Legionella pneumophila modulates the host cytoskeleton by an effector of transglutaminase activity

Abstract

The bacterial pathogen Legionella pneumophila delivers more than 330 effector proteins into host cells through its Dot/Icm type IV secretion system (T4SS) to facilitate its intracellular replication. A number of these effectors modulate organelle trafficking pathways to create a membrane-bound niche called the Legionella-containing vacuole (LCV). In this study, we found that L. pneumophila induces F-actin accumulation in the host cell cortex by its Dot/Icm substrate RavJ (Lpg0944). RavJ harbors a C₁₀₁H₁₃₈D₁₇₀ motif associated with human tissue transglutaminases (TGs). We show that RavJ catalyzes a covalent linkage between actin and members of the Motin family of proteins, including Angiomotin (AMOT) and Angiomotin-like 1 (AMOTL1), which are known to regulate cell migration and contribute to the formation of cellular structures such as endothelial cell junctions and tubes. Further study reveals that RavJ-induced crosslink between actin and AMOT occurs on its Gln₃₅₄ residue. Crosslink between actin and AMOT significantly reduces the binding between actin and its binding partner cofilin, suggesting that RavJ inhibits actin depolymerization. We also demonstrate that the metaeffector LegL1 directly interacts with RavJ to antagonize its TG activity, leading to reduced crosslinks between actin and Motin proteins. Our results reveal a novel mechanism of modulating the host actin cytoskeleton by L. pneumophila.

Keywords

Dot/Icm type IV secretion system / Legionella pneumophila / metaeffector LegL1 / transglutaminase activity

References

[1] Vogel JP, Andrews HL, Wong SK, Isberg RR. Conjugative transfer by the virulence system of Legionella pneumophila. Science. 1998; 279: 873-876.

[2] Qiu J, Luo ZQ. Legionella and Coxiella effectors: strength in diversity and activity. Nat Rev Microbiol. 2017; 15: 591-605.

[3] Qiu J, Luo ZQ. Hijacking of the host ubiquitin network by Legionella pneumophila. Front Cell Infect Microbiol. 2017; 7: 487.

[4] Isberg RR, O'Connor TJ, Heidtman M. The Legionella pneumophila replication vacuole: making a cosy niche inside host cells. Nat Rev Microbiol. 2009; 7: 13-24.

[5] Zhu W, Banga S, Tan Y, Zheng C, Stephenson R, Gately J, et al. Comprehensive identification of protein substrates of the Dot/Icm type IV transporter of Legionella pneumophila. PLoS One. 2011; 6: e17638.

[6] Huang L, Boyd D, Amyot WM, Hempstead AD, Luo ZQ, O'Connor TJ, et al. The E Block motif is associated with Legionella pneumophila translocated substrates. Cell Microbiol. 2011; 13: 227-245.

[7] Lockwood DC, Amin H, Costa TRD, Schroeder GN. The Legionella pneumophila Dot/Icm type IV secretion system and its effectors. Microbiology. 2022; 168: 001187.

[8] Pollard TD, Cooper JA. Actin, a central player in cell shape and movement. Science. 2009; 326: 1208-1212.

[9] Richard JF, Petit L, Gibert M, Marvaud JC, Bouchaud C, Popoff MR. Bacterial toxins modifying the actin cytoskeleton. Int Microbiol. 1999; 2: 185-194.

[10] Ono S. A plague of actin disassembly. J Biol Chem. 2017; 292: 8101-8102.

[11] Galán JE, Zhou D. Striking a balance: modulation of the actin cytoskeleton by Salmonella. Proc Natl Acad Sci USA. 2000; 97: 8754-8761.

[12] Liu Y, Zhu W, Tan Y, Nakayasu ES, Staiger CJ, Luo ZQ. A Legionella effector disrupts host cytoskeletal structure by cleaving actin. PLoS Pathog. 2017; 13: e1006186.

[13] Michard C, Sperandio D, Baïlo N, Pizarro-Cerdá J, LeClaire L, Chadeau-Argaud E, et al. The Legionella kinase LegK2 targets the ARP2/3 complex to inhibit actin nucleation on phagosomes and allow bacterial evasion of the late endocytic pathway. mBio. 2015; 6: e00354-15.

