii and human macrophages is due to the deletion of legK1. They also provide evidence that LegK1 is a major virulence factor of L. pneumophila that promotes bacterial growth in both amoeba and human macrophages.

Human Ago4 is a functionally relevant genetic target of LegK1 in infected macrophages

We next analysed whether LegK1 genetically targets human Ago functions to promote L. pneumophila virulence. Using available RNA-seq datasets [START_REF] Hruz | Genevestigator v3: a reference expression database for the meta-analysis of transcriptomes[END_REF] , we first noticed that human Ago2 and Ago4 mRNAs were the most abundant Ago transcripts in THP-1 cells (Figure 5e). This analysis supports recent findings showing that Ago4 is highly expressed in immune-related cells [START_REF] Adiliaghdam | A Requirement for Argonaute 4 in Mammalian Antiviral Defense[END_REF] . Consistently, we detected a higher Ago4 protein accumulation in THP-1 cells compared to HEK293T and A549 epithelial cells, which was also detected for Ago2, albeit to a lesser extent (Figure 5f). Based on these data, we decided to knock-out human Ago2 or Ago4 in THP-1 cells for further functional assays. A single guide RNA approach was used to generate CRISPR/Cas9-based deletions in the second exon of the Ago2 and in the third exon of the Ago4 loci. The resulting pools of ago2 -/-and ago4 -/-THP-1 lines were found respectively deprived of Ago2 and Ago4 proteins compared to THP-1 control cells (Figure 5g). Macrophages derived from the ago2 -/-and ago4 -/-THP-1 cells were infected with the L. pneumophila WT or DlegK1 strains and bacterial numbers were monitored at 24 hpi. A normal growth of the WT strain was achieved in ago2 -/-and ago4 -/-cells compared to control cells (Figure S4c), indicating that neither human Ago2 nor Ago4 control the growth of this bacterium in those conditions. Importantly, however, the growth defect of the Lpp DlegK1 strain was partially rescued in ago4 -/-macrophages (Figure 5g), indicating that the lack of Ago4 facilitates the growth of this bacterial mutant in macrophages. The latter rescue experiment demonstrates that human Ago4 is a functionally relevant genetic target of LegK1 in infected macrophages.

Discussion

Here, we showed that the type IV-secreted protein LegK1 from L. pneumophila can efficiently suppress RNA silencing in human cells (Figures 1 and2, Figure S1). More specifically, LegK1 dampened the silencing of the Ago2-dependent siRNA-based CXCR4 reporter (Figure 1). It also suppressed the silencing of a siRNA-based GFP reporter system, which depends, at least in part, on human Ago1 and Ago2 (Figure 1).

In addition, we found that LegK1 was able to efficiently suppress a miRNA-based GFP reporter system (Figure 1). Importantly, these silencing suppression effects relied on a W-dependent Ago-binding platform, which was critical for the interaction of LegK1 with human Ago proteins (Figures 1,2). Thus, these data indicate that LegK1 acts as a bona fide BSR by suppressing both siRNA-and miRNA-activities in human cells through a functional Ago-binding platform. The use of such Ago-binding platform is therefore not restricted to VSRs, as previously thought [START_REF] Aqil | The HIV-1 Nef Protein Binds Argonaute-2 and Functions as a Viral Suppressor of RNA Interference[END_REF][START_REF] Azevedo | Argonaute quenching and global changes in Dicer homeostasis caused by a pathogen-encoded GW repeat protein[END_REF][START_REF] Giner | Viral Protein Inhibits RISC Activity by Argonaute Binding through Conserved WG/GW Motifs[END_REF] , but can be additionally exploited by at least one human pathogenic bacterium.

LegK1 was not only capable to bind human Ago1, Ago2 and Ago4 through its Wmotifs, but was also recovered in protein complexes composed of TNRC6A, PABPC1 and DDX6 (Figure 2). Because these factors are part of the HMW-miRISC [START_REF] Duchaine | Mechanistic Insights into MicroRNA-Mediated Gene Silencing[END_REF][START_REF] Iwakawa | Life of RISC: Formation, action, and degradation of RNA-induced silencing complex[END_REF] , our data suggest that LegK1 interferes with the function of mature RISCs that are engaged in the silencing of sRNA targets. Intriguingly, we also noticed that the WS and LWG residues, which are present in the C-terminal domain of TNRC6 proteins, and known to be required for the direct interaction with components of deadenylase complexes [START_REF] Chen | A DDX6-CNOT1 complex and W-binding pockets in CNOT9 reveal direct links between miRNA target recognition and silencing[END_REF][START_REF] Braun | GW182 proteins directly recruit cytoplasmic deadenylase complexes to miRNA targets[END_REF][START_REF] Chekulaeva | miRNA repression involves GW182-mediated recruitment of CCR4-NOT through conserved W-containing motifs[END_REF][START_REF] Fabian | miRNA-mediated deadenylation is orchestrated by GW182 through two conserved motifs that interact with CCR4-NOT[END_REF][START_REF] Christie | Structure of the PAN3 pseudokinase reveals the basis for interactions with the PAN2 deadenylase and the GW182 proteins[END_REF] , are also part of the W283 (DVWSTG) and the W293 (LWGD) motifs of LegK1. These observations suggest that LegK1 might additionally use its W-motifs to physically interact with deadenylase complexes. This hypothesis is consistent with our co-immunoprecipitation data showing that LegK1 interacts with DDX6, a direct partner of CNOT1 [START_REF] Chen | A DDX6-CNOT1 complex and W-binding pockets in CNOT9 reveal direct links between miRNA target recognition and silencing[END_REF][START_REF] Mathys | Structural and biochemical insights to the role of the CCR4-NOT complex and DDX6 ATPase in microRNA repression[END_REF] . Based on these data, we propose that LegK1 might trigger silencing suppression by altering, at least in part, the function of HMW-RISCs. Such a mode of action would be well-adapted to rapidly repress RNA targets that fine-tune innate immune responses during L. pneumophila infections [START_REF] Herkt | A MicroRNA Network Controls Legionella pneumophila Replication in Human Macrophages via LGALS8 and MX1[END_REF] . It would also be analogous to the mode of action of the Sweet potato mild mottle virus (SPMMV) VSR P1, which was shown to interact with Nicotiana benthamiana AGO1 through W-motifs and to co-fractionate with RISC containing AGO1-loaded small RNAs 67 (Giner et al., 2010).

