Structurally Distinct Bacterial TBC-like GAPs Link Arf GTPase to Rab1 Inactivation to Counteract Host Defenses

Rab GTPases are frequent targets of vacuole-living bacterial pathogens for appropriate trafficking of the vacuole. Here we discover that bacterial effectors including VirA from nonvacuole Shigella flexneri and EspG from extracellular Enteropathogenic Escherichia coli (EPEC) harbor TBC-like dual-finger motifs and exhibits potent RabGAP activities. Specific inactivation of Rab1 by VirA/EspG disrupts ER-to-Golgi trafficking. S. flexneri intracellular persistence requires VirA TBC-like GAP activity that mediates bacterial escape from autophagy-mediated host defense. Rab1 inactivation by EspG severely blocks host secretory pathway, resulting in inhibited interleukin-8 secretion from infected cells. Crystal structures of VirA/EspG-Rab1-GDP-aluminum fluoride complexes highlight TBC-like catalytic role for the arginine and glutamine finger residues and reveal a 3D architecture distinct from that of the TBC domain. Structure of Arf6-EspG-Rab1 ternary complex illustrates a pathogenic signaling complex that rewires host Arf signaling to Rab1 inactivation. Structural distinctions of VirA/EspG further predict a possible extensive presence of TBC-like RabGAP effectors in counteracting various host defenses.

[1]  J. Kaper,et al.  Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector , 1991, Infection and immunity.

[2]  Steven M. Gallo,et al.  SnB: crystal structure determination via shake-and-bake , 1994 .

[3]  D. Lambright,et al.  TBC-domain GAPs for Rab GTPases accelerate GTP hydrolysis by a dual-finger mechanism , 2006, Nature.

[4]  C. Sasakawa,et al.  Shigella deploy multiple countermeasures against host innate immune responses. , 2011, Current opinion in microbiology.

[5]  R. Goody,et al.  Crystal structure of the GAP domain of Gyp1p: first insights into interaction with Ypt/Rab proteins , 2000, The EMBO journal.

[6]  R. Kramer,et al.  A Functional Genomic Yeast Screen to Identify Pathogenic Bacterial Proteins , 2008, PLoS pathogens.

[7]  F. Shao,et al.  A Bacterial Effector Targets Host Dh-ph Domain Rhogefs and Antagonizes Macrophage Phagocytosis , 2022 .

[8]  S. Pfeffer,et al.  Targeting Rab GTPases to distinct membrane compartments , 2004, Nature Reviews Molecular Cell Biology.

[9]  R. Orchard,et al.  The assembly of a GTPase–kinase signalling complex by a bacterial catalytic scaffold , 2011, Nature.

[10]  R. Scheller,et al.  Rab37 is a novel mast cell specific GTPase localized to secretory granules , 2000, FEBS letters.

[11]  D. Gallwitz,et al.  A yeast GTPase-activating protein that interacts specifically with a member of the Ypt/Rab family , 1993, Nature.

[12]  M. Colombo,et al.  Autophagosome Formation Depends on the Small GTPase Rab1 and Functional ER Exit Sites , 2010, Traffic.

[13]  W. Balch,et al.  Rab1 Interaction with a GM130 Effector Complex Regulates COPII Vesicle cis‐Golgi Tethering , 2001, Traffic.

[14]  L Orci,et al.  Regulation of protein secretion through controlled aggregation in the endoplasmic reticulum. , 2000, Science.

[15]  S. Pfeffer,et al.  Defining the boundaries: Rab GEFs and GAPs , 2009, Proceedings of the National Academy of Sciences.

[16]  Yawei Li,et al.  Isoform-selective effects of the depletion of ADP-ribosylation factors 1-5 on membrane traffic. , 2005, Molecular biology of the cell.

[17]  S. Pfeffer.  Unsolved mysteries in membrane traffic. , 2007, Annual review of biochemistry.

[18]  Abigail Clements,et al.  EspG of enteropathogenic and enterohemorrhagic E. coli binds the Golgi matrix protein GM130 and disrupts the Golgi structure and function , 2011, Cellular Microbiology.

