A bacterial YopJ-family acetyltransferase suppresses host immune response by Nε-acetylation of JAK1

Domain: chemistry
Published: 2026-02-18
Editorial outcome: minor_revision

Abstract
A common host response to pathogen infection involves the production of robust interferons or proinflammatory cytokines to activate the JAK-STAT pathway, thereby limiting pathogen replication. The bacterial pathogen Legionella pneumophila creates an intracellular niche and evades host immunity utilizing a cohort of effectors by diverse biochemical activities, thereby permissive for its intracellular replication. However, roles of the JAK-STAT pathway during bacterial infection remain elusive. Here, we identify for the first time that L. pneumophila acetyltransferase effector Lem17 acts as a negative regulator of the JAK-STAT signaling. Lem17 directly interacts with JAK1 through a JAK1-binding Box1-like motif, preventing its recruitment by cytokine receptors. As a YopJ-family acetyltransferase, Lem17 catalyzes Nε-lysine acetylation of JAK1 and impairs its kinase activity, thereby disrupting JAK1-mediated signaling transduction. Our findings provide insights into the mechanism by which L. pneumophila subverts host immunity through acetylation and underscore the role of the JAK-STAT pathway against bacterial infection.

Introduction
Protein acetylation, one of the most vital post-translational modifications (PTMs), is catalyzed by acetyltransferases that divert acetyl groups from acetyl co-enzyme A (acetyl-CoA) molecules to specific residues of substrates. Protein acetylation occurs on histone and non-histone proteins and plays a critical regulatory role in various cellular processes, e.g., signal transduction and immune response1. An accumulating body of evidence demonstrates that acetylation is one of the potent strategies for various intracellular pathogens to resist host immunity and promote their replication in host cells. One such example is found in bacterial virulence factors such as the Yersinia outer protein J (YopJ) family, which functions as Ser/Thr-acetyltransferases2. The primary function of these virulence factors is believed to play a pivotal role in evading the host immune response and establishing a persistent infection in host cells after bacterial infection3.

The innate immune system is the first line of host defense against pathogen infection. In response to pathogen infection, host cells produce robust cytokines such as interferons (IFNs) and interleukins4. The Janus kinase (JAK) /signal transducer and activator of transcription (STAT) pathway is important in orchestrating immune responses, which mediates the functions of IFNs and other cytokines5. IFNs bind to the cognate IFN receptor (IFNAR) and induce IFNAR dimerization to recruit JAKs, thus activating the JAK/STAT signaling. The activated JAKs phosphorylate downstream STATs. The phosphorylated STATs then form dimers and subsequently translocate into the nucleus, where they bind to DNA elements and trigger the transcription of IFN-stimulated genes6. These genes play an important role in establishing the immune response against pathogens7,8. Although the JAK/STAT pathway is well documented to be mainly activated through interferons for host defenses, many pathogens have evolved mechanisms to combat host defense and establish an efficient infection. Zika viruses (ZIKV) and Influenza A viruses (IAV) target JAK1 for proteasomal degradation, blocking JAK/STAT signaling and impairing IFN-mediated antiviral response9,10. Bacterial pathogen Shigella binds calmodulin (CaM) to inhibit the CaM/CaMKII pathway and block STAT1 phosphorylation, thereby dampening host IFN response11.

Legionella pneumophila, an intracellular bacterial pathogen, causes Legionnaires’ disease. This bacterium creates an intracellular niche and evades host immunity utilizing a large cohort of effectors by diverse biochemical activities, thereby permissive for its replication12,13. One of these effectors functions by mediating post-translational modifications that perturb host signaling pathways. Effector LnaB, as a phosphoryl-AMPylase, AMPylates the Src family of kinases, thereby inhibiting host immunoreceptor signaling14,15. Effector LphD, a eukaryotic-like histone deacetylase, targets H3K14ac and works in synergy with another effector, RomA, to promote H3K14 methylation, thereby counteracting host immune responses16,17. Notably, L. pneumophila encodes several effectors homologous to enzymes in either the eukaryotic Gcn5-related N-acetyltransferase family or prokaryotic Serine/Threonine acetyltransferase family. This suggests that effector-mediated acetylation may play a vital role in L. pneumophila infection. However, only L. pneumophila effector VipF has been identified as an acetyltransferase containing dual GNAT domains and targeted the eukaryotic eIF3 complex to modulate protein translation18,19. Nevertheless, the roles of these effectors in L. pneumophila infection have remained limited.

Here, we identified L. pneumophila effector Lem17 (Lpg1949) as a negative regulator of the JAK1-STAT pathway to attenuate the host immune response. Lem17 acts as an acetyltransferase that shuts down JAK1-STAT signaling through direct interactions and acetylation of JAK1, thereby facilitating the creation of a permissive intracellular environment for L. pneumophila replication in host cells.

Results
Lem17 is a bonafide acetyltransferase containing a conserved C-H-D motif
Bioinformatics analysis reveals that the YopJ family proteins are encoded by diverse bacterial pathogens of a wide range of hosts, particularly several proteins, including Lem17 (Lpg1949), Lpg2538, and Lpg2907, found in L. pneumophila species (Supplementary Fig. 1a). Notably, L. pneumophila deficient in the lpg1949 gene exhibited impaired replication within host cells, demonstrating that effector Lem17 contributes to the intracellular growth of L. pneumophila (Fig. 1a, b). Sequence analysis further revealed that Lem17 harbors a conserved C-H-D (C174-H105-D124) motif associated with the YopJ family members (Fig. 1c). To uncover whether Lem17 possesses an acetyltransferase activity, we ectopically expressed the wild-type Lem17 or its catalytic mutants (C174S, H104A and D124A) in HEK293T cells. Interestingly, we found that the global acetylation signaling increased in cells expressing Lem17 using an anti-pan acetyl-lysine antibody, instead, the indicated mutants are not (Fig. 1d). These results demonstrate that Lem17 is an acetyltransferase facilitating the intracellular growth of L. pneumophila.

Lem17 requires host inositol hexaphosphate for activation
Given the Ser/Thr acetyltransferase family members generally require an essential cofactor inositol hexaphosphate (IP6) as an activator20, we next examined whether Lem17 binds IP6. We found that IP6 shows high affinity for Lem17, with a binding constant (Kd) of approximately 2 μM (Fig. 2a). Specifically, Lem17 only binds acetyl-CoA in the presence of IP6 (Kd ≈ 20 μM), whereas no binding was detected in its absence (Fig. 2a). These results suggest that Lem17 requires IP6 for activation. To elucidate the IP6-mediated activation mechanism of Lem17, we have devoted significant efforts to determining the crystal structures of distinct forms: apo-Lem17, IP6-bound Lem17, and IP6-bound Lem17 in complex with the ligand CoA. However, our attempts have so far only succeeded in obtaining the IP6-bound Lem17 structure (Supplementary Fig. 1b). As the structural observation, Lem17 is divided into two subdomains: catalytic core domain (CD) and Lid domain (Lid). IP6 binds to a positive charge cleft in the YopJ-like domain, where is covered by the Lid domain, thus locking IP6 in place (Fig. 2b, c). The catalytically critical residues C174, H105 and D124 form a continuous platform (Fig. 2d), which may be the site for interactions between Lem17 and its host targets. Structural alignment reveals that Lem17 shares high structural similarity with the catalytic domain of the YopJ family members except for the additional Lid domain, for one example, HopZ121 (PDB ID: 5KLP) with RMSD of 3.4 Å and PopP222 (PDB ID: 5W3T) with RMSD of 3.6 Å, (Supplementary Fig. 1a, b). Specifically, residues within the conserved C-H-D motif exhibit an nearly identical spatial pattern (Supplementary Fig. 2c). The presence of this Lid domain may confer Lem17 with greater specificity or affinity for its host targets.

Moreover, structural alignment between the Lem17-IP6 complex and the AlphaFold3-predicted Lem17 model suggested that IP6 binding might cause a conformational change in its Lid domain (Supplementary Fig. 2d–f), potentially enabling the binding of ligand acetyl-CoA or substrates. In addition, structural data revealed that IP6 contacts Lem17 extensively via polar interactions (Fig. 2e and Supplementary Fig. 2g). Residues K248 and R222 coordinate with P1 in IP6 by hydrogen bonds. Residues K278 and R281 hydrogen bonds with P2 in IP6. Residues R45 and K46 coordinate with P3 and P4 in IP6 by hydrogen bonds. Residues R50, R53 and K187 coordinate with P5 in IP6 by hydrogen bonds. Residues K187 and K247 coordinate with P6 in IP6 by hydrogen bonds. As structural observations, alanine substitutions at the indicated residues severely impaired IP6 binding to Lem17 (Fig. 2f and Supplementary Fig. 3a–c). Consistent with these findings, these mutants deficient in IP6-binding exhibited a severe reduction of the global acetylation signaling in cells (Fig. 2g). Taken together, these data suggest that Lem17 is a bonafide acetyltransferase toward lysine residues. Of course, serine- or threonine-acetylation cannot be ruled out.

Lem17 mediates Nε-acetylation of host JAK1 that impairs its kinase activity
To elucidate the specific physiological function of Lem17 in the host, we next sought to identify host proteins that are targeted by Lem17. We ectopically expressed Flag-Lem17 in HEK293T cells and performed an immunoprecipitation (IP) after formaldehyde using an anti-Flag antibody, followed by mass spectrometric analysis, and another L. pneumophila effector Lpg2538 as a control (Fig. 3a and Supplementary Data 1). Since YopJ family members target host kinases, we focused on candidate proteins harboring kinase activity. The top 10 selected kinases are presented in Fig. 3b. We first examined their acetylated modification in cells using an anti-pan acetyl-lysine antibody. A distinct acetylation signal was observed at the position corresponding to Janus kinase 1 (JAK1) in the presence of Lem17, whereas no such signal was readily detectable for the other selected proteins examined (Fig. 3c). Consistently, we purified HA-tagged JAK1 by HA-beads after co-expression with Flag-Lem17 or its indicated mutant in HEK293T cells. Acetylated-JAK1 indeed was detectable in the IPed profile of cells expressing Flag-Lem17, while it was not in cells harboring the empty vector or Lem17 C174S mutant (Fig. 3c). In addition, the Flag-Lem17 or its C174S mutant were substantially enriched with JAK1 in the IPed fraction. Collectively, these data indicated that JAK1 is one host target of Lem17. In addition, we also examined the top 20 proteins from the proteomics data and found that Lem17 modified host protein EPB41L2 (Supplementary Fig. 4b, c). EPB41L2, which also contains a FERM domain, is required for dynein-dynactin complex and NUMA1 recruitment at the mitotic cell cortex during anaphase23. However, whether EPB41L2 engages in the JAK1-STAT pathway warrants further investigation.