We also found that the two W-motifs from the kinase domain of LegK1, namely W283 and W293, are essential for the binding of LegK1 to human Ago proteins (Figure 3). Interestingly, these tryptophan residues were found interspaced by 9 amino acids, which is a typical feature of functional W-motifs embedded in the ABD of TNRC6 proteins, which are in most cases separated by 8 to 14 amino acids from each other 29- 30,49 . This amino acid organization is thought to form a flexible linker that facilitates the interaction of W-motifs with the three W-binding pockets exposed at the surface of human Ago2 and Ago4 proteins, which are separated by a distance of 25 Å from each other [START_REF] Schirle | The Crystal Structure of Human Argonaute2[END_REF][START_REF] Sheu-Gruttadauria | Phase transitions in the assembly and function of human miRISC[END_REF][START_REF] Park | Human Argonaute2 and Argonaute3 are catalytically activated by different lengths of guide RNA[END_REF] . Based on these findings, we propose that the W283 and W293 motifs of LegK1 cooperate with each other to ensure a tight association between this bacterial protein and the W-binding pockets of human Ago proteins. It is also noteworthy that these W-motifs are not positioned in a disordered region, such as the ones typically found in the ABDs of TNRC6 proteins [START_REF] Till | A conserved motif in Argonaute-interacting proteins mediates functional interactions through the Argonaute PIWI domain[END_REF][START_REF] El-Shami | Reiterated WG/GW motifs form functionally and evolutionarily conserved ARGONAUTE-binding platforms in RNAi-related components[END_REF][START_REF] Takimoto | Mammalian GW182 contains multiple Argonaute-binding sites and functions in microRNA-mediated translational repression[END_REF][START_REF] Pfaff | Structural features of Argonaute-GW182 protein interactions[END_REF][START_REF] Karlowski | Genome-wide computational identification of WG/GW Argonaute-binding proteins in Arabidopsis[END_REF] . Instead, they are embedded in a structured kinase domain. Our data therefore imply to revisit in silico predictions of Ago-binding platforms located in structured domains, which were so far discarded from experimental validations [START_REF] Zielezinski | Integrative data analysis indicates an intrinsic disordered domain character of Argonaute-binding motifs[END_REF] .

To get further insights into the functional relevance of the W283 and W293 motifs of LegK1 in Ago-binding, we analyzed their sequence conservation across Legionella species. We found that the region corresponding to the L. pneumophila W293 motif, and its surrounding residues, are conserved among several Legionella species. The region corresponding to the W283 motif was also found conserved in L. norrlandica and L. waltersii species. In contrast, amino acid sequence variations were observed in the tryptophan and surrounding residues of the LegK1 protein sequences derived from the remaining analysed Legionella species. Furthermore, they prevented Agobinding of a peptide derived from the L. quateirensis LegK1 protein sequence (Figure 3). Therefore, the conservation of the W283 and W293 motifs, observed in some Legionella species, supports our experimental data showing that these W-motifs likely cooperate between each other to form a functional Ago-binding platform.

By disabling the two functional W-motifs from the Ago-binding platform of LegK1, we found that this effector was fully compromised in its ability to phosphorylate IkBa and to suppress RNA silencing (Figure 4). These data are consistent with the central role played by the kinase activity of LegK1 in RNA silencing suppression (Figure 1), and support a tight interconnection between the kinase and Ago-binding activities of LegK1. In addition, we discovered that the blockage of NF-kB signaling activation in human cells expressing LegK1 reduced the siRNA-directed silencing of the GFP reporter system (Figure 4). These data unveil an unexpected cross-talk between LegK1-mediated activation of NF-kB signaling and silencing suppression. They also highlight an interconnection between the kinase, RNA silencing suppression and NF-kB signaling activation activities of LegK1, which remains to be characterized in more depth in the future. It will notably be important to assess if cellular pools of human Ago proteins could be recovered in protein complexes containing NF-kB-related factors, and whether LegK1 acts on specific components of such complexes, besides IkBa and Ago proteins, to suppress RNA silencing while enhancing NF-kB signaling.

The long-lasting coevolution of Legionella in a wide range of amoebae and ciliated protozoa has shaped the Legionella genomes [START_REF] Burstein | Genome-Scale Identification of Legionella pneumophila Effectors Using a Machine Learning Approach[END_REF][START_REF] Gomez-Valero | More than 18,000 effectors in the Legionella genus genome provide multiple, independent combinations for replication in human cells[END_REF] . The presence of a large repertoire of eukaryotic-like proteins in the Legionella genomes indicates that these proteins must have been acquired through horizontal gene transfer from protist host organisms [START_REF] Gomez-Valero | More than 18,000 effectors in the Legionella genus genome provide multiple, independent combinations for replication in human cells[END_REF][START_REF] Cazalet | Evidence in the Legionella pneumophila genome for exploitation of host cell functions and high genome plasticity[END_REF][START_REF] De Felipe | Evidence for Acquisition of Legionella Type IV Secretion Substrates via Interdomain Horizontal Gene Transfer[END_REF][START_REF] Lurie-Weinberger | The origins of eukaryotic-like proteins in Legionella pneumophila[END_REF][START_REF] Gomez-Valero | Comparative analyses of Legionella species identifies genetic features of strains causing Legionnaires' disease[END_REF] . Accordingly, most of the virulence strategies used by L. pneumophila are required to replicate in both protists and human macrophages [START_REF] Mondino | Legionnaires' Disease: State of the Art Knowledge of Pathogenesis Mechanisms of Legionella[END_REF][START_REF] Escoll | From Amoeba to Macrophages: Exploring the Molecular Mechanisms of Legionella pneumophila Infection in Both Hosts[END_REF] . Consistently, we showed that the Lpp DlegK1 strain was altered in its ability to replicate in both A. castellanii and human macrophages (Figure 5), supporting a physiologically relevant role of LegK1 in L. pneumophila virulence. In addition, we found that the growth defect of the Lpp DlegK1 strain was partially rescued in Ago4-deficient human macrophages, (Figure 5). This finding suggests that the targeting of Ago proteins by LegK1 is likely a major virulence function of this effector, which might also be required to promote growth of L. pneumophila in its natural hosts. This assumption is supported by the fact that amoebae genomes like Dictyostelium discoideum, possess canonical Ago proteins, some of which are even experimentally characterized in RNA silencing [START_REF] Zhang | RNA interference in Entamoeba histolytica: implications for parasite biology and gene silencing[END_REF] .