[14] Guo Z, Stephenson R, Qiu J, Zheng S, Luo ZQ. A Legionella effector modulates host cytoskeletal structure by inhibiting actin polymerization. Microb Infect. 2014; 16: 225-236.

[15] Franco IS, Shohdy N, Shuman HA. The Legionella pneumophila effector VipA is an actin nucleator that alters host cell organelle trafficking. PLoS Pathog. 2012; 8: e1002546.

[16] Zhang Q, Wan M, Kudryashova E, Kudryashov DS, Mao Y. Membrane-dependent actin polymerization mediated by the Legionella pneumophila effector protein MavH. PLoS Pathog. 2023; 19: e1011512.

[17] Chambers KA, Scheck RA. Bacterial virulence mediated by orthogonal post-translational modification. Nat Chem Biol. 2020; 16: 1043-1051.

[18] Mukherjee S, Liu X, Arasaki K, McDonough J, Galán JE, Roy CR. Modulation of Rab GTPase function by a protein phosphocholine transferase. Nature. 2011; 477: 103-106.

[19] Tan Y, Arnold RJ, Luo ZQ. Legionella pneumophila regulates the small GTPase Rab1 activity by reversible phosphorylcholination. Proc Natl Acad Sci USA. 2011; 108: 21212-21217.

[20] Müller MP, Peters H, Blümer J, Blankenfeldt W, Goody RS, Itzen A. The Legionella effector protein DrrA AMPylates the membrane traffic regulator Rab1b. Science. 2010; 329: 946-949.

[21] Lee PC, Machner MP. The Legionella effector kinase LegK7 hijacks the host Hippo pathway to promote infection. Cell Host Microbe. 2018; 24: 429-438.e6.

[22] Qiu J, Sheedlo MJ, Yu K, Tan Y, Nakayasu ES, Das C, et al. Ubiquitination independent of E1 and E2 enzymes by bacterial effectors. Nature. 2016; 533: 120-124.

[23] Black MH, Osinski A, Park GJ, Gradowski M, Servage KA, Pawłowski K, et al. A Legionella effector ADP-ribosyltransferase inactivates glutamate dehydrogenase. J Biol Chem. 2021; 296: 100301.

[24] Fu J, Zhou M, Gritsenko MA, Nakayasu ES, Song L, Luo ZQ. Legionella pneumophila modulates host energy metabolism by ADP-ribosylation of ADP/ATP translocases. eLife. 2022; 11: e73611.

[25] Gan N, Nakayasu ES, Hollenbeck PJ, Luo ZQ. Legionella pneumophila inhibits immune signalling via MavC-mediated transglutaminase-induced ubiquitination of UBE2N. Nat Microbiol. 2019; 4: 134-143.

[26] Lorand L, Graham RM. Transglutaminases: crosslinking enzymes with pleiotropic functions. Nat Rev Mol Cell Biol. 2003; 4: 140-156.

[27] Nimchuk ZL, Fisher EJ, Desveaux D, Chang JH, Dangl JL. The HopX (AvrPphE) family of Pseudomonas syringae type III effectors require a catalytic triad and a novel N-terminal domain for function. Mol Plant Microbe Interact. 2007; 20: 346-357.

[28] Zhang L, Krachler AM, Broberg CA, Li Y, Mirzaei H, Gilpin CJ, et al. Type III effector VopC mediates invasion for Vibrio species. Cell Rep. 2012; 1: 453-460.

[29] Urbanus ML, Quaile AT, Stogios PJ, Morar M, Rao C, Di Leo R, et al. Diverse mechanisms of metaeffector activity in an intracellular bacterial pathogen, Legionella pneumophila. Mol Syst Biol. 2016; 12: 893.

[30] Rybalko VY, Pham CB, Hsieh PL, Hammers DW, Merscham-Banda M, Suggs LJ, et al. Controlled delivery of SDF-1α and IGF-1: CXCR4⁺ cell recruitment and functional skeletal muscle recovery. Biomater Sci. 2015; 11: 1475-1486.

[31] Huang T, Zhou Y, Zhang J, Cheng ASL, Yu J, To KF, et al. The physiological role of Motin family and its dysregulation in tumorigenesis. J Transl Med. 2018; 16: 98.