JAK1, which belongs to the JAK family of non-receptor tyrosine protein kinases, has four domains. Starting from the carboxyl terminus, these include: a tyrosine kinase domain (TK) that phosphorylates substrates, a pseudokinase domain (PK) that regulates the activity of the TK domain, a Src-homology 2 (SH2) domain and a four-point-one, ezrin, radixin, moesin domain (FERM) domain5. We next analyzed Lem17-targeted acetylation sites on JAK1 using mass spectrometry. This analysis revealed multiple lysine residues were modified by Nε-acetylation, including K205, K213, K263, and K493 in the FERM-SH2 domain, and K911, K941 and K953 in the TK domain (Fig. 3d, Supplementary Figs. 5, 6 and Supplementary Data 2). Consistent with these findings, strong acetylation signal was detectable in JAK1 and its FERM-SH2 and TK domains in the IPed profiles (Supplementary Fig. 4d). Alanine substitutions in the FERM domain (individual: K205; triple: K205A/K213A/K267A) or the TK domain (individual: K911; triple: K911A/K941A/K953A) caused detectable decreases in JAK1 acetylation. Notably, the quadruple substitution (K205A/K267A/K911A/K941A) exhibited a substantial decrease in acetylation signal (Fig. 3e). In addition, due to the non-expression of the K213A and K267A mutations in cells, we further constructed these substitutions in the truncated FERM-SH2 and TK domains of JAK1, respectively. Reduced acetylation signals were detected for the K213A and K267A substitutions within the FERM-SH2 domain (Supplementary Fig. 4e). These results suggest that Lem17 could acetylate multiple residues on JAK1.

Notably, these acetylated residues cluster in two functional domains: the FERM-SH2 domain, which mediates cytokine-associated receptor binding, and the kinase domain (TK), which is essential for substrate phosphorylation. Specifically, residues K267 and K493, positioned adjacent to the JAK1-IFNλR1 interaction interface, potentially disrupt cytokine receptor binding, whereas K911, K941 and K953, localized within the N-lobe of the kinase domain, are likely to impair kinase activity (Supplementary Fig. 4f). Consistently, Lem17-mediated acetylation of JAK1 led to a significantly reduced kinase activity (Fig. 3f). Although Lem17 belongs to the Ser/Thr acetyltransferase family characterized by acetylating substrates’ Ser/Thr residues, MS analysis failed to detect any Ser/Thr-acetylated residues in JAK1. This discrepancy may arise from differences in the electrostatic potentials surrounding their C-H-D catalytic triads: Lem17 exhibits a negatively charged microenvironment, whereas YopJ family proteins such as PopP222 and HopZ1a21 display a distinct bias toward positively charged regions (Fig. 3g).

Lem17 blocks JAK1 recruitment to cytokine receptors
To elucidate the mechanism by which Lem17 recognizes and acetylates JAK1, we have made extensive efforts to resolve the structure of the Lem17-JAK1 complex; however, these attempts have thus far been unsuccessful. Therefore, we predicted the structural model of the Lem17-JAK1 complex (Supplementary Fig. 7a, b), and showed that the Lid domain of Lem17 binds to the FERM domain of JAK1 through polar and hydrophobic interactions (Fig. 4a and Supplementary Fig. 7c, d). To validate this model, we performed alanine substitutions at residues involved in the hydrophobic interaction between JAK1 and Lem17 and conducted the co-immunoprecipitation assay. As the predicted structural observation, alanine mutations in JAK1 significantly reduced its affinity for Lem17, particularly in the LVL (L190A/V194A/L195A) and VFF (V243A/F244A/F247A) mutants (Fig. 4b). Similarly, alanine mutation F368A/I371A/F375A (termed FIF) in Lem17 also markedly impaired its interaction with JAK1 (Fig. 4c). It notes that the predicted JAK1-Lem17 complex structure is limited by a moderate ipTM score, despite our point-variant data supports this prediction. In addition, Lem17 was observed in the IPed profile of cells harboring the full-length and FERM-SH2 of JAK1 (Supplementary Fig. 7e). These results indicate that the FERM domain of JAK1 plays a dominant role in binding to Lem17. Further sequence alignment revealed that three hydrophobic residues in Lem17 (F368, I371, F375) are conserved and correspond to characteristic residues of the PxxLxF motif within the Box1 of JAK1-associated cytokine receptor cytoplasmic domains (Fig. 4d). This conserved PxxLxF motif, corresponding to each cytokine receptor, is documented to be functionally essential for JAK1 binding24.

Subsequently, structural alignment of the Lem17-JAK1 complex with the JAK1-IFNλR1 complex revealed that the binding interface between Lem17 and JAK1 sterically clashes with the Box1 motif of IFNR1, which resides within the FERM domain of JAK1 (Fig. 4e and Supplementary Fig. 7f). This clash primarily involves three hydrophobic residues in Lem17 Lid domain (F368, I371, and F375) that occupy equivalent spatial positions to three residues in IFNλR1 (P264, L267, and F269) (Fig. 4f). Consistently, the mutant FIF in Lem17 impaired the intracellular replication of L. pneumophila (Fig. 4g). These results suggested that Lem17 might sterically hinder JAK1 from recognizing its cognate cytokine receptors by preemptively occupying the cytokine receptor’s binding pocket before JAK1 engagement. To clarify this hypothesis, we immunoprecipitated endo-JAK1 in IFN-λ-primed HEK293T cells with ectopically expressing Flag-lem17. Immunoprecipitation profiles demonstrated reduced enrichment of the JAK1-upstream IFNAR2 and JAK1-downstream STAT1, while these proteins were significantly enriched in Lem17-deficient cells (Fig. 4h). Collectively, these results indicated that Lem17 impairs the ability of JAK1 to recognize its associated cytokine receptors through a dual mechanism involving direct interaction and acetylation.

Lem17 dampens the JAK1 signaling that facilitates bacterial intracellular replication
Given that JAKs function as the central signaling hub in the JAK-STAT pathway, transducing the cytokine-mediated extracellular stimuli into intracellular STAT activation response5,25, we subsequently detected the role of Lem17 in the JAK1-mediated signaling transduction. Phosphorylation levels of STAT1 and STAT2 were strongly elevated in the cells primed with IFN-β. However, this increase was remarkably suppressed by the ectopic expression of Lem17. In contrast, the C174S mutant reversed the suppression caused by Lem17 (Fig. 5a). Consistently, we performed RNA-sequencing (RNA-seq) analysis on total RNA isolated from IFN-primed cells ectopically expressing Lem17 or an empty vector. The distinct gene expression profiles (comprising approximately 500 differentially expressed genes) were observed in Lem17-expressing cells compared to those transfected with empty vector (Fig. 5b). Gene Ontology (GO) analysis revealed that these genes are enriched in multiple signaling pathways, including the interferon-mediated antiviral pathway (Fig. 5c). Genes associated with interferon-mediated antiviral pathways – including those involved in the ISG15 antiviral mechanism, antiviral mechanism by IFN-stimulated genes, and interferon signaling, demonstrate significant enrichment in negatively correlated phenotypes, as evidenced by negative normalized enrichment scores (Fig. 5d, e and Supplementary Fig. 8a). Collectively, these results demonstrate that Lem17 dampens the JAK signaling.

A common host response to pathogen infection is the production of IFNs or proinflammatory cytokines to limit pathogen replication. Similarly, host cells produce robust proinflammatory cytokines like interferon and interleukins, particularly IFN-β and IL-6, in response to L. pneumophila infection (Supplementary Fig. 8b–e). These cytokines exert antibacterial effects through their downstream JAK-STAT pathway26. As expected, JAK1 knockdown promoted the intracellular proliferation of L. pneumophila in both HeLa and U937 cells (Fig. 5f and Supplementary Fig. 8f–h). Therefore, these results suggest that Lem17 disrupts JAK1-mediated signal transduction, which contributes to the intracellular replication of L. pneumophila in host cells.

Methods
Cell lines
HEK293T, HeLa, U937 and mouse RAW264.7 cells purchased from the ATCC were cultured in Dulbecco’s modified minimal Eagle’s medium (DMEM) supplemented with 10% FBS at 37°C. All mammalian cell lines were regularly checked for potential mycoplasma contamination by the universal mycoplasma detection kit from ATCC (Cat# 30-1012K).

Bacterial strains
All L. pneumophila strains were derived from the Philadelphia-1 strain Lp0237. The bacteria were grown on a charcoal-yeast extract (CYE) plate or in N-(2-acetamido) 2-aminoethanesulfonic acid (ACES)–buffered yeast extract (AYE) broth with additional thymidine (200 μg/mL) for the thymidine auxotrophic mutant and its derivatives. The Lp02Δlem17 mutant was described earlier38.

Escherichia coli (E. coli) strains DH5α and BL21 Rosetta DE3 were grown in lysogeny broth (LB) shaking at 37°C. Antibiotics were added to culture media at the following concentrations: ampicillin (100 μg/mL), kanamycin (30 μg/mL), and streptomycin (50 μg/mL), unless specified. All strains were stored at -80°C in LB supplemented with 10% (v/v) glycerol.

Plasmid constructs
To express Lem17 in mammalian cells, the ORF of lem17 was amplified from L. pneumophila genome and inserted into vectors pCMV-3×Flag or pCMV-HA at EcoRI/NotI sites. To express JAK1 in mammalian cells, the coding regions of JAK1 or its fragments were amplified from a human cell complementary DNA library, digested with XhoIII/NotI, and inserted into vector pcDNA3.1 with an HA-tag or pCMV-3×Flag.

To produce Lem17 protein in E. coli, the lem17 gene and its fragments were amplified from L. pneumophila genome, digested with BamHI/NheI, and inserted into vector pET-his harboring a 6×His-tag.