Here, by searching for the presence of Ago-like factors in the recently released genome sequence of the A. castellanii strain used in this study [START_REF] Matthey-Doret | Chromosome-scale assemblies of Acanthamoeba castellanii genomes provide insights into Legionella pneumophila infection-related chromatin reorganization[END_REF] , we recovered two candidate Ago-like proteins composed of canonical PIWI and PAZ domains (Figure S4d). In contrast, none of the NF-kB-related domains were retrieved from the genomes of amoebae, including the A. castellanii strain used herein [START_REF] Escoll | From Amoeba to Macrophages: Exploring the Molecular Mechanisms of Legionella pneumophila Infection in Both Hosts[END_REF][START_REF] Matthey-Doret | Chromosome-scale assemblies of Acanthamoeba castellanii genomes provide insights into Legionella pneumophila infection-related chromatin reorganization[END_REF] . Based on the lack of NF-kB-related genes in amoebae genomes, and on the presence of canonical Ago proteins in these natural hosts, we propose that the ability of LegK1 to suppress Ago activities is likely the primary virulence function of this bacterial effector.

Antiviral RNA interference (RNAi) has been extensively characterized in plants, fungi, insects and worms [START_REF] Hamilton | A Species of Small Antisense RNA in Posttranscriptional Gene Silencing in Plants[END_REF][START_REF] Lu | Animal virus replication and RNAi-mediated antiviral silencing in C elegans[END_REF][START_REF] Wang | RNA interference directs innate immunity against viruses in adult Drosophila[END_REF][START_REF] Ding | RNA-based antiviral immunity[END_REF][START_REF] Jin | Recent advances in understanding plant antiviral RNAi and viral suppressors of RNAi[END_REF] . In mammals, emerging evidence indicates that antiviral RNAi also operates in embryonic stem cells, adult stem cells and progenitor cells, in which the antiviral interferon (IFN) response -known to repress RNAi-is less active [START_REF] Maillard | Inactivation of the type I interferon pathway reveals long double-stranded RNA-mediated RNA interference in mammalian cells[END_REF][START_REF] Van Der Veen | The RIG-I-like receptor LGP2 inhibits Dicerdependent processing of long double-stranded RNA and blocks RNA interference in mammalian cells[END_REF][START_REF] Xu | Zika virus infection induces RNAi-mediated antiviral immunity in human neural progenitors and brain organoids[END_REF][START_REF] Poirier | An isoform of Dicer protects mammalian stem cells against multiple RNA viruses[END_REF] . In these tissues/cell types, canonical antiviral sRNAs readily accumulate during viral infections and inhibit viral replication in an Ago2-dependent manner [START_REF] Li | Induction and suppression of antiviral RNA interference by influenza A virus in mammalian cells[END_REF][START_REF] Qiu | Human Virus-Derived Small RNAs Can Confer Antiviral Immunity in Mammals[END_REF][START_REF] Xu | Zika virus infection induces RNAi-mediated antiviral immunity in human neural progenitors and brain organoids[END_REF] .

Interestingly, mouse Ago4 was also recently shown to restrict the replication of different viruses in IFN-competent cells, including influenza A virus (IAV) [START_REF] Adiliaghdam | A Requirement for Argonaute 4 in Mammalian Antiviral Defense[END_REF] .

Importantly, mouse Ago4 was also shown to dampen IAV replication in vivo, further supporting a key role of this factor in antiviral resistance [START_REF] Adiliaghdam | A Requirement for Argonaute 4 in Mammalian Antiviral Defense[END_REF] . In the present study, we unveiled a novel function of human Ago4 in host-bacterial interactions. We first showed that Ago4 -and Ago2 to a lesser extent-were the most expressed Ago genes in immune-related cells (Figure 5). Significantly, although a normal growth of the Lpp WT strain was observed in human ago2 -/-and ago4 -/-macrophages, we found that the growth defect of the Lpp DlegK1 mutant was partially rescued in Ago4-deficient cells (Figure 5). This provides genetic evidence that LegK1 must suppress the function of Ago4 to promote growth of L. pneumophila in IFN-competent macrophages. These findings therefore extend the roles of human Ago4 to antibacterial defense. They are thus consistent with previous reports in plants showing that AGO1 and AGO2 proteins orchestrate antibacterial defense, beyond their well-described antiviral functions [100][101][102][103] .

In conclusion, this study reports on the first bacterial effector that directly suppresses RNA silencing by physically interacting with Ago proteins through W-motifs.

Intriguingly, we have also retrieved W-motifs in an appropriate sequence context in effectors from various human pathogenic bacteria, fungi and parasites (Figure S5).

These observations suggest that a wide range of non-viral mammalian pathogens and parasites might have evolved an analogous mode of action to promote virulence. It will thus be interesting to establish if the discoveries made on LegK1 hold true for other virulence/parasitic factors from non-viral mammalian pathogens/parasites. It will also be worth developing innovative strategies to counteract this potentially widespread virulence mechanism in order to control infectious and parasitic diseases.

Methods

Human cell lines and culture

HeLa cells (ATCC ® CCL-2™), control (CTL), ago1 -/-, ago2 -/-, ago1 -/-/ago2 -/-and dicer - /-CRISPR/Cas9-based HeLa cell lines 104 (this study), and Human Embryonic Kidney 1640 medium (Dominique Dutscher, L0498-500). The percentages of editing efficiency after expansion were of 97% and 96% for THP-1 ago2 -/-and ago4 -/-cell pools, respectively. All media were supplemented with 10% fetal bovine serum. Cells were maintained at 37°C in a humidified 5% CO2 atmosphere. In addition, all human cell lines were regularly tested negative for mycoplasma contamination.

Acanthamoeba castellanii culture

Acanthamoeba castellanii (ATCC ® 50739) was cultured in PYG 712 medium (2% proteose peptone, 0.1% yeast extract, 0.1 M glucose, 4 mM MgSO4, 0.4 M CaCl2, 0.1% sodium citrate dihydrate, 0.05 mM Fe (NH4)2(SO4)2 x 6H2O, 2.5 mM NaH2PO3, 2.5 mM K2HPO3) at 20°C for 72 h prior to harvesting for L. pneumophila infection.

Bacterial strains and mutant constructions

Legionella pneumophila strain Paris WT (CIP 107629T) and DlegK1 (Dlpp1439) mutant were cultured in N-(2-acetamido)-2-aminoethanesulfonic acid (ACES)buffered yeast extract broth (BCY) or on ACES-buffered charcoal-yeast (BCYE) extract agar 105 . When needed antibiotics were added: for L. pneumophila: gentamycin 12.5 μg/ml, chloramphenicol 10 μg/ml. All strains were grown at 37°C. The DlegK1 mutant was constructed by replacing the gene of interest with a gentamycin resistance cassette in the chromosome. The mutant allele was constructed using a three-step PCR technique 106 (Table S1). Briefly, the gene of interest was inactivated by introduction of a gentamycin resistance cassette into the chromosome. Therefore, three overlapping fragments, corresponding to the upstream region, the antibiotic cassette and the downstream region of the gene of interest, were amplified Transformants were selected by plating on BCYE containing chloramphenicol.