[32] Bratt A, Wilson WJ, Troyanovsky B, Aase K, Kessler R, Van Meir EGV, et al. Angiomotin belongs to a novel protein family with conserved coiled-coil and PDZ binding domains. Gene. 2002; 298: 69-77.

[33] Berger KH, Merriam JJ, Isberg RR. Altered intracellular targeting properties associated with mutations in the Legionella pneumophila dotA gene. Mol Microbiol. 1994; 14: 809-822.

[34] Berger KH, Isberg RR. Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila. Mol Microbiol. 1993; 7: 7-19.

[35] Zhao B, Li L, Lu Q, Wang LH, Liu CY, Lei Q, et al. Angiomotin is a novel Hippo pathway component that inhibits YAP oncoprotein. Genes Dev. 2011; 25: 51-63.

[36] McGough A, Pope B, Chiu W, Weeds A. Cofilin changes the twist of F-actin: implications for actin filament dynamics and cellular function. J Cell Biol. 1997; 138: 771-781.

[37] Schutt CE, Myslik JC, Rozycki MD, Goonesekere NCW, Lindberg U. The structure of crystalline profilin-β-actin. Nature. 1993; 365: 810-816.

[38] Ezezika OC, Younger NS, Lu J, Kaiser DA, Corbin ZA, Nolen BJ, et al. Incompatibility with formin Cdc12p prevents human profilin from substituting for fission yeast profilin. J Biol Chem. 2009; 284: 2088-2097.

[39] Pfaendtner J, De La Cruz EM, Voth GA. Actin filament remodeling by actin depolymerization factor/cofilin. Proc Natl Acad Sci USA. 2010; 107: 7299-7304.

[40] Revenu C, Athman R, Robine S, Louvard D. The co-workers of actin filaments: from cell structures to signals. Nat Rev Mol Cell Biol. 2004; 5: 635-646.

[41] Colonne PM, Winchell CG, Voth DE. Hijacking host cell highways: manipulation of the host actin cytoskeleton by obligate intracellular bacterial pathogens. Front Cell Infect Microbiol. 2016; 6: 107.

[42] Meconi S, Jacomo V, Boquet P, Raoult D, Mege JL, Capo C. Coxiella burnetii induces reorganization of the actin cytoskeleton in human monocytes. Infect Immun. 1998; 66: 5527-5533.

[43] Aguilera M, Salinas R, Rosales E, Carminati S, Colombo MI, Berón W. Actin dynamics and Rho GTPases regulate the size and formation of parasitophorous vacuoles containing Coxiella burnetii. Infect Immun. 2009; 77: 4609-4620.

[44] Campbell S, Richmond SJ, Yates PS. The effect of Chlamydia trachomatis infection on the host cell cytoskeleton and membrane compartments. J Gen Microbiol. 1989; 135: 2379-2386.

[45] Kumar Y, Valdivia RH. Actin and intermediate filaments stabilize the Chlamydia trachomatis vacuole by forming dynamic structural scaffolds. Cell Host Microbe. 2008; 4: 159-169.

[46] Gouin E, Egile C, Dehoux P, Villiers V, Adams J, Gertler F, et al. The RickA protein of Rickettsia conorii activates the Arp2/3 complex. Nature. 2004; 427: 457-461.

[47] Jeng RL, Goley ED, D'Alessio JA, Chaga OY, Svitkina TM, Borisy GG, et al. A Rickettsia WASP-like protein activates the Arp2/3 complex and mediates actin-based motility. Cell Microbiol. 2004; 6: 761-769.

[48] Oda T, Iwasa M, Aihara T, Maéda Y, Narita A. The nature of the globular- to fibrous-actin transition. Nature. 2009; 457: 441-445.

[49] Martinez-Quiles N, Rohatgi R, Antón IM, Medina M, Saville SP, Miki H, et al. WIP regulates N-WASP-mediated actin polymerization and filopodium formation. Nat Cell Biol. 2001; 3: 484-491.

[50] Kühn S, Mannherz HG. Actin: structure, function, dynamics, and interactions with bacterial toxins. Curr Top Microbiol Immunol. 2017; 399: 1-34.