For jak1-knockout HeLa cells, the sense and antisense chains of human jak1 gRNA were annealed, and then inserted into vector pLentiCRISPR-v2 that was pre-digested with BsmBI, to generate pLentiCRISPR-Δjak1.

All expression constructs for Lem17 or JAK1 point mutations were generated by the modified site-directed mutagenesis protocol with corresponding primers. All primers used in this study are synthesized by a local company (Beijing Tsingke Biotech Co., Ltd, Chain) and listed in Supplementary Table 1.

L. pneumophila preparation and infection
L. pneumophila used for infection were grown in AYE broth to their post-exponential phase, as judged by both optical density [optical density at 600 nm (OD600), 3.2 to 4.0] and bacterial motility.

For intracellular growth assays, cells were seeded into 12- or 24-well plates or 24 at 5×105 per well respectively. Cells infected with L. pneumophila at a multiplicity of infection (MOI) of 0.05 were synchronized at 2 hours by washing three times with fresh medium to remove all extracellular bacteria. Infected macrophages were continued to culture at 37°C in the presence of 5% CO2. To measure the bacterial growth, cells were lysed with 0.02% digitonin. Colony forming unit of infected bacteria was determined by plating a series of lysate dilutions on solid CYET medium. HeLa cells were transfected with the plasmid expressing the FcγΙΙ receptor for 20-24h before infection. U937 cells were differentiated in fresh medium by phorbol myristate acetate (1 μM) for 12h.

For the transcription assay, mouse RAW264.7 cells were seeded into 6-well plates at 2×106 and 1×106 per well, respectively, for 1 day before infection. Cells infected with L. pneumophila at a multiplicity of infection (MOI) of 50 were synchronized at 2h by washing three times with fresh medium to remove all extracellular bacteria. Infected macrophages were continued to culture at 37°C in the presence of 5% CO2.

Formaldehyde-crosslinking in vivo and mass spectrometry
HeLa cells were plated in six 10-cm dishes at a density of 2×106 cells per dish, three of which were transfected with plasmid pCMV-3×Flag-Lem17, while the other three were transfected with empty vector pCMV-3×Flag vector as a control. The cells after 18h of transfection were washed twice by PBS, and cross-linked with 1% (v/v) formaldehyde for 10 min at 37°C, and terminated by 1.25 mM glycine for 10 min at room temperature. The cells were then harvested by centrifugation at 1500×g for 3 min, washed twice by PBS, and lysed by sonication for 1 min in a lysis buffer, containing 50 mM Tris (pH 7.5), 150 mM NaCl, 10% glycerol, 1% NP-40, 0.2 mM EDTA, and 1 mM PMSF. The cell lysate was rocked for another 4h at 4°C and centrifuged at 16,200×g for 30 min. Supernatant of the lysate was collected into a new tube and incubated with 50 μL of the Flag affinity resins at 4°C overnight. The affinity resin was then collected by centrifugation at 400×g for 2 min, washed in lysis buffer three times, and boiled for 10 min with 1.25× SDS loading buffer. All IPed proteins were separated on 12% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) gel and further identified by mass spectrometric analysis.

Co-Immunoprecipitation
HEK293T or HeLa cells were plated at a density of 2×106 cells per 100 mm dish and transfected with plasmid pCMV-3×Flag-Lem17 or pCMV-HA-JAKs using a liposomal transfection reagent (Yeasan). The cells were collected at 24h post-transfection and were incubated for 30 min on ice with 1.0 mL of a lysis buffer after sonication. The cell lysis supernatant was collected by centrifugation at 16,200×g for 10 min, and was transferred to a new 15 ml tube. The supernatant was incubated with 20 μL of the Flag affinity resins in IP buffer including 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.02% Triton X-100, 10% glycerol, 0.2 mM EDTA at 4°C for 6h, followed by three washes with the IP buffer. The captured proteins were eluted with 30 μL of Flag peptide (50 μg/mL) for 30 min on ice. The elution was boiled with 1.25× SDS loading buffer and separated on 12% SDS-PAGE, followed by immunoblotting with appropriate antibodies.

Immunoprecipitation
HeLa cells were plated in ten 10-cm dishes at a density of 2×106 cells per dish, and five of which were transfected with plasmid pCMV-3×Flag-Lem17 or its mutants, while the other five were transfected with empty vector pCMV-3×Flag vector as a control. The cells were collected at 24h post-transfection and were incubated for 30 min on ice with 1.0 ml of a lysis buffer after sonication. The cell lysis supernatant was collected by centrifugation at 16,200×g for 10 min, and was transferred to a new 15 ml tube. The supernatant was incubated with 20 μL of the Flag affinity resins in IP buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.02% Triton X-100, 10% glycerol, 0.2 mM EDTA at 4°C for 6h followed by three washes with the IP buffer. The captured proteins were eluted with 30 μL of Flag peptide (50 μg/mL) for 30 min on ice. The elution was boiled with 1.25× SDS loading buffer and separated on 12% SDS-PAGE, followed by immunoblotting with appropriate antibodies.

Liquid chromatography-tandem mass spectrometry analysis
After separation by SDS–PAGE, gel slices containing the protein detected by CBB stain were digested using 20 ng/L trypsin in 25 mM NH4HCO3 overnight at 37°C39. The digested peptides were analyzed on a C18 reversed-phase column connected to a UPLC (Acquity, Waters) coupled to an Orbitrap mass spectrometer (Q-Exactive Plus, Thermo Fisher Scientific). The mass spectrometer was operated in positive ion and standard data-dependent acquisition mode with the Advanced Peak Detection function activated for the top 10n. The resolution of the Orbitrap mass analyzer was set to 60,000 and 15,000 for MS1 and MS2, respectively. The full scan MS1 spectra were collected in the mass range of 350-1400 m/z, with an isolation window of 1.2 m/z and a fixed first mass of 100 m/z for MS2. The spray voltage was set at 2.2 kV and the Automatic Gain Control (AGC) target was 300% for MS1 and standard for MS2, respectively. Maximum ion injection time was set as 50 ms for MS1 and 110 ms for MS2, respectively. Dynamic exclusion was set with an exclusion duration of 6 s.

For protein identification, the raw data were processed with the software MaxQuant40 (version 1.6.3.3) against the Homo sapiens database (Uniprot, UP000005640) or L. pneumophila database (Uniprot, UP000000609). For acetylation identification, the raw data were converted to peak lists using DeconMSn41 and submitted for blind post-translational modification search using MODa42 against. Post-translational modification candidates were confirmed by manual inspection, looking for consistent mass shifts in b and y fragment series, and by reprocessing the data with MaxQuant, considering the specific modifications. MaxQuant was set to search with the following parameters: peptide tolerance at 10 ppm, MS/MS tolerance at 0.02 Da, carbamidomethyl (C) as a fixed modification, oxidation (M) as a variable modification, and a maximum of two missed cleavages. The false-discovery rates (FDR) were controlled at <1%.

Quantitative real-time PCR analysis
Total RNA was isolated from RAW264.7 cells infected with L. pneumophila using RNAiso Plus (Takara, 9109) as per the manufacturer’s instructions. Then reverse transcribed into cDNA by PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, RR047A). Real-time PCR was performed with TB Green® Premix Ex Taq™ (Tli RNaseH Plus) (Takara, RR420A) in QuantStudio 6 Flex Real-Time PCR System (ABI) according to the standard procedure. The fold change expression (-ΔΔCt) was calculated after normalization with GAPDH expression.

Generation of CRISPR-Cas9 genome-edited cell lines
The lentiviral particles were packaged by co-transfecting 1.5 μg of pLentiCRISPR-Δjak1 together with 1.5 μg of lentiviral packaging plasmids (pMDL:pVSV-G:pREV=0.5:0.3:0.2) into HEK293T cells plated at 2×106 cells each well by liposome transfection reagent Turbofect (Thermo Fisher Scientific). The cells were changed into fresh medium after 8 to 12h and continued to culture for another 24–48h. The supernatant containing lentivirus particles was then centrifuged to remove cell debris at 1500×g for 5 min at room temperature. A new batch of HeLa cells were infected for 48h by the lentivirus in 60-mm dishes and continued to culture for several generations with puromycin (2 μg/mL) until the knockout cell line was obtained. The efficiency of the jak1 knockout was evaluated by Western blot using anti-JAK1 antibody.

JAK1 knockdown in U937 cells by shRNA
shRNAs were designed to target human JAK1 using a design tool provided by Sigma (https://www.sigmaaldrich.cn/). The corresponding sequences were cloned into the pLKO.1-puro vector, and the insert sequences were verified by Sanger sequencing. The knockdown procedure was performed following the protocol from our previous method38. The lentiviral particles were packaged by co-transfecting 1.5 μg of pLKO-JAK1 together with 1 μg of lentiviral packaging plasmids (psPAX2:pMD2G=3:1) into HEK293T cells plated at 2×106 cells each well by liposome transfection reagent. The cells were changed into fresh medium after 8 to 12h and continued to culture for another 24–48h. The supernatant containing lentivirus particles was then centrifuged to remove cell debris at 1500×g for 5 min at room temperature. U937 cells were infected by the lentivirus in the presence of 10 µg/mL polybrene. 48h post-infection, the cells were selected with 2 μg/mL puromycin to generate stable pools. After the mock-infected control cells were completely eliminated, the knockdown efficiency of each shRNA construct was evaluated by Western Blot using anti-JAK1 antibody. The cell line exhibiting the most potent JAK1 knockdown was selected for subsequent experiments. Primers for cloning shRNA-JAK1 as follows:
F: 5′-CCGGGAGACTTCCATGTTACTGATTCTCGAGAATCAGTAACATGGAAGTCTCTTTTTG-3′
R: 5′-AATTCAAAAAGAGACTTCCATGTTACTGATTCTCGAGAATCAGTAACATGGAAGTCTC-3′

Kinase activity assay of JAK1
Flag-tagged JAK1 was purified from HEK293T cells co-expressing either Lem17 or its mutant C174S. The IRS-1 peptide (KKHTDDGYMPMSPGVA) synthesized by a local company (Sangon Biotech, China) was used as the substrate in the kinase activity assay. The kinase reaction was performed in a reaction buffer containing 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 10 mM MgCl₂. Briefly, 100 μL of reaction mixture containing 1 mM ATP, 1 mM IRS-1 peptide and 0.1 μM purified JAK1 was incubated at 37°C for 1h. Then, 100 μL of ATP detection reagent was added, followed by incubation at room temperature for another 30 min. The remaining amount of ATP in the reaction was monitored using an ATP Assay Kit (Beyotime, S0027) according to its manual, with a fluorescence microplate reader (Thermo Fisher Varioskan LUX, USA). Results are indicated as luminescence, where greater luminescence indicates lower kinase activity of JAK1.