Expression plasmids and constructions

The expression vectors for bacterial candidates were generated using GATEWAY technology. Briefly, PCR amplification (Table S1 Lauriane Quenee, Institute Biosafety Officer at Caltech, Los Angeles, USA). Finally, the coding sequence of legK1 (lpp1439) was PCR amplified using gDNA of the Legionella pneumophila strain Paris. All resulting PCR products were introduced into the pENTR/D-TOPO entry vector (Invitrogen, K240020). They were sequenced and then recombined into the GATEWAY binary destination vector pPURO-Flag-HA (gift from Dr. Sebastien Pfeffer, IBMC, Strasbourg, France) using LR clonase (Invitrogen, 11791020), hereinafter referred to as pFlag, allowing an expression under the control of the cytomegalovirus (CMV) immediate early promoter. The molecular characterization of LegK1 was then done in the HEK293T cell line, using a pPURO-2xFlag-HA, hereinafter referred to as p2xFlag. This plasmid was constructed from pFlag expression vector, by insertion of a second Flag using Gibson assembly (New England BioLabs, E5510S). The legK1 coding sequence was then inserted from pENTR/D-TOPO-LegK1 into p2xFlag through the GATEWAY technology, as previously described. Point mutations were introduced in this plasmid to generate the kinase-dead mutant (LegK1-KA), the mutants on the three putative W-motifs (LegK1-3WF), on the two functional W-motifs (LegK1-W283F-W293A) or on the individual Wmotifs (LegK1-W283F, -W293F, -W293A). All these point mutations were carried out using site-directed mutagenesis by PCR with appropriate primers containing the desired nucleotide changes (Table S1), and followed by selection with DpnI digestion.

For siRNA-guided GFP silencing reporter, the plasmid pPURO-Flag-HA-eGFP was cloned in this study using GATEWAY technology, as previously described, and was hereinafter referred to as pFlag-eGFP. The miRNA-guided GFP silencing reporter, referred to as AutomiG, was kindly provided by Pr. Christophe Antoniewski 76 (Institut de Biologie Paris Seine, Paris, France). A vector expressing TNRC6B-derived peptide (T6B) was used as positive control of the siRNA-guided GFP silencing reporter, and was kindly provided by Pr. Gunter Meister [START_REF] Hauptmann | Biochemical isolation of Argonaute protein complexes by Ago-APP[END_REF] (Universität Regensburg, Regensburg, Germany).

For the co-immunoprecipitation assays, a plasmid expressing human Ago2 fused to GFP in the N-terminal region of Ago2 was obtained from Addgene (11590). For

Human cell transfections

For the detection of recombinant LegK1 WT or mutant proteins by Western blot analysis, HEK293T cells were seeded in 24-well plates at a density of 1.4x10 5 cells per well, and were transiently transfected for 48h with either 500 ng of p2xFlag-LegK1-WT, -KA or -3WF plasmids using JetPrime (Polyplus, 114-15), according to the manufacturer's instructions.

For the characterization of the siRNA-based luciferase reporter, control (CTL), ago1 -/- , ago2 -/-, ago1 -/-/ago2 -/-and dicer -/-HeLa cell lines were transfected with Lipofectamine For the co-immunoprecipitation assays, HEK293T cells were seeded in 10 cm 2 dishes at a density of 6.2x10 6 cells per well and were transiently transfected during 48h with 10 µg of pFlag-eGFP or p2xFlag-LegK1-WT, -KA, -3WF using JetPrime. For the coimmunoprecipitation assay followed by RNAse A treatment (Thermo Scientific, R1253), HEK293T cells were seeded in the same manner and were co-transfected for 48h with 10 µg of pGFP-Ago2-WT and 10 µg of pFlag-T6B or 14 µg of p2xFlag-LegK1 using Lipofectamine 2000. To determine the catalytic activity of LegK1 mutants in the kinase assay, HEK293T cells were seeded in 6 cm 2 dishes at a density of 8.4x10 5 cells per well, and were transiently transfected for 48h with 4 µg of pFlag-eGFP or p2xFlag-LegK1-WT or p2xFlag-LegK1 mutants using JetPrime. Finally, for the mass spectrometry analysis, HeLa cells were seeded in 15 cm 2 dishes at a density of 1.4x10 7 cells per well and were transfected for 24h with 20 µg of pFlag-eGFP, p2xFlag-LegK1-WT or -KA using JetPrime.

A. castellanii and THP-1 infection assays

A. castellanii were washed once with Infection Buffer (PYG 712 medium without proteose peptone, glucose and yeast extract) and seeded in 25 cm 2 flasks at a concentration of 4x10

Dual-luciferase silencing reporter analyses

The initial screening in HeLa cells was carried out using a siRNA-guided luciferase silencing reporter, consisting in the expression of the pGL3-CXCR4-2p vector [START_REF] Doench | siRNAs can function as miRNAs[END_REF]

GFP-based silencing reporter system analyses

The suppression activities on siRNA-and miRNA-guided GFP silencing reporters were both determined by Western blot analyses. Transfected cells were washed with 1X PBS and lysed in 100 µL of 1X Laemmli Loading Buffer. For each condition, 40 µL of samples were loaded on SDS-PAGE and subjected to Western Blot analysis. In some experiments, cells were transiently co-transfected with siRNA-guided silencing reporters and p2xFlag-LegK1 or pFlag empty control plasmid or pFlag-YopM, to ensure that each transfection received the same amount of total DNA. For the inhibition of NF-kB signaling, cells were then treated for 1h with 25 µg/mL of Caffeic Acid Phenethyl Ester inhibitor (CAPE, Santa Cruz Biotechnology, sc-200800) or DMSO. Cell extracts were subjected to Western blot analysis, as described previously.

Quantification of eGFP protein from Western blot analysis was performed by densitometric analysis, using the Fiji (ImageJ) software, and was then normalised to the YopM-siCTL or empty vector-siCTL conditions.

