Molecular Mechanism Of Rho GTPase Fatty Acylation By The MARTX Toxin Effector RID From Vibrio Cholerae

Multifunctional-autoprocessing repeats-in-toxin(MARTX)toxins are a family of large toxins extensively distributed in bacterial pathogens and playing important roles in bacterial pathogenesis.MARTX toxin is secreted by TISS,then insert into the host cell plasma membrane where they can be autocleaved by their conserved inositol hexaphosphate-activating cysteine protease domain to produce multiple effector domains into the cytosol of host cells.These effectors could modulate the host signaling pathways.MARTX is required for the virulence of Vibrio Cholerae.RID,a conserved effector of MARTX toxins,disrupts the host actin cytoskeleton with an unknown mechanism.In this study we revealed that RID specifically targets and inactivates the host Racl small GTPase through a posttranslational modification.The unknown modification of Racl by RID caused a downward shift of Racl on SDS-PAGE gels.And the RID-catalyzed modification disrupts the cycling of Racl between the cell membrane and cytosol.To uncover the enzymatic activity of RID,we carried out structural studies.After many attempts on RID proteins from different MARTX toxins,we successfully determined the crystal structure of full length RID from V,vulnificus MARTX toxin.Structural studies revealed that RID adopts a twisted "U'-shaped architecture with an N-terminal membrane localization domain which binds to PtdIns(4,5)P2.The C-terminal catalytic domain of RID has significant structural similarities with a human acyltransferase HRASLS3.Biochemical studies and mass spectrometry analysis revealed that RID fatty-acylates Rac1 on the C-terminal lysine residues in the polybasic region with acyl-CoAs containing long fatty chains as its ligands.The fatty-acylation of Racl by RID requires the prenylation of Rac1 on its C-terminus.RID also fatty-acylates other members of Rho family GTPases,such as RhoA and Cdc42,but has the substrate preference to Rac1.We also observed that the fatty acylation by RIDvc inhibited Rac1 activation by different types of guanine nucleotide exchange factors.RIDvc-modified Rac1 also could not activate the down stream kinase PAKl.During infection,RID inhibited Rho GTPase-mediated cellular processes including phagocytosis,reactive oxygen species production,and cell migration.Our results show that RID is a fatty acyltransferase that modifies the lysine residues in the C-terminal polybasic regions to inactivate Rho GTPases.The catalytic domain of RID shares sequence identity with the type ? effectors IcsB from Shigella species and BopA from Burkholderia species.Long-chain fatty acylation has been shown to occur on the lysine residues of some cytokines and histone proteins in mammalian cells.However,the enzyme for this modification has not been identified.Our study describes an enzyme that represents a family of bacterial toxins or effectors able to catalyze lysine fatty acylation on host proteins.