Protein expression and purification
E. coli strain BL21 (DE3) transformed with an expression construct was cultivated in LB medium at 37°C till its optical density OD600 of 0.8, and induced by 400 μM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 18°C overnight. The cell pellets were harvested by centrifugation at 3470×g for 15 min and stored at -20°C.

His6-tagged proteins were purified using the following protocol. The cell pellets were suspended in a lysis buffer containing 50 mM Tris pH 8, 500 mM NaCl, 10% (v/v) glycerol, 5 mM β-mercaptoethanol, 20 mM imidazole and protease inhibitors, and lysed using sonication on ice. The cell lysate was centrifuged for 30 min at 34,572×g and its supernatant was collected and incubated with nickel sepharose beads for 1h at 4°C. The beads were washed by the lysis buffer and eluted by the lysis buffer added with a gradient of imidazole: 50, 100, 200, and 500 mM. The best fractions that contained our protein of interest were pooled, concentrated, and run through a Superdex 200 size-exclusion column (GE Healthcare) in buffer containing 20 mM Tris-HCl pH 8.0, 150 mM NaCl and 2 mM DTT. The best fractions of protein peak were pooled, concentrated to 10 mg/mL with an Amicon Centrifugal filter (Millipore), flash-frozen in liquid nitrogen and stored at -80°C for crystallization and activity assay. Protein concentration was measured at A280 and calculated using their theoretical extinction coefficients.

Protein crystallization and structural determination
The truncated Lem1715-446 was pre-incubated with 1 mM IP6 for 30 min on ice and the crystallization was performed at 16°C using a sitting drop diffusion method by mixing 0.4 μL of protein (10 mg/mL) with an equal volume of reservoir solution. The crystals initially grew in 0.1 M MES pH 6.0, 5% (w/v) polyethyleneglycol PEG 3000, and 30% (v/v) PEG 200 at 16°C after 3 days. After optimization, the best crystallization buffer was 0.1 M MES pH 6.2, 25% PEG 200 and 5% PEG 3,000. The Lem17-IP6 binary complex crystals were cryoprotected by brief soaking in the crystallization buffer with an additional 1 mM IP6 and flash-frozen in liquid nitrogen. Diffractions of crystals were collected at beamline BL02U1 of Shanghai Synchrotron Radiation Facility (SSRF) (Shanghai, China). Datasets were indexed, integrated, and scaled with xia2 software43. Lem17-IP6 binary structure was determined by the molecular replacement method with program Phaser in PHENIX44, and the AlphaFold3 model of Lem1715-320 was employed as a template. The subsequent model was manually built using COOT45 and refined with the program Refine in PHENIX44. The data collection and structure refinement statistics are summarized in Supplementary Table 2. Structural figures were generated using PyMol (http://pymol.org/2/).

Isothermal titration calorimetry (ITC)
The ITC experiments were performed using a microcalorimeter Affinity ITC (Waters, USA) at 20°C. The Lem17 protein or its mutants purified above were diluted into a binding buffer including 50 mM Tris-HCl (pH 8.0), 150 mM NaCl to a final concentration of 50 μM. IP6 or Acetyl-CoA stocks were also diluted in the same buffer at a final concentration of 500 μM, separately. Titrations are set for 20 injections of 2 μL each with 200-s intervals except from 0.2 μL of the first injection. Baseline subtraction and data analysis are performed using NanoAnayze. Heat spikes are integrated and fit with 1:1 binding model. The first injection of each experiment is excluded from the analysis.

To test the impact of IP6 on acetyl-CoA binding to Lem17, Lem17 was pre-incubated with IP6 at a molar ratio of 1:5 for 60 min before the ITC experiments. The ITC experiment was performed as above.

RNA-seq analysis
Total RNA was isolated using R (Qiagen) following the manufacturer’s instructions. DNase I in column digestion was included to ensure RNA quality. RNA library preparation was performed by using NEBNext@ UItraTM Directional RNA Library Prep Kit for Illumina (E7420L), and paired-end sequencing was performed with Illumina HiSeq 4000 platform. Low-quality reads were cleaned through FastQC (version 0.11.9) and Fastp (version 0.22.0), two packages for sequence pre-disposal. Processed reads were aligned to the human reference genome hg38 using STAR (version 2.7.11b). The alignment results were processed using the featureCount (version 2.0.6) for gene quantification with the option -primary (count primary alignments only) -B (only count read pairs that have both ends aligned) -C (do not count read pairs that have their two ends mapping to different chromosomes or mapping to the same chromosomes but on different strands) -Q 20 (the minimum mapping quality score 20) -s 0 (unstranded). Differential gene analysis was performed using limma (version 3.54.1), using P-value < 0.05 and FC ≥ 1 as cutoff. Then, volcano plots were drawn using the ggplot2 (version 3.5.1) package of R software to depict these differentially expressed genes. Reactome enrichment analysis was performed using the R package clusterProfiler (version 4.6.2) and ReactomePA (version 1.42.0). GSEA analysis was performed using the R package GSVA (version 1.46.0).

Antibodies and immunoblotting
For immunoblotting, samples resolved by SDS-PAGE were transferred onto 0.2 μm Polyvinylidene Fluoride (PVDF) membranes (Millipore, 0000183267), which were blocked with 5% non-fat milk at room temperature for 1h prior to being incubated with the appropriate primary antibodies: anti-DYKDDDDK tag (Proteintech, 66008-4-Ig), 1:5000; anti-HA (Proteintech, 51064-2-AP), 1:5000; anti-JAK1 (CST, 3344T), 1:1000; anti-STAT1 (Abcam, ab109320), 1:2000; anti-STAT1 (phospho Y701) (Abcam, ab109457), 1:1000; anti-STAT2 (Abcam, ab32367), 1:2000; anti-STAT2 (phospho Y690) (Abcam, ab191601), 1:1000; anti-Acetyllysine (PTM, PTM-105RM), 1: 2000; anti-ICDH (Sigma-Aldrich, ABS2090), 1:10000; anti-IFNAR2 (Proteintech, 10522-1-AP), 1: 1000; anti-GAPDH (Proteintech, 60004-1-Ig), 1:5000; anti-Lem17 (prepared from Biodragon), 1:1000. Membranes were then incubated with appropriate secondary antibodies conjugated to HRP (Proteintech, SA00001-2 or SA00001-1, 1:5000), and scanned by an imaging system (ImageQuant 800, Cytiva).

Bioinformatic identification of members of the YopJ family
We retrieved the YopJ protein from the Pfam database (ID: PF03421) and obtained all sequences under this record. Additionally, protein sequences from Legionella pneumophila were included. Sequences were deduplicated at 90% identity using CD-HIT, followed by manual removal of excessively long and short sequences, resulting in a final set of 240 sequences. Corresponding protein structures were acquired by querying the AlphaFold Database and performing predictions using AlphaFold3. Multiple structural alignments were conducted with USalign. The resulting structure-based sequence alignment served as a reference profile, which was then used to guide multiple sequence alignment with Clustal Omega. Phylogenetic analysis was performed using FastTree 2.1.11 with 1000 bootstrap replicates. Results were visualized in iTOL v6, with branch coloring applied according to bacterial family classifications.