Western blot analyses

Cells were washed with 1X PBS and lysed either in 1X Laemmli Loading Buffer or in Radio-ImmunoPrecipitation Assay (RIPA) buffer, for which the protein concentration was determined using the Bradford reagent (Bio-Rad, 5000006EDU). Approximately 100 µg of proteins were denatured 5 min at 95°C and then subjected to SDS-PAGE and Western blot analyses, according to standard procedures. The detection of proteins of interest was performed using the following primary antibodies: anti-Ago1

Purification of recombinant proteins

All recombinant proteins were expressed from the E. coli strain BL21(DE3) codon plus (ThermoFisher, EC0114) in 1L of Terrific broth (TB). Cultures were grown at 37°C until they reached an OD600 of 2 and protein production was induced with 1 mM Isopropylβ-D-thiogalactopyranoside (IPTG), followed by overnight growth at 18°C with shaking at 180 rpm. Bacterial cells expressing His6-SUMO-LegK1

RNA extractions and RT-qPCR

For gene expression analyses, total RNAs were isolated by phenol-chloroform extraction using TRIzol Reagent (Thermo Fisher Scientific, 15-596-018), according to the manufacturer's instructions. Approximately 0.5 μg of RNAs were then digested by DNAse I (Promega, M6101) at 37°C for 50 min to remove the genomic DNA, followed by 10 min at 65°C to inactivate the DNAse. DNAse-digested RNAs were reversetranscribed into complementary DNA using qScript cDNA Supermix (Quanta Biosciences, 733-1178). The cDNAs were quantified with SYBR Green qPCR mix (Takyon, Eurogentec, UF-NSMT-B0701) and gene-specific primers (Table S1) in 384well plates, following a protocol of heating at 95°C for 10 min, 45 cycles of denaturation at 95°C for 10 sec, and annealing at 60°C for 40 sec. A melting curve was performed at the end of the amplification, and transcript levels were then normalised to the abundance of GAPDH transcripts.

PRM data acquisition method

HeLa cells were seeded in 15 cm 2 dishes at a density of 1.4x10 S2). The acquisition list was generated from the peptides obtained from the mix samples (5 replicates) of each condition, based on the data-dependent acquisition (DDA) results (data not shown).

PRM data analyses

The PRM data were analysed using Skyline 4.1 (MacCoss Lab Software, Seattle, Washington [https://skyline.ms/project/home/software/Skyline/begin.view]). Fragment ions for each targeted mass (Table S3) were extracted and peak areas were

Alignment of orthologous protein sequences and phylogeny

The protein sequence of LegK1 (from the lpp1439 gene) from L. pneumophila strain Paris was used as a reference to retrieve homologous LegK1 protein sequences from Legionella species (taxid: 445). To this end, a BLASTP (Basic Local Alignment Search Tool) was performed on the NCBI (National Center for Biotechnology Information)

website. An identity cut-off of 40%, an Expect (E)-value cut-off of 10 -5 and a minimum percentage match length of subject and query of 65% were used. The set of homologous protein sequences were then aligned using ClustalW2 on Geneious 10.2.6 software. Tree was conducted using Neighbor-joining.

Heatmap of Ago transcripts

The RNA-sequencing results were generated in the Cancer Cell Line Encyclopedia (CCLE, Primary ID: E-MTAB-2770) and are reported as linear values, retrieved from Genevestigator.

  293T cells (HEK293T) cells (ATCC® CRL-3216™) were cultured in high glucose Dulbecco's modified Eagle's medium (DMEM) (containing 4.5 g/l of glucose) (Dominique Dutscher, L0103-500). CTL and ago2 -/-CRISPR/Cas9-based HeLa cell lines were previously generated 104 . LentiCRISPRv2-Ago1 and LentiCRISPRv2-Dicer expression plasmids expressing single-guide RNA targeting exon 3 of Ago1 (AAACTCATACACAGGCTTGCGATC) or Dicer (AAACCTGATCTGATAGGACAGCTC) genes respectively, were used to transfect HEK293T cells using Lipofectamine LTX (Life technologies, A12621). Forty-eight hours later, viral particles were harvested and used to transduce HeLa cells. Cells were selected in medium supplemented with puromycin for 15 days. Data acquisition and data analysis were performed on the Cochin Cytometry and Immunobiology core Facilities. Single cells were isolated from lentiCRISPRv2-Ago1 or lentiCRISPRv2-Dicer transduced cells using a BD Biosciences FACSAria III. Expression of Ago1 or Dicer in the cell clones was analyzed by SDS-PAGE and immunoblotting. One cell clone knockout was selected for further analysis. A549 cells (ATCC ® CCL-185) were cultured in Ham's F-12K Nutrient Mixture (Kaighn's Modification) (Corning, 10-025-CVR). THP-1 (ATCC ® TIB-202™) and THP-1 ago2 -/-and ago4 -/-cell pools (Synthego®, this study) were cultured in Gibco Roswell Park Memorial Institute (RPMI)

  independently and purified on agarose gels. The three resulting PCR products were mixed at the same molar concentration (15 nM) and a second PCR with flanking primer pairs was performed. The resulting PCR product, the gentamycin resistance cassette flanked by 500 bp regions homologous to legK1 was introduced into L. pneumophila by natural competence for chromosomal recombination. Strains that had undergone allelic exchange were selected by plating on BCYE containing gentamycin and the mutant was verified by PCR and whole genome sequencing. Bacterial growth assays were performed by seeding the indicated strains in wells of a microtiter plate (TPP) containing 200µl of BYE. The microtiter plate was incubated with shaking at 37°C for 48 hours in a Tecan Infinte multiwell plate reader (Tecan Group). Optical density measurements were taken at 600nm every 30 min. For complementation, the L. pneumophila WT and DlegK1 mutant strains were transformed by electroporation (2.5kV, 200Ω and 25µF) with 100 ng of the empty plasmid or pBC-KS-LegK1.

  recombinant protein purification, a DNA fragment encoding LegK1 amino acids 2 to 386 was amplified by PCR, digested with NotI and XhoI enzymes and inserted into pET22-His6-SUMO plasmid 107 , kindly given by Dr. Hervé Le Hir (IBENS, Paris, France). This construct allows the expression of LegK1 2-386 recombinant protein in fusion with an amino-terminal SUMO tag and a polyhistidine tag. The pET22-His6-SUMO-LegK1 2-386 -KA and pET22-His6-SUMO-LegK1 2-386 -3WF were generated using site-directed mutagenesis by PCR. The full-length GST-Ago2 was purified from the pGST-Ago2 plasmid, obtained from Addgene (24317). A construct including only the PIWI domain of Ago2 and a fraction of MID domain was generated by PCR amplification of Ago2 amino-acids 517 to 659. PIWI 517-859 DNA was digested by BamHI and HindIII enzymes and cloned into pET28-CBP-His6, kindly given by Dr. Hervé Le Hir, allowing a fusion with the Calmodulin Binding Protein (CBP) and a polyhistidine tag in amino-terminal and in carboxyl-terminal ends, respectively. Finally, the fulllength IkBa was purified from pET28-CBP-IkBa-His6 plasmid. IkBa DNA was PCR amplified from pBABE-GFP-IkBa-wt (Addgene, 15263) and was digested and cloned into pET28-CBP-His6, as described for pET28-CBP-PIWI 517-859 -His6. The negative control protein TAP, consisting of the CBP and His6 tags, was purified from a plasmid pET28 derivative, kindly provided by Dr. Hervé Le Hir 107 . For complementation assay, the full-length legK1 gene was cloned under the control of its own promoter into pBC-KS (Stratagene, 212215) using SacI and KpnI restriction enzymes. All constructions were confirmed by Sanger sequencing.