[51] Horwitz MA. The Legionnaires’ disease bacterium (Legionella pneumophila) inhibits phagosome-lysosome fusion in human monocytes. J Exp Med. 1983; 158: 2108-2126.

[52] Derré I, Isberg RR. Legionella pneumophila replication vacuole formation involves rapid recruitment of proteins of the early secretory system. Infect Immun. 2004; 72: 3048-3053.

[53] Swanson MS, Isberg RR. Association of Legionella pneumophila with the macrophage endoplasmic reticulum. Infect Immun. 1995; 63: 3609-3620.

[54] Kagan JC, Roy CR. Legionella phagosomes intercept vesicular traffic from endoplasmic reticulum exit sites. Nat Cell Biol. 2002; 4: 945-954.

[55] Shin S, Roy CR. Host cell processes that influence the intracellular survival of Legionella pneumophila. Cell Microbiol. 2008; 10: 1209-1220.

[56] Heidtman M, Chen EJ, Moy MY, Isberg RR. Large-scale identification of Legionella pneumophila Dot/Icm substrates that modulate host cell vesicle trafficking pathways. Cell Microbiol. 2009; 11: 230-248.

[57] He C, Li C, Liu Y,Chen TT, Li C, Chu X, et al. Modulation of host ATP levels by secreted bacterial effectors. Nat Commun. 2025; 16: 4675.

[58] Iyer S, Das C. The unity of opposites: strategic interplay between bacterial effectors to regulate cellular homeostasis. J Biol Chem. 2021; 297: 101340.

[59] Luo ZQ, Isberg RR. Multiple substrates of the Legionella pneumophila Dot/Icm system identified by interbacterial protein transfer. ‘Proc Natl Acad Sci USA. 2004; 101: 841-846.

[60] Conover GM, Derré I, Vogel JP, Isberg RR. The Legionella pneumophila LidA protein: a translocated substrate of the Dot/Icm system associated with maintenance of bacterial integrity. Mol Microbiol. 2003; 48: 305-321.

[61] Luo ZQ, Farrand SK. Signal-dependent DNA binding and functional domains of the quorum-sensing activator TraR as identified by repressor activity. Proc Natl Acad Sci USA. 1999; 96: 9009-9014.

[62] McCloskey A, Perri K, Chen T, Han A, Luo ZQ. The metaeffector MesI regulates the activity of the Legionella effector SidI through direct protein-protein interactions. Microb Infect. 2021; 23: 104794.

[63] Xu L, Shen X, Bryan A, Banga S, Swanson MS, Luo ZQ. Inhibition of host vacuolar H⁺-ATPase activity by a Legionella pneumophila effector. PLoS Pathog. 2010; 6: e1000822.

[64] Sheedlo MJ, Qiu J, Tan Y, Paul LN, Luo ZQ, Das C. Structural basis of substrate recognition by a bacterial deubiquitinase important for dynamics of phagosome ubiquitination. Proc Natl Acad Sci USA. 2015; 112: 15090-15095.

[65] Wang SH, Wang A, Liu PP, Zhang WY, Du J, Xu S, et al. Divergent pathogenic properties of circulating coxsackievirus A6 associated with emerging hand, foot, and mouth disease. J Virol. 2018; 92: 10-1128.

[66] Hubber A, Arasaki K, Nakatsu F, Hardiman C, Lambright D, De Camilli P, et al. The machinery at endoplasmic reticulum-plasma membrane contact sites contributes to spatial regulation of multiple Legionella effector proteins. PLoS Pathog. 2014; 10: e1004222.

[67] Connelly KE, Hedrick V, Paschoal Sobreira TJ, Dykhuizen EC, Aryal UK. Analysis of human nuclear protein complexes by quantitative mass spectrometry profiling. Proteomics. 2018; 18: e1700427.

[68] Mohallem R, Aryal UK. Regulators of TNFα mediated insulin resistance elucidated by quantitative proteomics. Sci Rep. 2020; 10: 20878.

[69] Cox J, Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol. 2008; 26: 1367-1372.

RIGHTS & PERMISSIONS

2025 The Author(s). mLife published by John Wiley & Sons Australia, Ltd on behalf of Institute of Microbiology, Chinese Academy of Sciences.