Brett Geissler

Identification of a conserved membrane localization domain within numerous large bacterial protein toxins

Vibrio cholerae is the causative agent of the diarrheal disease cholera. Many virulence factors contribute to intestinal colonization and disease including the Multifunctional Autoprocessing RTX toxin (MARTXVc). The Rho-inactivation domain (RID) of MARTXVc is responsible for inactivating the Rho-family of small GTPases, which leads to depolymerization of the actin cytoskeleton. Based on a deletion analysis of RID to determine the minimal functional domain, we have identified a subdomain at the N terminus of RID that is homologous to the membrane targeting C1 domain of Pasteurella multocida toxin. A GFP fusion to this subdomain from RID colocalized with a plasma membrane marker when transiently expressed within HeLa cells and can be found in the membrane fraction following subcellular fractionation. This C1-like subdomain is present in multiple families of bacterial toxins, including all of the clostridial glucosyltransferase toxins and various MARTX toxins. GFP-fusions to these homologous domains are also membrane associated, indicating that this is a conserved membrane localization domain (MLD). We have identified three residues (Y23, S68, R70) as necessary for proper localization of one but not all MLDs. In addition, we found that substitution of the RID MLD with the MLDs from two different effector domains from the Vibrio vulnificus MARTX toxin restored RID activity, indicating that there is functional overlap between these MLDs. This study describes the initial recognition of a family of conserved plasma membrane-targeting domains found in multiple large bacterial toxins.

Plasma membrane association of three classes of bacterial toxins is mediated by a basic-hydrophobic motif

Plasma membrane targeting is essential for the proper function of many bacterial toxins. A conserved fourhelical bundle membrane localization domain (4HBM) was recently identified within three diverse families of toxins: clostridial glucosylating toxins, MARTX toxins and Pasteurella multocida‐like toxins. When expressed in tissue culture cells or in yeast, GFP fusions to at least one 4HBM from each toxin family show significant peripheral membrane localization but with differing profiles. Both in vivo expression and in vitro binding studies reveal that the ability of these domains to localize to the plasma membrane and bind negatively charged phospholipids requires a basic‐hydrophobic motif formed by the L1 and L3 loops. The different binding capacity of each 4HBM is defined by the hydrophobicity of an exposed residue within the motif. This study establishes that bacterial effectors utilize a normal host cell mechanism to locate the plasma membrane where they can then access their intracellular targets.

Identification of a His-Asp-Cys catalytic triad essential for function of the Rho-inactivation domain (RID) of Vibrio cholerae MARTX toxin

Background: The Vibrio cholerae MARTX toxin Rho inactivation domain (RIDVc) currently has an unknown mechanism of action. Results: Residues His-2782, Leu-2851, Asp-2854, and Cys-3022 are shown to be essential for RIDVc activity. Conclusion: His-Asp-Cys form a catalytic triad necessary for enzymatic modification of the cellular target. Significance: RID effectors are Clan CE cysteine endopeptidases essential for Rho GTPase inactivation. Vibrio cholerae is the causative agent of the severe diarrheal disease cholera. For V. cholerae to colonize the intestinal epithelium, accessory toxins such as the multifunctional autoprocessing repeats-in-toxin (MARTXVc) toxin are required. MARTX toxins are composite toxins comprised of arrayed effector domains that carry out distinct functions inside the host cell. Among the three effector domains of MARTXVc is the Rho inactivation domain (RIDVc) known to cause cell rounding through inactivation of small RhoGTPases. Using alanine scanning mutagenesis in the activity subdomain of RIDVc, four residues, His-2782, Leu-2851, Asp-2854, and Cys-3022, were identified as impacting RIDVc function in depolymerization of the actin cytoskeleton and inactivation of RhoA. Tyr-2807 and Tyr-3015 were identified as important potentially for forming the active structure for substrate contact but are not involved in catalysis or post translational modifications. Finally, V. cholerae strains modified to carry a catalytically inactive RIDVc show that the rate and efficiency of MARTXVc actin cross-linking activity does not depend on a functional RIDVc, demonstrating that these domains function independently in actin depolymerization. Overall, our results indicate a His-Asp-Cys catalytic triad is essential for function of the RID effector domain family shared by MARTX toxins produced by many Gram-negative bacteria.

Genetic determination of essential residues of the Vibrio cholerae actin cross‐linking domain reveals functional similarity with glutamine synthetases

Actin cross‐linking domains (ACDs) are distinct domains found in several bacterial toxins, including the Vibrio cholerae MARTX toxin. The ACD of V. cholerae (ACDVc) catalyses the formation of an irreversible iso‐peptide bond between lysine 50 and glutamic acid 270 on two actin molecules in an ATP‐ and Mg/Mn2+‐dependent manner. In vivo, cross‐linking depletes the cellular pool of G‐actin leading to actin cytoskeleton depolymerization. While the actin cross‐linking reaction performed by these effector domains has been significantly characterized, the ACDVc catalytic site has remained elusive due to lack of significant homology to known proteins. Using multiple genetic approaches, we have identified regions and amino acids of ACDVc required for full actin cross‐linking activity. Then, using these functional data and structural homology predictions, it was determined that several residues demonstrated to be important for ACDVc activity are conserved with active‐site residues of the glutamine synthetase family of enzymes. Thus, the ACDs are a family of bacterial toxin effectors that may be evolutionarily related to ligases involved in amino acid biosynthesis.