  2000 (Invitrogen, 11668019). One day before transfection, HeLa cell lines were trypsinized, resuspended in DMEM, and seeded in 48-well plates at a density of 7x10 4 cells per well. Cells were transiently co-transfected for 48h with 100 ng of pGL3-CXCR4-2p, 50 ng of pRL-TK as transfection control, and 250 pM of AllStars Eurofins Genomics). For the initial screening of bacterial effectors, WT HeLa cells were co-transfected as previously described, with an addition of 400 ng of vector expressing a bacterial candidate or HBx as positive control. Firefly and Renilla luciferase expressions were determined as described in the Dual-luciferase silencing reporter analyses section. For the siRNA-based GFP sensor, HeLa and HEK293T cell lines were seeded in 24-well plates at the same density as mentioned above. Cells were co-transfected for 48h with 200 ng of pFlag-eGFP and 30 pmol of GFP RNA duplex (RNA GFP duplex I, Thermoscientific, P-002048-01-20) or AllStars Negative Control siRNA, and with 1 µg of p2xFlag-LegK1-WT, -KA, -3WF, -W283F, -W293F, -W293A, -W283F-W293A, pFlag-YopM or empty vector as negative control, or pFlag-T6B as positive control using Lipofectamine 2000. GFP proteins levels were next analysed by Western blot. For the miRNA-based GFP sensor, HEK293T cells were seeded in 24-well plates at the same density as previously described and cells were co-transfected with 200 ng of AutomiG and 1µg of plasmid expressing either WT or LegK1 mutants, YopM or T6B using Lipofectamine 2000 for 48h. GFP protein levels were further analysed by Western blot analyses.

  , along with CXCR4 RNA duplexes. In parallel, a Renilla luciferase control reporter vector was used, corresponding to the pRL-TK. Transfected cells were washed with 1X PBS and then lysed in 65 µL of 1X Passive Lysis Buffer (Dual-Luciferase Reporter Assay System, Promega, E1910) by shaking for 15 min at room temperature. Firefly and Renilla activities were measured in 10 µL of cell lysate with the Luciferase reporter assay system, according to the supplier's protocol. Briefly, bioluminescence was initiated by automatic injection of 30 µL of Luciferase Assay Reagent II. After a 1second shaking and an additional 1-second delay, Firefly luciferase emission signals were recorded on TriStar LB 941 Multimode Microplate Reader luminometer (Berthold Technologies), using a 10-second measurement period for each condition. The Renilla luciferase emission signals were then measured after injection of 30 µL of Stop & Glo Reagent, in the same manner as for Firefly luminescence detection. All measurements were carried out in three technical replicates for each independent experiment. Firefly luminescence expression was corrected with the Renilla control, the luminescence value of siCXCR4 condition was then normalised on siCTL condition, and further normalised by the negative control condition, corresponding to the CTL HeLa cell line or eGFP transfected WT HeLa cells.

(

  D84G10, Cell Signaling Technology, 5053), anti-Ago2 (C34C6, Cell Signaling Technology, 2897), anti-Ago3 (4B1-F6, Active Motif, 39787), anti-Ago4 (D10F10, Cell Signaling Technology, 6913), anti-DDX6 (Bethyl, A300-460A), anti-Dicer (Cell Signaling Technology, 3363), anti-Flag (M2, Sigma-Aldrich, F1804), anti-GAPDH (14C10, Cell Signaling Technology, 2118), anti-GFP (D5.1, Cell Signaling Technology, 2956), anti-PABPC1 (Atlas Antibodies, HPA045423), anti-phospho-IκBα (Ser32) (14D4, Cell Signaling Technology, 2859), anti-TNRC6A (Bethyl, A302-329A) and antia-Tubulin (DM1A, Cell Signaling Technology, 3873). Both antibodies were diluted 1000×. As secondary antibodies, anti-mouse (Cell Signaling Technology, 7076S) or anti-rabbit (Cell Signaling Technology, 7074S) were used and proteins were detected with Agrisera ECL SuperBright (Agrisera, AS16 ECL-S-100) using a LAS 4000 mini (GE Healthcare).Co-immunoprecipitation analysesCells were transiently transfected in 10 cm 2 dishes, harvested at 48h post-transfection, washed twice with ice cold 1X PBS and lysed with Lysis Buffer (20 mM, Tris-HCl [pH 7.4], 150 mM NaCl, 2 mM EDTA, 0.5% NP-40, 1 mM DTT, 1X protease inhibitor [EDTA-free complete Protease Inhibitor Cocktail, Roche, 11873580001] and 1:100 phosphatase inhibitor [Sigma-Aldrich, P5726]). In some experiments, cell lysates were treated with 100 ng/µL of RNase A (ThermoFisher, R1253) for 1h at 37°C. The RNAse A treatment was checked on agarose gel by loading some input extract (data not shown). For each condition, 30 µL of input were collected and the concentration was measured using the Bradford reagent in order to load 100 µg of input proteins for the following analysis. Thirty μl of Dynabeads Protein G (Invitrogen, 10003D) were incubated with 7 µg of anti-FLAG M2 antibody (Sigma-Aldrich, F1804) for 1h30 at 4°C in 1X PBS containing 0.1% of Tween20. Dynabeads Protein G linked to the antibodies were then incubated overnight with cell lysates at 4°C under agitation. The immunoprecipitates were washed three times with IP Buffer (50 mM Tris-HCl (pH 7.4), from 150 mM to 250 mM NaCl, 0.05% NP-40, 1X protease inhibitor and phosphatase inhibitor) and eluted in 4X Laemmli Loading Buffer. Protein samples were denatured 5 min at 95°C, resolved on SDS-PAGE gel and analysed by Western blot.