Data analysis
The significant difference analysis of one-way analysis of variance (ANOVA) and Student’s t test were calculated all in GraphPad Prism 8 software (San Diego, USA). P < 0.05 was considered a significant difference.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Chen et al. describes the identification and functional characterization of Lem17, a novel effector from Legionella pneumophila. Lem17 was identified through sequence similarity searches using the known effector YopJ as a query. Although it shares homology with other members of the YopJ family—most of which function as O-acetyltransferases—Lem17 acts as an N-acetyltransferase. Like its relatives, Lem17 is activated by inositol hexaphosphate (IP6). Using MS-based proteomics, the authors show that Lem17 acetylates several host proteins, including the immune kinase JAK1. Lem17-mediated Nε-acetylation occurs on multiple lysine residues within the FERM-SH2 domain of JAK1, impairing its function. Overall, this is a high-quality study that broadens our understanding of YopJ family substrate specificity and reveals a new mechanism by which L. pneumophila subverts host immunity—through lysine acetylation of JAK1. However, to strengthen the manuscript and fully support the authors’ conclusions, both experimental and editorial issues must be addressed.\n\nMajor points\na) The authors used mass-spectrometry-based peptide mass fingerprinting to identify Lem17 targets and map the sites of acetylation. While supplementary tables summarize the data, the manuscript is missing representative MS and MS/MS spectra that support the correct identification of these proteins and the sites of modification. Moreover, it does not appear that the peptides identified from JAK1 (Lem17+ background) were compared to those from a wild-type background. A faint band appears to be visible in the acetyl-lysine blot (Fig. 3e). Any background acetylation that might exist in JAK1 should be noted to assign Lem17 target sites unambiguously.\nAdditionally, the authors have not deposited their mass-spectrometry proteomics (pull-down) data to a public repository and included an accession code in the data availability section. This is required for publication.\nb) The data presented show that Lem17 binds multiple targets in the cell (Fig. 3ab), of which only Jak1 was found to have the highest extent of acetylation when co-expressed with Lem17 (extended Fig. 4a). However, this contrasts with Fig. 1d, which shows multiple acetylated proteins above background when Lem17 is expressed. While the authors chose the top 10 kinases from the proteomics data to follow up on based on precedent, do the authors have any indication of what the other proteins acetylated in Fig. 1d could be, and if they’re relevant for the subversion of the JAK STAT pathway? Moreover, it is surprising that these other Lem17-acetylated proteins are not also present in extended Fig. 4a. Why is this the case?\nc) The authors propose a model where JAK1 function is perturbed through a dual mechanism involving both lysine acetylation and Lem17 binding, where both are proposed to interfere with cytokine binding. However, support for this model is incomplete and not entirely justified.\nUsing an Alphafold-3 model (for which validation and confidence statistics aren’t shown), the authors propose that the FIF region within the Lid domain of Lem17 interacts with the FERM domain of JAK1 and created an Ala mutant to confirm this interaction. While this result is promising, confirmation of this interface can only be complete if equivalent mutations are made in the FERM domain. There is a strong possibility that the FIF of Lem17 binds elsewhere. The reasons for my thoughts on this are based on Fig 3d, which shows that the FERM domain is acetylated at four Lys positions along its sequence. None of these Lys residues are remotely close to the active site of the Lem17 in the predicted complex. If this model is accurate, I would anticipate the active site of Lem17 to reside somewhere near these residues. Additional mutagenesis, binding, or biochemical studies are necessary to fully characterize the interaction interface between Lem17 and the FERM domain, thereby supporting the claim that Lem17 directly impairs JAK1 function through these interactions.\nAgain, for the predicted structural models the authors do not include any quality measures for the predictions. For example, if an AlphaFold model is used the authors must include pLDDT and PAE plots for the reader to access the quality of the predictions.\nd) The authors overstate many of their conclusions. Lem17 clearly only has a subtle/minor phenotype related to bacterial infection. However, this is common for Legionella effectors as it is known in the field that one effector is often insufficient for virulence. Given their data, the authors need to temper statements that Lem17 is critical or essential throughout the paper.\ne) The writing and sentence structure of the paper needs significant improvement. There are many grammatically awkward sentences, including many sentences that are intelligible.\n\nMinor points:\nThere are many editorial issues throughout the manuscript. Below is a summary of some, but not all of the issues identified.\nLine 62 – Incorrect spelling of dimer\nLine 74 – It's unclear what this sentence connects to. Please revise.\nLine 77: The sentence seems to have grammatical errors “Chief Among them are mediated the…”\nLine 95: For Legionella it is common that multiple effectors are required to establish the intracellular environment. The authors should temper their claims that Lem17 creates the environment. Especially as Figure 1b shows that deletion of Lem17 only has a minor affect.\nLine 99-114: again, the authors should temper their claim that Lem17 is required and essential for intracellular growth. The deletion only shows a minor effect on growth. It must be reiterated that it is widely known in the Legionella effector field that multiple effectors are required to produce disease phenotypes and establish the intracellular niche.\nLine 112 – The language referring to the mutants is unclear. Please revise.\nLine 119-122. The sentence is awkward as written.\nITC experiments with Lem17 + IP6: coA. The kd is only 2 uM. The authors co-incubate Lem17 with 1.25-time excess of IP6 to Lem17. From panel A, it is clear that a molar ratio of 2 IP6 to Lem17 is required for saturation. The authors should consider repeating the Lem17+IP6:CoA with higher IP6 concentration to saturate Lem17.\nLines 132-136. The authors should report an rmsd of the backbone alignments and/or a Dali-score to strengthen the claim that the structures are similar. Also, the cylinder alpha-helix cartoons makes it difficult to access the structural similarity. Would ribbons be clearer for the extended data?\nAlso, please cite the PDBid and paper for the YopJ structure in the figure legends and main text in this section.\nLines 136-137. Did the authors mean greater substrate specificity or affinity for IP6?\nLines 139-140. If you use an AlphaFold model you must include the pLDDT score and a figure of the model coloured by score.\nLines 143-147. P1-6 are not defined or labeled on the structure.\nFor the point mutants, a quality control should be shown to demonstrate that they are not mis-folded. Could the authors include a gel of the purification results and a folding control such as size-exclusion chromatography?\nFigure 2: The omit map density if very hard to see from the figure. Can it be made clearer? I would also suggest using the refined 2Fo-Fc maps for the main figure, and creating an extended data figure where very clear Fo-Fc maps are shown that are made from refinements where the ligand has been omitted.\nLin 144 -- Replace binds with bonds.\nLine 155 – Sentence needs revising.\nFigure 3: please be sure that the PDBs used in Figure 3g have their papers cited in the main text.\nLine 184 – correct inexpression.\nLines 189–190 - cytokines-associated receptor binding. Should be “cytokine-associated receptor binding.\nLine 204: “resolve structure of the Lem17-JAK1 complex” Sholud be resolve the structure of the Lem17-JAK1 complex.\nLine 249: “removed such this limit” Please revise.\nLine 244: “Such these genes enrich” please revise.\nLine 263-272. The JAK1 knockout increased Legionella proliferation in Figure 5F. However, the authors did not include an experiment of Legionella with a Lem17 deletion in this experiment. That should be also included here. If possible they should also do a Legionella strain harboring an Lem17 point variant or truncation they show decreased the ability of Lem17 to bind JAK1.\nLine 271: “promoting the bacterial replication” please revise.\nExtended Fig. 4b and 6C -- The labelling of the blot is confusing. Do the +/- signs refer to FLAG-tagged truncations or HA-Lem17? The way it is labelled indicates that the variable input is HA-Lem17. But since HA-Lem17 is present in the input panel, I assume these +/- signs indicate the FLAG-tagged proteins. Please clarify.\nCrystallographic table\nDo not say Rsym or Rmerge. Although sometimes used interchangeable they are technically different. The authors can just report Rmerge.\nI would suggest also reporting Rpim.\nThe authors must report CC(1/2)", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "In the manuscript by Chen, et al., the authors analyze the Legionella pneumophila Lem17 protein, which is translocated by the bacterial Type IV Secretion system (T4SS) and shows homology to the Yersinia YopJ protein, which inactivates kinases via an acetylation reaction. The authors identified interacting proteins by MS, after pulldown, and then focused only on kinases in the pulldown, finally looking for kinases acetylated by Lem17. The authors then focused on Jak1, which was the 383rd-most abundant protein in the pulldowns, but the only overproduced kinase of the group that was aceylated. They show direct interaction with Jak1 and Lem17, and mutants predicted by docking experiments were defective for binding, based on the crystal structure of Lem17 they produced and the presumed binding to the FERM domain of Jak1. The authors argue that Lem17 interfere with Jak1 immune signaling, as a number of interferon-regulated proteins are reduced after overproduction of Lem17 and exposure to IFN-B. They show that IFN-B is produced in RAW cells in response to Legionella (repeating previous observations of Opitz and Vance), although the effects of loss of Lem17 are unclear. Finally they show that Lem17 mutants grow better in HeLa cells knocked out for Jak1, however, the literature is ambiguous as to whether there is an interferon response in HeLa cells, leaving a disconnect between figs. 5e and f.\n1) Although the biochemistry is often technically excellent, I am very dubious about JAK1 being a primary target, and even how they identified it as a primary target is a little unclear. There were 382 other proteins that were more abundant in their pulldowns, so they decided that only kinases were relevant. In addition, the primary hosts for amoebae do not have Jak kinases. Finally, the connection between better growth in HeLa cells (possibly an IFN-nonproducer) and the data from RAW cells is totally unclear. To try to resolve this issue a little, I took the structure of the JAK1 FERM domain to query against the alpha-fold database of Dictyostelium using DALI, as amoebae are the primary hosts. The top hits were Talin-B (fyqa-A), Talin-A (f5b3-A) and unconventional Myosin I (fxjm-A). If you look at the authors XL file, which appears to be the MS dataset from this pool down, Talin-1 is #14 in abundance and an unconventional myosin is #64. Is this because there was no evidence of acetylation in the pulldown via MS? If so, I still think the authors have to go back and check some obvious more abundant candidates and make sure they are not substrates.