[19]  P. De Camilli,et al.  The association of epsin with ubiquitinated cargo along the endocytic pathway is negatively regulated by its interaction with clathrin. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[20]  P. Novick,et al.  A Rab GAP cascade defines the boundary between two Rab GTPases on the secretory pathway , 2009, Proceedings of the National Academy of Sciences.

[21]  D. Lambright,et al.  Legionella pneumophila proteins that regulate Rab1 membrane cycling , 2007, Nature.

[22]  D. Gallwitz,et al.  Identification of the catalytic domains and their functionally critical arginine residues of two yeast GTPase‐activating proteins specific for Ypt/Rab transport GTPases , 1999, The EMBO journal.

[23]  Hiroshi Sagara,et al.  Escape of Intracellular Shigella from Autophagy , 2005, Science.

[24]  J. Schmitzová,et al.  Significance of GTP Hydrolysis in Ypt1p-regulated Endoplasmic Reticulum to Golgi Transport Revealed by the Analysis of Two Novel Ypt1-GAPs* , 2002, The Journal of Biological Chemistry.

[25]  B. Spiller,et al.  Structural and functional studies indicate that the EPEC effector, EspG, directly binds p21-activated kinase. , 2011, Biochemistry.

[26]  P. Novick,et al.  Role of Rab GTPases in membrane traffic and cell physiology. , 2011, Physiological reviews.

[27]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[28]  B. Finlay,et al.  Modulation of Host Cytoskeleton Function by the Enteropathogenic Escherichia coli and Citrobacter rodentium Effector Protein EspG , 2005, Infection and Immunity.

[29]  H. Stenmark.  Rab GTPases as coordinators of vesicle traffic , 2009, Nature Reviews Molecular Cell Biology.

[30]  M. Scidmore,et al.  Manipulation of Rab GTPase Function by Intracellular Bacterial Pathogens , 2007, Microbiology and Molecular Biology Reviews.

[31]  D. Lambright,et al.  Rab GEFs and GAPs. , 2010, Current opinion in cell biology.

[32]  Gordon W. Lathrop.  The Assembly , 1941 .

[33]  D. Klionsky,et al.  Antibacterial autophagy occurs at PI(3)P-enriched domains of the endoplasmic reticulum and requires Rab1 GTPase , 2011, Autophagy.

[34]  M. Watarai,et al.  Identification of a novel virulence gene, virA, on the large plasmid of Shigella, involved in invasion and intercellular spreading , 1995, Molecular microbiology.

[35]  C. Sasakawa,et al.  EspG, a Novel Type III System-Secreted Protein from Enteropathogenic Escherichia coli with Similarities to VirA of Shigella flexneri , 2001, Infection and Immunity.

[36]  M. Goldberg,et al.  Structural and functional studies indicate that Shigella VirA is not a protease and does not directly destabilize microtubules. , 2008, Biochemistry.

[37]  Katrin Rittinger,et al.  Structure at 1.65 Å of RhoA and its GTPase-activating protein in complex with a transition-state analogue , 1997, Nature.

[38]  A. Barnekow,et al.  The Golgi matrix protein GM130: a specific interacting partner of the small GTPase rab1b , 2001, EMBO reports.

[39]  J. Dixon,et al.  A Yersinia Effector and a Pseudomonas Avirulence Protein Define a Family of Cysteine Proteases Functioning in Bacterial Pathogenesis , 2002, Cell.

[40]  W. Kabsch,et al.  The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants. , 1997, Science.

[41]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[42]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[43]  D. Holden,et al.  Bacterial interactions with the eukaryotic secretory pathway. , 2005, Current opinion in microbiology.

[44]  A. Wlodawer,et al.  Novel fold of VirA, a type III secretion system effector protein from Shigella flexneri , 2008, Protein science : a publication of the Protein Society.

[45]  C. Sasakawa,et al.  Microtubule-Severing Activity of Shigella Is Pivotal for Intercellular Spreading , 2006, Science.

[46]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.