Large-scale Structural Rearrangement of a Serine Hydrolase from Francisella Tularensis Facilitates Catalysis

Background: Acyl protein thioesterases control protein S-acylation at cellular membranes. Results: FTT258 is a serine hydrolase with broad substrate specificity that binds to bacterial membranes and exists in two distinct conformations. Conclusion: Conformational changes in FTT258 are correlated with catalytic activity. Significance: Structural rearrangement dually regulates the membrane binding and catalytic activity of acyl protein thioesterases. Tularemia is a deadly, febrile disease caused by infection by the Gram-negative bacterium, Francisella tularensis. Members of the ubiquitous serine hydrolase protein family are among current targets to treat diverse bacterial infections. Herein we present a structural and functional study of a novel bacterial carboxylesterase (FTT258) from F. tularensis, a homologue of human acyl protein thioesterase (hAPT1). The structure of FTT258 has been determined in multiple forms, and unexpectedly large conformational changes of a peripheral flexible loop occur in the presence of a mechanistic cyclobutanone ligand. The concomitant changes in this hydrophobic loop and the newly exposed hydrophobic substrate binding pocket suggest that the observed structural changes are essential to the biological function and catalytic activity of FTT258. Using diverse substrate libraries, site-directed mutagenesis, and liposome binding assays, we determined the importance of these structural changes to the catalytic activity and membrane binding activity of FTT258. Residues within the newly exposed hydrophobic binding pocket and within the peripheral flexible loop proved essential to the hydrolytic activity of FTT258, indicating that structural rearrangement is required for catalytic activity. Both FTT258 and hAPT1 also showed significant association with liposomes designed to mimic bacterial or human membranes, respectively, even though similar structural rearrangements for hAPT1 have not been reported. The necessity for acyl protein thioesterases to have maximal catalytic activity near the membrane surface suggests that these conformational changes in the protein may dually regulate catalytic activity and membrane association in bacterial and human homologues.

A novel phosphatidylinositol 4,5-bisphosphate binding domain mediates plasma membrane localization of ExoU and other patatin-like phospholipases

Background: The Pseudomonas aeruginosa cytotoxin ExoU localizes to the plasma membrane in eukaryotic cells. Results: ExoU and related proteins utilize a conserved four-helical bundle to bind the lipid phosphatidylinositol 4,5-bisphosphate for localization. Conclusion: The membrane localization domain of ExoU represents a novel phosphoinositide binding domain. Significance: This is the first report of a four-helical bundle with specificity for phosphatidylinositol 4,5-bisphosphate. Bacterial toxins require localization to specific intracellular compartments following injection into host cells. In this study, we examined the membrane targeting of a broad family of bacterial proteins, the patatin-like phospholipases. The best characterized member of this family is ExoU, an effector of the Pseudomonas aeruginosa type III secretion system. Upon injection into host cells, ExoU localizes to the plasma membrane, where it uses its phospholipase A2 activity to lyse infected cells. The targeting mechanism of ExoU is poorly characterized, but it was recently found to bind to the phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a marker for the plasma membrane of eukaryotic cells. We confirmed that the membrane localization domain (MLD) of ExoU had a direct affinity for PI(4,5)P2, and we determined that this binding was required for ExoU localization. Previously uncharacterized ExoU homologs from Pseudomonas fluorescens and Photorhabdus asymbiotica also localized to the plasma membrane and required PI(4,5)P2 for this localization. A conserved arginine within the MLD was critical for interaction of each protein with PI(4,5)P2 and for localization. Furthermore, we determined the crystal structure of the full-length P. fluorescens ExoU and found that it was similar to that of P. aeruginosa ExoU. Each MLD contains a four-helical bundle, with the conserved arginine exposed at its cap to allow for interaction with the negatively charged PI(4,5)P2. Overall, these findings provide a structural explanation for the targeting of patatin-like phospholipases to the plasma membrane and define the MLD of ExoU as a member of a new class of PI(4,5)P2 binding domains.

Bacterial Toxin Effector-Membrane Targeting: Outside in, then Back Again

Pathogenic bacteria utilize multiple approaches to establish infection and mediate their toxicity to eukaryotic cells. Dedicated protein machines deposit toxic effectors directly inside the host, whereas secreted toxins must enter cells independently of other bacterial components. Regardless of how they reach the cytosol, these bacterial proteins must accurately identify their intracellular target before they can manipulate the host cell to benefit their associated bacteria. Within eukaryotic cells, post-translational modifications and individual targeting motifs spatially regulate endogenous host proteins. This review focuses on the strategies employed by bacterial effectors to associate with a frequently targeted location within eukaryotic cells, the plasma membrane.