\n2) One other major point, it is totally unclear why the authors chose to make a knockout in HeLa cells, given that their whole argument is that the targeting of JAK1 is due to blocking the IFN response. They have to show that the mutant actually blocks an IFN response or IFN production in HeLa cells or make the mutation in phagocytic cell line that produces or responds to IFN. There are numerous reports of knockouts in phagocytic cell lines, and even primary macrophages can be manipulated easily by RNAi, so this knockout is quite unconvincing that this has anything to do with the responses that the authors argue are important.\n3) Fig. 1b: it is inaccurate to say that Lem17 “is essential” for intracellular growth. After one cyle of growth within macrophages (24 hours pi) the mutant does not seem statistically different from WT, consistent with a number of studies looking for defects after one cycle of growth. Its possible that after 48 hrs there is a statistically significant defect, but there is no statistical verification of that claim. At any rate, using the term essential is an exaggeration, given the mild defects observed."}, "author_response": "Point-by point responses to reviewers’ comments\nReviewer #1:\nThe manuscript by Chen et al. describes the identification and functional\ncharacterization of Lem17, a novel effector from Legionella pneumophila. Lem17 was\nidentified through sequence similarity searches using the known effector YopJ as a\nquery. Although it shares homology with other members of the YopJ family—most of\nwhich function as O-acetyltransferases—Lem17 acts as an N-acetyltransferase. Like\nits relatives, Lem17 is activated by inositol hexaphosphate (IP6). Using MS-based\nproteomics, the authors show that Lem17 acetylates several host proteins, including\nthe immune kinase JAK1. Lem17-mediated Nε-acetylation occurs on multiple lysine\nresidues within the FERM-SH2 domain of JAK1, impairing its function. Overall, this is\na high-quality study that broadens our understanding of YopJ family substrate\nspecificity and reveals a new mechanism by which L. pneumophila subverts host\nimmunity—through lysine acetylation of JAK1. However, to strengthen the manuscript\nand fully support the authors’ conclusions, both experimental and editorial issues must\nbe addressed.\nMajor points:\na) The authors used mass-spectrometry-based peptide mass fingerprinting to identify\nLem17 targets and map the sites of acetylation. While supplementary tables\nsummarize the data, the manuscript is missing representative MS and MS/MS spectra\nthat support the correct identification of these proteins and the sites of modification.\nMoreover, it does not appear that the peptides identified from JAK1 (Lem17+\nbackground) were compared to those from a wild-type background. A faint band\nappears to be visible in the acetyl-lysine blot (Fig. 3e). Any background acetylation that\nmight exist in JAK1 should be noted to assign Lem17 target sites unambiguously.\nAdditionally, the authors have not deposited their mass-spectrometry proteomics (pull-\ndown) data to a public repository and included an accession code in the data\navailability section. This is required for publication.\nResponse: Thank you for your important comments. According to your comments, we\nhave included the MS/MS spectra of Lem17-targeted acetylated sites as Extended\nData Fig. 5 (Results shown below) in the revised manuscript. To help assign Lem17-\ntargeted sites, we also have provided a revised Supplementary Table S4. This table\nlists the peptides identified from JAK1 in the Lem17-expressing group compared with\nthe vector control group. In addition, we have deposited our mass-spectrometry data\nto the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org)\nand included the accession code PXD070394 in the Data Availability in the revised\nmanuscript.\nExtended Data Figure 5 Identification of Lem17-targeted acetylation sites in JAK1 by\nrepresentative MS/MS spectra. Acetylated lysine (K ) in each peptide is highlighted in red.\nAc\nb) The data presented show that Lem17 binds multiple targets in the cell (Fig. 3ab), of\nwhich only Jak1 was found to have the highest extent of acetylation when co-\nexpressed with Lem17 (extended Fig. 4a). However, this contrasts with Fig. 1d, which\nshows multiple acetylated proteins above background when Lem17 is expressed.\nWhile the authors chose the top 10 kinases from the proteomics data to follow up on\nbased on precedent, do the authors have any indication of what the other proteins\nacetylated in Fig. 1d could be, and if they’re relevant for the subversion of the JAK\nSTAT pathway? Moreover, it is surprising that these other Lem17-acetylated proteins\nare not also present in extended Fig. 4a. Why is this the case?\nResponse: We appreciate your insightful comments. We fully agree with the reviewers’\ncomments that Lem17 likely targets multiple host proteins, as supported by our data\nin Fig.1d. Here, we would like to clarify that we did NOT claim JAK1 to be the primary\nhost substrate of Lem17. In our manuscript, we have indicated that our study focused\non candidate proteins harboring kinase activity, because Lem17 belongs to the YopJ\nfamily, which is known to be involved in immune regulation through targeting host\nkinase-mediated pathways (Science.2006. PMID: 16728640; PNAS. 2006. PMID:\n17116858; PNAS. 2012. PMID: 22802624; Cell Host Microbe. 2012. PMID: 22520462;\nCell Host Microbe. 2008. PMID: 18407067; J Boil Chem. 2007. PMID: 17881352;\nMicrobiology. 2006. PMID: 16946275).\nAdditionally, among the top 20 candidates of Lem17 identified in our screening, the\neffector Lem17 also modified host protein EPB41L2. These results have been included\nas Extended Data Figure 4b and 4c in the revised manuscript. EPB41L2, a protein that\nalso contains a FERM domain. It is required for dynein-dynactin complex and NUMA1\nrecruitment at the mitotic cell cortex during anaphase (Cell.2013. PMID: 23870127).\nHowever, whether EPB41L2 engages in the JAK1-STAT pathway or Lem17 regulates\nEPB41L2’s function remains an interesting question that warrants further investigation.\nExtended Data Figure 4b and 4c: Screening the acetylated target by Lem17. HEK293T cells\nwere co-transfected to express HA-Lem17 and its potential target for 24 h and the samples\nwere stained with the appropriate antibodies. The acetylated proteins are marked by a red star.\nc) The authors propose a model where JAK1 function is perturbed through a dual\nmechanism involving both lysine acetylation and Lem17 binding, where both are\nproposed to interfere with cytokine binding. However, support for this model is\nincomplete and not entirely justified.\nUsing an Alphafold-3 model (for which validation and confidence statistics aren’t\nshown), the authors propose that the FIF region within the Lid domain of Lem17\ninteracts with the FERM domain of JAK1 and created an Ala mutant to confirm this\ninteraction. While this result is promising, confirmation of this interface can only be\ncomplete if equivalent mutations are made in the FERM domain. There is a strong\npossibility that the FIF of Lem17 binds elsewhere. The reasons for my thoughts on this\nare based on Fig 3d, which shows that the FERM domain is acetylated at four Lys\npositions along its sequence. None of these Lys residues are remotely close to the\nactive site of the Lem17 in the predicted complex. If this model is accurate, I would\nanticipate the active site of Lem17 to reside somewhere near these residues.\nAdditional mutagenesis, binding, or biochemical studies are necessary to fully\ncharacterize the interaction interface between Lem17 and the FERM domain, thereby\nsupporting the claim that Lem17 directly impairs JAK1 function through these\ninteractions.\nAgain, for the predicted structural models the authors do not include any quality\nmeasures for the predictions. For example, if an AlphaFold model is used the authors\nmust include pLDDT and PAE plots for the reader to access the quality of the\npredictions.\nResponse: We appreciate these insightful and useful comments. To address these\nconcerns regarding the predicted complex model of Lem17 and JAK1, we have\nconstructed equivalent mutations in the FERM domain involved in the FIF region of\nLem17 binding and performed Co-IP experiments. Consistent with the effects of\nmutations in the FIF region of Lem17, the equivalent mutations in the FERM domain\nsignificantly impaired the interactions between JAK1 and Lem17. We have included\nthis result as Fig.4b in the revised manuscript as follows. These results strongly\nsupport the existence of a specific interface between Lem17 and the FERM domain of\nJAK1, confirming that Lem17 directly impairs JAK1 function through these interactions.\nNevertheless, we do not exclude the possibility that the FIF region of Lem17 may also\nbind to other sites on JAK1.\nFig.4a and 4b: a, Close-up view of hydrophobic interaction between Lem17 and JAK1. JAK1\n(green) and Lem17 (Cyan) are shown in cartoon representation. Residues are depicted in sticks.\nb, Validation of residues involved in hydrophobic interaction between JAK1 and Lem17 by co-\nimmunoprecipitation. WT: Wild type JAK1; FF: F247A/F251A; LVL: L190A/V194A/L195A; VFF:\nV243A/F244A/F247A. The data represent three independent experiments.\nIn addition, we have included the pLDDT and PAE plots of the predicted AlphaFold\nmodel of JAK1-Lem17 complex as Extended Data Fig. 6a and 6b in the revised\nmanuscript as follows.\nExtended Data Fig. 6a and 6b: a, The predicted JAK1-Lem17 complex models colored by\nthe per-residue local Distance Difference Test (IDDT) scores. b, The PAE metrics for the\npredicted JAK1-Lem17 complex models show the predicted relative position error for each\nresidue sequence, with low-confidence values in white and high-confidence values in green.\nd) The authors overstate many of their conclusions. Lem17 clearly only has a\nsubtle/minor phenotype related to bacterial infection. However, this is common for\nLegionella effectors as it is known in the field that one effector is often insufficient for\nvirulence. Given their data, the authors need to temper statements that Lem17 is\ncritical or essential throughout the paper.\nResponse: We appreciate your insightful comments. We completely agree with you.\nWe have revised the related descriptions throughout the manuscript.\ne) The writing and sentence structure of the paper needs significant improvement.\nThere are many grammatically awkward sentences, including many sentences that are\nintelligible.\nResponse: We would like to thank you for the thorough review of our work. We have\ncarefully reviewed our manuscript with help from colleagues who have excellent\ncommand of the English language, which has greatly improved its clarity.\nMinor points:\nThere are many editorial issues throughout the manuscript. Below is a summary of\nsome, but not all of the issues identified.\n1. Line 62 – Incorrect spelling of dimer.\nResponse: Thankyou for pointing out this. We have corrected it in the revised\nmanuscript.\n2. Line 74 – It's unclear what this sentence connects to. Please revise.\nResponse: Thank you. We have rewritten this sentence (Lines 73-74) in the revised\nmanuscript as follows:\n“Legionella pneumophila, an intracellular bacterial pathogen, causes Legionnaires’\ndisease.”\n3. Line 77: The sentence seems to have grammatical errors “Chief Among them are\nmediated the…”\nResponse: Thank you for you pointing this issue out this. We have rewritten this\nsentence in the revised manuscript (Lines 76-78) as follows:\n“One of these effectors functions by mediating post-translational modifications that\nperturb host signaling pathways.”\n4. Line 95: For Legionella it is common that multiple effectors are required to establish\nthe intracellular environment. The authors should temper their claims that Lem17\ncreates the environment. Especially as Figure 1b shows that deletion of Lem17 only\nhas a minor affect.