  integrated. The peptide areas were log2 transformed and the mean log2-area was normalised by the mean area of five Ago2 peptides (ELLIQFYK, SGNIPAGTTVDTK, SIEEQQKPLTDSQR, VELEVTLPGEGK and VLQPPSILYGGR) using software R 3.1.0. On each phospho-peptide, a linear model was used to estimate the mean fold change between the conditions, its 95% confidence interval and the p-value of the twosided associated t-test. The p-values were adjusted with the Benjamini-Hochberg procedure 108 . Data are available via ProteomeXchange with identifier PXD037279 (Username: reviewer_pxd037279@ebi.ac.uk; Password: xzomxsb6) 109 .Prediction of W-motifsW-motifs were predicted using the Wsearch algorithm[START_REF] Zielezinski | Integrative data analysis indicates an intrinsic disordered domain character of Argonaute-binding motifs[END_REF] , which scores the W-motifs according to a Position-Specific Scoring Matrice (PSSM) derived from experimentally validated Ago-binding motifs from animals. Briefly, the protein sequences of all bacterial organisms tested were retrieved from Uniprot (UniProt Consortium, 2018) and subjected to Wsearch either on the website or using the python script. The score of the different positive motifs were then added together, corresponding to the final score. For the first pre-selection, we applied an arbitrary cut-off greater than six on the average W-scores of all motifs present in the candidate protein sequence. Among the bacterial candidate proteins exhibiting the highest W-score, we further selected known secreted virulence factors or putative virulence factors predicted to be secreted using PSORTb 3.0 (https://www.psort.org/psortb/) 110 .

Figure 1 .

 1 Figure 1. LegK1 suppresses siRNA-and miRNA-guided gene silencing through

Figure 2 .d

 2 Figure 2. LegK1 interacts with components of the miRISC and directly binds to

Figure 3 .

 3 Figure 3. The W283 and W293 motifs of LegK1 bind to human Ago proteins and

Figure 4 .

 4 Figure 4. The kinase, RNA silencing suppression and NF-kB signaling activation

Figure 5 .a

 5 Figure 5. LegK1 is a key virulence factor from L. pneumophila that promotes

  ) of Lysteriolysin O and a-Hemolysin coding sequences was performed using pAD-hly-Myc plasmid and Staphylococcus aureus SH1000 genomic DNA as templates, respectively (gift from Dr. Alice Lebreton,

IBENS, Paris, France). BepB coding sequence was amplified from Bartonella Henselae gDNA, ExoY from Pseudomonas aeruginosa PAO1 strain, NleH1 from Escherichia coli O157:H7 gDNA (gift from Dr. Julie Guignot, Institut Cochin, Paris, France), Pertussis toxin from Bordetella pertussis Tohama gDNA (gift from Dr. Benoit Garin, Institut Pasteur, Paris, France), YopM from Yersinia pestis gDNA (gift from Dr.

  Some cell lysate were collected and the concentration was measured using the Bradford reagent in order to load 80 µg of input proteins for the following analysis. Cells were incubated at 4°C under rotation for 30 min and the supernatant was harvested by centrifugation at 16,000 g for 10 min at 4°C. Beads-coupled peptides were incubated in the presence of cell lysates for 1 hour at 4°C After three washing steps of the beads with IP Buffer, the proteins were eluted in 4X Laemmli Loading Buffer and resolved on SDS-PAGE. The presence of Ago proteins was detected by Western blot analyses.

	Bacterial cells expressing GST-Ago2 recombinant protein were collected by of 2X BB Buffer (40 mM Hepes pH 7.5, 83 mM NaCl, 1 mM MgAc2, 0.2% NP-40, 11.7% temperature in the presence of 30 µL of Dynabeads MyOne™ Streptavidin T1 (Thermo
	centrifugation, resuspended in Lysis Buffer (1.5X PBS, 1 mM MgAc2, 1mM DTT, 0.1% glycerol, 2 mM DTT) was added. After 20 min of interaction at 30°C under rotation, 12 Fisher Scientific, 65601). The beads were then washed once in 0.1% PBS Tween and
	NP-40, 10% glycerol), and lysed by sonication during 4 min on ice. Lysate was clarified μL of Glutathione Sepharose 4B were added. Beads were washed three times with twice in IP Buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 0.05% NP-40,
	by high-speed centrifugation and then purified on 500 µL of Glutathione Sepharose 500 μL of 1X BB 250/10 Buffer (20 mM HEPES pH 7.5, 250 mM NaCl, 1 mM MgAc2, 1/100 Phosphatase inhibitor and 1X protease inhibitor). HEK293T cells were washed
	4B resin (Merck, GE-17-0756-01). Beads were pre-equilibrated in Lysis Buffer and 0,2 % NP-40, 10 % glycerol, 1 mM DTT), and then proteins were eluted by incubation twice with ice cold 1X PBS and scraped with Lysis Buffer (see the Co-
	supernatant was incubated with resin for 2h at 4°C. GST fusion proteins linked to the with 20 μL of Elution Buffer (50 mM Tris•HCl (pH 7.5), 250 mM KCl, 1 mM MgAc2, 1 immunoprecipitation analyses section).
	beads were washed once with Lysis Buffer, then once with Wash Buffer (1.5X PBS, mM DTT, 0.1 % NP-40, 10 % glycerol, 10 mM reduced Glutathione) for 5 min at 30
	250 mM KCl, 1 mM MgAc2, 1 mM DTT, 0.1 % NP-40, 10 % glycerol), and finally with °C, under shaking at 1,400 rpm. Finally, proteins bound to GST beads were eluted
	Lysis Buffer. Proteins were eluted in Elution Buffer (50 mM Tris pH8.0, 250 mM KCl, with Elution Buffer for 5 min at 30 °C, under shaking at 1,400 rpm. 4X Laemmli Loading
	1 mM MgAc2, 1 mM DTT, 0.1 % NP-40, 10 % glycerol, 10 mM reduced glutathione). Buffer was added and proteins were separated onto SDS-PAGE gel and analysed by
	Proteins were subjected to overnight dialysis using the same dialysis tubing as Western blot.
	mentioned above into Dialysis Buffer (10 mM HEPES pH7.5, 250 mM KCl, 1 mM
	MgAc2, 10 % glycerol, 2 mM DTT) before storage at -80°C. All recombinant protein For binding assays using CBP pull-down, proteins were prepared according to the
	concentrations were determined using the Bradford reagent. Proteins were analysed same protocol as mentioned above. In some experiments, from 5 to 15 μg of human
	by Coomassie Blue staining after SDS-PAGE. cell extracted RNAs were incubated with protein samples. After 20 min of interaction
	Buffer, and supernatant was incubated with resin for 2h at 4°C. His fusion proteins at 30°C under rotation, 12 μL of Calmodulin Affinity Resin were added. Beads were
	linked to the resin were washed once with Lysis Buffer, then once with Wash Buffer In vitro interaction assays washed three times with 500 μL of 1X BB 250/10 Buffer (20 mM Hepes pH=7.5, 250
	(1.5X PBS, 250 mM NaCl, 1 mM MgAc2, 0.1 % NP-40, 50 mM imidazole, 10 % Glutathione Sepharose 4B resin and Calmodulin Affinity Resin (Agilent Technologies, mM NaCl, 2 mM MgAc2, 2 mM imidazole, 2 mM CaCl2, 0.1 % NP-40, 10 % glycerol, 1
	glycerol), and finally with Lysis Buffer. Proteins were eluted in Elution Buffer (1.5X 214303) were washed twice with Blocking Buffer (20 mM Hepes pH 7.5, 150 mM NaCl, mM DTT). Proteins were eluted by incubation with 20 μL of Elution Buffer (10 mM
	PBS, 1 mM MgAc2, 0.1 % NP-40, 150 mM imidazole, 10 % glycerol). Excess imidazole 0.1 % NP-40) and then blocked into Blocking Buffer supplemented with 20 mM NaCl, Tris•HCl (pH 7.5), 250 mM KCl, 1 mM MgAc2, 1 mM DTT, 0.1 % NP-40, 10 % glycerol,
	was removed by overnight dialysis using Spectrum™ Labs Spectra/Por™ 2 12-14 kD 2 mg/mL glycogen carrier, 10 mg/mL tRNA and 10 mg/mL BSA during 2h at 4°C. 20 mM EGTA) for 5 min at 30 °C, under shaking at 1,400 rpm. 4X Laemmli Loading
	MWCO (FisherScientific, 15310762) into Dialysis Buffer (1.5X PBS, 1 mM MgAc2, 10 Subsequently, beads were washed twice with Blocking Buffer and resuspended into Buffer was added and proteins were separated onto SDS/PAGE gel and analysed by
	% glycerol, 2 mM DTT) before storage at -80°C. Storage Buffer (10 mM HEPES pH 7.5, 250 mM NaCl, 1 mM EDTA). For in vitro Western blot.
	interaction assays using GST pull-down, 5 µg of each purified protein were incubated
	in B1.5 Buffer (1.5X PBS, 1 mM MgAc2, 2 mM DTT, 10% glycerol). The same volume