\nResponse: Thank you for this insightful point. We have rewritten this sentence in the\nrevised manuscript (Lines 94-96) as follows:\n“…, thereby facilitating the creation of a permissive intracellular environment for L.\npneumophila replication in host cells.”\n5. Line 99-114: again, the authors should temper their claim that Lem17 is required\nand essential for intracellular growth. The deletion only shows a minor effect on growth.\nIt must be reiterated that it is widely known in the Legionella effector field that multiple\neffectors are required to produce disease phenotypes and establish the intracellular\nniche.\nResponse: Thank you for this insightful point. We have rewritten related sentences in\nthe revised manuscript as follows:\nLines 98-99: “Lem17 is a bona fide acetyltransferase containing a conserved C-H-D\nmotif”\nLines 105-106: “…, demonstrating that effector Lem17 contributes to the intracellular\ngrowth of L. pneumophila (Fig.1a, b).”\nLines 113-114: “These results demonstrate that Lem17 is an acetyltransferase\nfacilitating the intracellular growth of L. pneumophila.”\n6. Line 112 – The language referring to the mutants is unclear. Please revise.\nResponse: Thank you for raising this point. We have added the specific information\nof mutants in the revised manuscript (Lines 109-110) as follows:\n“…, we ectopically expressed the wild-type Lem17 or its catalytic mutants (C174S,\nH104A and D124A) in HEK293T cells.”\n7. Line 119-122. The sentence is awkward as written.\nResponse: According to your comment, we have rewritten the sentence in the revised\nmanuscript (Lines 117-122) as follows:\n“We next examined whether Lem17 binds IP6. We found that IP6 shows high affinity\nfor Lem17, with a binding constant (Kd) of approximately 2M (Fig.2a). Specifically,\nLem17 only binds acetyl-CoA in the presence of IP6 (Kd ≈20 M ), whereas no binding\nwas detected in its absence (Fig.2a). These results suggest that Lem17 requires IP6\nfor activation.”\n8. ITC experiments with Lem17 + IP6: coA. The kd is only 2 uM. The authors co-\nincubate Lem17 with 1.25-time excess of IP6 to Lem17. From panel A, it is clear that\na molar ratio of 2 IP6 to Lem17 is required for saturation. The authors should consider\nrepeating the Lem17+IP6:CoA with higher IP6 concentration to saturate Lem17.\nResponse: Thank you for your insightful suggestion. According to your suggestion,\nwe have repeated ITC experiments that a molar ratio of IP6 to Lem17 (5:1). The results\nare included in Fig.2a and the relevant method has been corrected (Line 576) in the\nrevised manuscript as follows:\nFig. 2a: ITC experiments verifying that IP6 is required for Lem17 binding the ligand CoA.\nRaw\nITC curves (top panel) and binding isotherms with fitting curves (bottom panel) of Lem17\ntitration by IP6. Lem17+IP6: Lem17 is saturated with IP6 at a 1: 5 molar ratio for 60 min before\ntitration. The data represent three independent experiments; the data are mean ± s.d.\n9. Lines 132-136. The authors should report an rmsd of the backbone alignments\nand/or a Dali-score to strengthen the claim that the structures are similar. Also, the\ncylinder alpha-helix cartoons makes it difficult to access the structural similarity. Would\nribbons be clearer for the extended data?\nResponse: Thank you for your useful suggestion. According to your suggestion, we\nhave redrawn Extended Data Fig. 2b in the revised manuscript as follows:\nExtended Data Fig. 2b: Structural alignment of Lem17 to the YopJ family used theC atoms\nof the Lem17-CD domain.\n10. Also, please cite the PDB id and paper for the YopJ structure in the figure legends\nand main text in this section.\nResponse: Thank you for you raising this point. We have added the related\ninformation in the revised manuscript.\n11. Lines 136-137. Did the authors mean greater substrate specificity or affinity for IP6?\nResponse: We have rewritten related descriptions in the revised manuscript as follows:\nLines 131-132: “…, which may be the site for interactions between Lem17 and its host\ntargets.”\nLines 138-139: “The presence of this Lid domain may confer Lem17 with greater\nspecificity or affinity for its host targets.”\n12. Lines 139-140. If you use an AlphaFold model you must include the pLDDT score\nand a figure of the model coloured by score.\nResponse: Thank you for your professional suggestion. We have included the pLDDT\nscore and a figure of the model coloured by score as Extended Data Fig. 2d and 2e in\nthe revised manuscript as follows:\nExtended Data Fig. 2d and 2e: d, The predicted Lem17 model colored by the per-residue\nlocal Distance Difference Test (IDDT) scores. e, The PAE metrics for the predicted Lem17\ncomplex models show the predicted relative position error for each residue sequence, with low-\nconfidence values in white and high-confidence values in green\n13. Lines 143-147. P1-6 are not defined or labeled on the structure.\nResponse: Thank you for raising this point. We have redrawn Figure 2e in the revised\nmanuscript as follows:\nFig. 2e: The IP6-binding pocket. Residues important for IP6 binding were shown as sticks.\nHydrogen bonds were marked by blue dashed lines. The refined 2mFo-Fc map (colored in pink)\nof IP6 is contoured at the 2.0σ level. The six phosphate groups of IP6 are designated the labels\nP1 to P6.\n14. For the point mutants, a quality control should be shown to demonstrate that they\nare not mis-folded. Could the authors include a gel of the purification results and a\nfolding control such as size-exclusion chromatography?\nResponse: Thank you for your useful comment. We have included size-exclusion\nchromatography and gels of the purification results as Extended Data Fig. 3b and 3c\nin the revised manuscript as follows:\nExtended Data Fig. 3b and 3c: b, Gel filtration profiles of Lem17 and its mutants by\nsize-exclusion chromatography. c, The peak fractions from panel b were visualized by\nSDS-PAGE and Coomassie blue staining.\n15. Figure 2: The omit map density if very hard to see from the figure. Can it be made\nclearer? I would also suggest using the refined 2Fo-Fc maps for the main figure, and\ncreating an extended data figure where very clear Fo-Fc maps are shown that are\nmade from refinements where the ligand has been omitted.\nResponse: We appreciate your insightful comments. According to your suggestions,\nwe have redrawn Figure 2d, 2e and included the Fo-Fc map of IP6 as Extended Data\nFig. 2g in the revised manuscripts as follows:\nFig.2d: The conserved residues C174, H105 and D124 in the structure of Lem17. Residues\nare represented as sticks with the annealing omit map contoured at the 2.0σ level and proteins\nwere depicted in cyan cartoon.\nFig.2e: The IP6-binding pocket. Residues important for IP6 binding were shown as sticks.\nHydrogen bonds were marked by blue dashed lines. The refined 2mFo-Fc map (colored in pink)\nof IP6 is contoured at the 2.0σ level.\nExtended Data Fig.2g: The Fo-Fc map (colored in green) of IP6 contoured at the 5.0σ level.\n16. Line 144 -- Replace binds with bonds.\nResponse: Thank you. We have corrected it (Line 146) in the revised manuscript.\n17. Line 155 – Sentence needs revising.\nResponse: According to your comment, we have revised this sentence (Lines 159-\n160) in the revised manuscript as follows:\n“To elucidate the specific physiological function of Lem17 in the host, we next sought\nto identify host proteins that are targeted by Lem17.”\n18. Figure 3: please be sure that the PDBs used in Figure 3g have their papers cited\nin the main text.\nResponse: Thank you for you pointing out this. We have added citations of these\npapers (Lines 219-221) in the revised manuscript.\n“…, whereas YopJ family proteins such as PopP222 and HopZ1a21 display a distinct\nbias toward positively charged regions (Fig. 3g).”\n19. Line 184 – correct inexpression.\nResponse: The term “inexpression” has been changed to “non-expression” (Line 201)\nin the revised manuscript.\n20. Lines 189–190 - cytokines-associated receptor binding. Should be “cytokine-\nassociated receptor binding.\nResponse: According to your comment, “cytokines-associated receptor binding” has\nbeen changed to “cytokine-associated receptor binding” (Line 208) in the revised\nmanuscript.\n21. Line 204: “resolve structure of the Lem17-JAK1 complex” Should be resolve the\nstructure of the Lem17-JAK1 complex.\nResponse: Thank you. According to your comment, “resolve structure of the Lem17-\nJAK1 complex” has been changed to “resolve the structure of the Lem17-\nJAK1 complex” (Line 224) in the revised manuscript.\n22. Line 249: “removed such this limit” Please revise.\nResponse: Thank you for you pointing out this point. We have revised it (Lines 270-\n271) in the revised manuscript as follows:\n“In contrast, the C174S mutant reversed the suppression caused by Lem17 (Fig.5a).”\n23. Line 244: “Such these genes enrich” please revise.\nResponse: Thank you for raising this point. We have revised it (Lines 276-278) in the\nrevised manuscript as follows.\n“Gene Ontology (GO) analysis revealed that these genes are enriched in multiple\nsignaling pathways,”\n24. Line 263-272. The JAK1 knockout increased Legionella proliferation in Figure 5F.\nHowever, the authors did not include an experiment of Legionella with a Lem17\ndeletion in this experiment. That should be also included here. If possible they should\nalso do a Legionella strain harboring an Lem17 point variant or truncation they show\ndecreased the ability of Lem17 to bind JAK1.\nResponse: We appreciate your insightful comments. We have repeated this\nexperiment by using Lem17-deleted and FIF mutant (which has impaired JAK1-binding\nability) Legionella strains and incorporated the results as Figure 4g in the revised\nmanuscript as follows:\nFig. 4g: Effect of the important residues of Lem17 in JAK1 interaction on bacterial replication.\nBacteria of the indicated strains were used to infect macrophage-like U937 cells at an MOI of\n0.05 and the growth of the bacteria was determined at the indicated time points. The data\nrepresent three independent experiments; the data are mean ± s.d.\n25. Line 271: “promoting the bacterial replication” please revise.\nResponse: Thank you for you pointing out this point. We have revised it (Lines 293-\n294) in the revised manuscript as follows:\n“…, which contributes to the intracellular replication of L. pneumophila in host cells.”\n26. Extended Fig. 4b and 6C -- The labelling of the blot is confusing. Do the +/- signs\nrefer to FLAG-tagged truncations or HA-Lem17? The way it is labelled indicates that\nthe variable input is HA-Lem17. But since HA-Lem17 is present in the input panel, I\nassume these +/- signs indicate the FLAG-tagged proteins. Please clarify.\nResponse: Thank you for you pointing out these issues. We have revised the labeling\nof Extended Fig.4d and 6e in the revised manuscript as follows:\n27. Crystallographic table: Do not say Rsym or Rmerge. Although sometimes used\ninterchangeable they are technically different. The authors can just report Rmerge. I\nwould suggest also reporting Rpim. The authors must report CC(1/2).\nResponse: Thank you for your professional and insightful comments. According to\nyour comments, we have now included the values of Rpim and CC(1/2) in the updated\ncrystallographic table in the revised manuscript.\nReviewer #3:\nIn the manuscript by Chen, et al., the authors analyze the Legionella pneumophila\nLem17 protein, which is translocated by the bacterial Type IV Secretion system (T4SS)\nand shows homology to the Yersinia YopJ protein, which inactivates kinases via an\nacetylation reaction. The authors identified interacting proteins by MS, after pulldown,\nand then focused only on kinases in the pulldown, finally looking for kinases acetylated\nby Lem17. The authors then focused on Jak1, which was the 383rd-most abundant\nprotein in the pulldowns, but the only overproduced kinase of the group that was\naceylated. They show direct interaction with Jak1 and Lem17, and mutants predicted\nby docking experiments were defective for binding, based on the crystal structure of\nLem17 they produced and the presumed binding to the FERM domain of Jak1. The\nauthors argue that Lem17 interfere with Jak1 immune signaling, as a number of\ninterferon-regulated proteins are reduced after overproduction of Lem17 and exposure\nto IFN-B. They show that IFN-B is produced in RAW cells in response to Legionella\n(repeating previous observations of Opitz and Vance), although the effects of loss of\nLem17 are unclear. Finally they show that Lem17 mutants grow better in HeLa cells\nknocked out for Jak1, however, the literature is ambiguous as to whether there is an\ninterferon response in HeLa cells, leaving a disconnect between figs. 5e and f.\n1) Although the biochemistry is often technically excellent, I am very dubious about\nJAK1 being a primary target, and even how they identified it as a primary target is a\nlittle unclear. There were 382 other proteins that were more abundant in their\npulldowns, so they decided that only kinases were relevant. In addition, the primary\nhosts for amoebae do not have Jak kinases. Finally, the connection between better\ngrowth in HeLa cells (possibly an IFN-nonproducer) and the data from RAW cells is\ntotally unclear. To try to resolve this issue a little, I took the structure of the JAK1 FERM\ndomain to query against the alpha-fold database of Dictyostelium using DALI, as\namoebae are the primary hosts. The top hits were Talin-B (fyqa-A), Talin-A (f5b3-A)\nand unconventional Myosin I (fxjm-A). If you look at the authors XL file, which appears\nto be the MS dataset from this pool down, Talin-1 is #14 in abundance and an\nunconventional myosin is #64. Is this because there was no evidence of acetylation in\nthe pulldown via MS? If so, I still think the authors have to go back and check some\nobvious more abundant candidates and make sure they are not substrates.\nResponse: Thank you for your insightful comments. We fully agree with the reviewers’\ncomments that Lem17 likely targets multiple host proteins, as supported by our data\nin Fig.1d. Here, we would like to clarify that we did NOT claim JAK1 to be the primary\nhost substrate of Lem17. In our manuscript, we have indicated that our study focused\non candidate proteins harboring kinase activity, because Lem17 belongs to the YopJ\nfamily, which is known to be involved in immune regulation through targeting host\nkinase-mediated pathways (Science.2006. PMID: 16728640; PNAS. 2006. PMID:\n17116858; PNAS. 2012. PMID: 22802624; Cell Host Microbe. 2012. PMID: 22520462;\nCell Host Microbe. 2008. PMID: 18407067; J Boil Chem. 2007. PMID: 17881352;\nMicrobiology. 2006. PMID: 16946275).\nAdditionally, among the top 20 candidates of Lem17 identified in our screening, the\neffector Lem17 also modified host protein EPB41L2 (Results shown below panel a\nand b). EPB41L2, a protein that also contains a FERM domain. It is required for dynein-\ndynactin complex and NUMA1 recruitment at the mitotic cell cortex during anaphase\n(Cell.2013. PMID: 23870127). Whether EPB41L2 engages in the JAK-STAT pathway\nor Lem17 regulates EPB41L2’s function remains an interesting question that warrants\nfurther investigation. Moreover, the reviewer performed a structural analysis of the\nJAK1 FERM domain using DALI to query the Alpha-Fold database of Dictyostelium,\nwhich identified Talin and unconventional Myosin I as the top hits. This result may\nprovide further support for the notion that Lem17 interacts with JAK1. Despite these\ntwo proteins also contain a FERM domain, we did not detect its interactions with Lem17\n(Results shown below panel c), likely due to differences in their sequence and\nstructural features from those of JAK1.\nFinally, we would like to note that JAK1 is not the only protein targeted by Legionella\neffectors that is absent in amoebae. A salient example is the targeting of linear ubiquitin\nchains involved in immunity by the L. pneumophila deubiquitinase effector RavD (Nat\nMicrobiol.2019; PMID: 31110362). Amoebae does NOT produce linear ubiquitin chains.\nSimilarly, L. pneumophila effectors LnaB and LegK1 are known to activate the host\nNF-κB signaling, thereby modulating macrophage defense and inflammatory\nresponses (Cell Microbiol.2010. PMID: 20148897; Proc Natl Acad Sci USA.2009.\nPMID: 19666608; J Exp Med.2006. PMID: 16940169). NF-kB signaling does NOT exist\nin amoebae. It is of interest to note that a case of probable person-to-person\ntransmission of L. pneumophila has been reported (NEJM.2016; PMID: 26840151).\nTaken together, these observations suggest that the host range of L. pneumophila is\nbeyond amoebae (Nat Rev Microbiol. 2017; PMID: 28713154).\nFigure legend: a and b, Screening the acetylated target by Lem17. HEK293T cells were co-\ntransfected to express HA-Lem17 and its potential target for 24 h and the samples were stained\nwith the appropriate antibodies. The acetylated proteins are marked by a red star. c, Validation\nof interaction between Lem17 with Talin-1 and myosin by co-Immunoprecipitation.\n2) One other major point, it is totally unclear why the authors chose to make a knockout\nin HeLa cells, given that their whole argument is that the targeting of JAK1 is due to\nblocking the IFN response. They have to show that the mutant actually blocks an IFN\nresponse or IFN production in HeLa cells or make the mutation in phagocytic cell line\nthat produces or responds to IFN. There are numerous reports of knockouts in\nphagocytic cell lines, and even primary macrophages can be manipulated easily by\nRNAi, so this knockout is quite unconvincing that this has anything to do with the\nresponses that the authors argue are important.\nResponse: We appreciate these important comments. To address these concerns,\nwe generated a JAK1-knockout cell line from the macrophage-like U937 cells and\nrepeated the relevant experiments to validate our findings. Consistent with our earlier\nresults in HeLa cells, JAK1-knockout promotes the intracellular replication of L.\npneumophila (Results shown below panel a). In addition, we also demonstrated that\nJAK1 knockout in HeLa cells impairs the IFN response (Results presented below panel\nb), which is consistent with previous reports (Cell reports.2024.PMID: 38733583; Mol\nCell Biol.2016.PMID: 27044867).\nNotably, HeLa cells have been commonly used to study type I IFN signaling. For\nexample, studies have confirmed that HeLa cells, in response to IFNs, express both\nsubunits (IFNAR1 and IFNAR2) of the type I interferon receptor on their surface, which\nactivates the JAK-STAT signaling and leads to STATs activation (Cell.2024. PMID:\n39672162; Sci Signal.2021. PMID: 34813358; J Mol Biol.2019. PMID: 31207241; J\nCell Biol.2015. PMID: 26008745; J Cell Biol.2014. PMID: 25385185; Mol. Cell.\nBiol.1990. PMID: 2118988). Additionally, HeLa cells are known to be capable of\nproducing interferon (IFN) under stimulatory conditions, as evidenced by multiple\nstudies (Cell Rep.2021. PMID: 33406424; Singal Transduct Target Ther.2021. PMID:\n34732709; J Mol Boil.2019. PMID: 31207241; J Immunol.2015. PMID: 25070851).\nFinally, while phagocytic cell lines such as Raw264.7, U937, and THP1, are commonly\nused in studies of Legionella pneumophila infection, non-phagocytic cells, including\nHeLa cells ectopically expressing the Fc receptor, have been extensively employed\nfor experiments involved in Legionella pneumophila infection (Nat Commun.2024.\nPMID: 39349471; Nat Commun.2017. PMID: 28504273; Cell Rep.2025. PMID:\n39661521; iScience.2020. PMID: 32622269; Cell Host Microbe. 2019. PMID:\n31540829).\nCollectively, HeLa cells serve as a common model for studying L. pneumophila\ninfection and type I IFN signaling.\nFigure legend: a, JAK1-knockdown in U937 cells promotes the intracellular replication of L.\npneumophila. Bacteria of the indicated strains were used to infect macrophage-like U937 cells\nat an MOI of 0.05 and the growth of the bacteria was determined at the indicated time points.\nThe data represent three independent experiments; the data are mean ± s.d. b, JAK1-knockout\nin HeLa cells impairs the IFN response. HeLa cells were treated with 10 ng/mL IFN- for 6 h.\nThe specific JAK1 inhibitor ABT-494 (Upadacitinib) was used as a positive control and was\nincubated with HeLa cells for 2 h before IFN- treatment. The samples were probed with the\nappropriate antibodies. GAPDH was probed as a loading control.\n3) Fig. 1b: it is inaccurate to say that Lem17 “is essential” for intracellular growth. After\none cyle of growth within macrophages (24 hours pi) the mutant does not seem\nstatistically different from WT, consistent with a number of studies looking for defects\nafter one cycle of growth. Its possible that after 48 hrs there is a statistically significant\ndefect, but there is no statistical verification of that claim. At any rate, using the term\nessential is an exaggeration, given the mild defects observed.\nResponse: We appreciate your insightful comments. We completely agree with you.\nAccording to your comments, we have revised the related descriptions throughout the\nmanuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have significantly improved the manuscript and addressed my concerns. There are only two last minor points that could be addressed.\n\nExtended Data Figure 5 is very low resolution and small fonts.\n\nExtended Data Figure 6\nThe authors have included the AlphaFold prediction of the complex which shows that AlphaFold3 has limited confidence. The ipTM of 0.46 is defined by AlphaFold3 as a poor prediction. Additionally, the interface regions are clearly in the confident ranges, but nothing very high. The PAE plot in the 360 residue region is more encouraging.\nWhile the prediction is acceptable and the authors have point-variant data to support the complex, the authors should at least briefly mention the limits of the complex prediction in the main text.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Point-by point responses to reviewers’ comments\nReviewer #1:\nThe authors have significantly improved the manuscript and addressed my concerns.\nThere are only two last minor points that could be addressed.\n1. Extended Data Figure 5 is very low resolution and small fonts.\nResponse: Thank you for your important comments. We have redrawn and enhanced\nthe resolution of Extended Data Fig. 5 (now Supplementary Figure 5 and 6, shown\nbelow) in the revised manuscript, and hope this meets your needs.\nSupplementary Figure 5 The representative MS/MS spectra of Lem17-targeted acetylation\nsites in JAK1 FERM-SH2 domain. Acetylated lysine (K ) in each peptide is highlighted in red.\nAc\nSupplementary Figure 6 The representative MS/MS spectra of Lem17-targeted acetylation\nsites in JAK1 kinase domain. Acetylated lysine (K ) in each peptide is highlighted in red.\nAc\n2. Extended Data Figure 6\nThe authors have included the AlphaFold prediction of the complex which shows that\nAlphaFold3 has limited confidence. The ipTM of 0.46 is defined by AlphaFold3 as a\npoor prediction. Additionally, the interface regions are clearly in the confident ranges,\nbut nothing very high. The PAE plot in the 360 residue region is more encouraging.\nWhile the prediction is acceptable and the authors have point-variant data to support\nthe complex, the authors should at least briefly mention the limits of the complex\nprediction in the main text.\nResponse: We appreciate your insightful comments. According to your suggestion,\nwe have added a description of the structural limitation of our predicted JAK1-Lem17\ncomplex (Lines 236-238) in the revised manuscript as follows.\n“It notes that the predicted JAK1-Lem17complex structure is limited by a moderate\nipTM score, despite our point-variant data supports this prediction.”"}]

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