, His6-SUMO-LegK1-KA

, CBP-PIWI 517-859 -His6, CBP-IkBa-His6 and His6-TAP were collected by centrifugation, resuspended in Lysis Buffer (1.5X PBS, 1 mM MgAc2, 0,1 % NP-40, 20 mM imidazole, 10 % glycerol), and lysed by sonication during 4 min on ice. Lysate was clarified by high-speed centrifugation (18,000 rpm) and then purified on 250 µL of Ni-NTA resin (Thermo Fisher Scientific, 88221). Resin was pre-equilibrated in Lysis

Acknowledgments

We thank Dr. Alice Lebreton, Dr. Julie Guignot, Dr. Benoit Garin, Dr. Lauriane Quenee, Dr. Clément Carré, Pr. Gunter Meister, Dr. Hervé Le Hir for providing the Staphylococcus aureus SH1000 genomic DNA, Pseudomonas aeruginosa PAO1 strain and Escherichia coli O157:H7 gDNA, Bordetella pertussis Tohama gDNA, Yersinia pestis gDNA, AutomiG reporter, a vector expressing T6B, pET22-6His-SUMO and pET28-CBP-6His, respectively. We thank Dr. Sophie Goudey for the generation of KO HeLa cell lines. We also thank all members of the Navarro lab for their inputs, discussions and critical reading of the manuscript, as well as Dr. Alice Lebreton, Dr.

Michael Mourez, and Miss Isabelle Barbosa for helpful advices and discussions. This work was supported by the European Research council (281749, Silencing & Immunity, to L.N.), a grant from the European Molecular Biology Organization (EMBO) Young Investigator Program (to L.N.), an Action Thématique et Incitative sur Programme (ATIP)/Avenir Grant from the Fondation Bettencourt Schueller (FBS, to

Data availability

All materials generated in this study are available upon request. Mass spectrometry data are available via ProteomeXchange with identifier PXD037279. Sequencing data corresponding to the mutant DlegK1 have been submitted to Zenodo (https://zenodo.org/record/7190772). All data reported in this paper will be shared by the lead contact upon request.

Correspondence and requests for materials should be addressed to Lionel Navarro (lionel.navarro@ens.psl.eu).

Kinase assays

In vitro phosphorylation of 1 µg of purified GST-Ago2 or CBP-IkBa-His6 recombinant proteins was performed in the presence of 1 µg of His6-SUMO-LegK1 or His6-SUMO-LegK1 -KA in 20 µl of phosphorylation buffer, containing 25 mM Tris HCl (pH 7.5), 200 mM NaCl, 10 mM MgCl2, and 50 μM unlabeled ATP. Reactions were split in two, and in one half 10 µCi [γ-32P] ATP were added. The phosphorylation reaction was performed for 1h at 30°C and stopped by adding 4X Laemmli Loading Buffer. A Western blot analysis was carried out on unlabeled samples. Labeled samples were analysed by SDS-PAGE, which was exposed to a phosphor screen and visualised by Typhoon FLA 9500 biomolecular imager.

The kinase activity of LegK1 mutant versions was determined using IkBa recombinant protein. For this, HEK293T cells were seeded in 6 cm 2 dishes and transfected with either pFlag-eGFP, p2xFlag-LegK1-WT, -KA, -3WF, -W283F, -W293F, -W293A or -W283F-W293A. Cells were lysed and subjected to a Flag immunoprecipitation. The resulting immunoprecipitates were incubated with Phosphorylation Buffer (250 mM Tris-HCl pH 7.5, 50 mM MnCl2, 50 mM DTT) and 1 µg of purified CBP-IkBa-His6 recombinant proteins for 30 min at 30°C. Proteins were mixed with 4X Laemmli Loading Buffer and then analysed by Western blot using the phospho-IκBα (Ser32) (14D4) antibody.

Peptide pull-down assays

Biotinylated peptides (synthetised by Genscript) were resuspended in DMSO, and heated at 40°C for 5 min. For binding assays, 10 µg of peptides were diluted into 200 µL of PBS containing 0.1% of Tween20, and were incubated for 30 min at room

Quantification and statistical analysis

Experimental results were shown as standard error of the mean (SEM) for luciferase reporter and qPCR experiments and as standard deviation (SD) for infection assays.

Statistical analysis was carried out using the Prism Software (GraphPad Prism 9.0).

For statistical comparison between two groups or several conditions, an unpaired t test or one-way analysis of variance (ANOVA) of biological replicates were used, respectively. For infection assays, a Wilcoxon test was used. A value of P < 0.05 was considered statistically significant. All statistical tests are specified in the respective