Intracellular localization of type III-delivered Pseudomonas ExoS with endosome vesicles

ExoS (453 amino acids) is a bi-functional type III cytotoxin produced by Pseudomonas aeruginosa. Residues 96-219 include the Rho GTPase-activating protein (RhoGAP) domain, and residues 234-453 include the 14-3-3-dependent ADP-ribosyltransferase domain. Earlier studies also identified an N-terminal domain (termed the membrane localization domain) that comprises residues 51-77 and includes a novel leucine-rich motif that targets ExoS to the perinuclear region of cultured cells. There is limited information on how ExoS or other type III cytotoxins enter and target intracellular host proteins. Type III-delivered ExoS localized to both plasma membrane and perinuclear region, whereas ExoS(DeltaMLD) was localized to the cytosol. Plasma membrane localization of ExoS was transient and had a half-life of approximately 20 min. Type III-delivered ExoS co-immunoprecipitated 14-3-3 proteins and Rab9, Rab6, and Rab5. Immunofluorescence experiments showed that ExoS colocalized with Rab9, Rab6, and Rab5. Fluorescent energy transfer was detected between ExoS and 14-3-3 proteins but not between ExoS and Rabs proteins. Together, these results indicate that type III-delivered ExoS localizes on the host endosomes and utilizes multiple pathways to traffic from the plasma membrane to the perinuclear region of intoxicated host cells.     

Ezrin/radixin/moesin proteins are high affinity targets for ADP-ribosylation by Pseudomonas aeruginosa ExoS

Pseudomonas aeruginosa ExoS is a bifunctional type III-secreted cytotoxin. The N terminus (amino acids 96-233) encodes a GTPase-activating protein activity, whereas the C terminus (amino acids 234-453) encodes a factor-activating ExoS-dependent ADP-ribosyltransferase activity. The GTPase-activating protein activity inactivates the Rho GTPases Rho, Rac, and Cdc42 in cultured cells and in vitro, whereas the ADP-ribosylation by ExoS is poly-substrate-specific and includes Ras as an early target for ADP-ribosylation. Infection of HeLa cells with P. aeruginosa producing a GTPase-activating protein-deficient form of ExoS rounded cells, indicating the ADP-ribosyltransferase domain alone is sufficient to elicit cytoskeletal changes. Examination of substrates modified by type III-delivered ExoS identified a 70-kDa protein as an early and predominant target for ADP-ribosylation. Matrix-assisted laser desorption ionization mass spectroscopy identified this protein as moesin, a member of the ezrin/radixin/moesin (ERM) family of proteins. ExoS ADP-ribosylated recombinant moesin at a linear velocity that was 5-fold faster and with a K(m) that was 2 orders of magnitude lower than Ras. Moesin homologs ezrin and radixin were also ADP-ribosylated, indicating the ERMs collectively represent high affinity targets of ExoS. Type III delivered ExoS ADP-ribosylated moesin and ezrin (and/or radixin) in cultured HeLa cells. The ERM proteins contribute to cytoskeleton dynamics, and the ability of ExoS to ADP-ribosylate the ERM proteins links ADP-ribosylation with the cytoskeletal changes associated with ExoS intoxication.     

Plasma membrane localization affects the RhoGAP specificity of Pseudomonas ExoS

Pseudomonas aeruginosa ExoS (453 amino acids) is a bifunctional type III cytotoxin, comprising a Rho GTPase-activating protein domain (RhoGAP), and a 14-3-3 dependent ADP-ribosyltransferase domain. In addition, ExoS contains a membrane localization domain (termed MLD, residues 51-77) which localizes and traffics ExoS within intoxicated host cells. While membrane localization has been shown to be essential for ExoS to ADP-ribosylate Ras, the relationship between intracellular localization and expression of RhoGAP activity has not been addressed. In this study, loss of MLD function was observed to abolish expression of ExoS RhoGAP activity in HeLa cells. One mutation within the MLD (R56, R63, D70 mutated to N, RRD-->N) diminished plasma membrane localization and altered the cell rounding phenotype elicited by ExoS RhoGAP. In addition, cell rounding caused by ExoS-MLD(RRD-->N) was reversed by dominant active Rac1, but not dominant active Cdc42, indicating a switch in ExoS RhoGAP substrate specificity. Mutation of the C-terminal polybasic region abolished the ability of dominant active Rac1 to protect HeLa cells from expression of the RhoGAP activity of ExoS-MLD(RRD-->N). This study shows the importance of membrane localization in the targeting of Rho GTPases by ExoS RhoGAP.     

The RhoGAP activity of the Yersinia pseudotuberculosis cytotoxin YopE is required for antiphagocytic function and virulence

A variety of pathogenic bacteria use type III secretion pathways to translocate virulence proteins into host eukaryotic cells. YopE is an important virulence factor that is translocated into mammalian cells via a plasmid-encoded type III system in Yersinia spp. YopE action in mammalian cells promotes the disruption of actin filaments, cell rounding and blockage of phagocytosis. It was reported recently that two proteins with sequence similarity to YopE, SptP of Salmonella typhimurium and ExoS of Pseudomonas aeruginosa, function as GTPase-activating proteins (GAPs) for Rho GTPases. YopE contains an 'arginine finger' motif that is present in SptP, ExoS and other Rho GAPs and is essential for catalysis by this class of proteins. We show here that a GST-YopE fusion protein stimulated in vitro GTP hydrolysis by the Rho family members Cdc42, RhoA and Rac1, but not by Ras. Conversion of the essential arginine in the arginine finger motif to alanine (R144A) eliminated the in vitro GAP activity of GST-YopE. Infection assays carried out with a Yersinia pseudotuberculosis strain producing YopER144A demonstrated that GAP function was essential for the disruption of actin filaments, cell rounding and inhibition of phagocytosis by YopE in HeLa cells. Furthermore, the GAP function of YopE was important for Y. pseudotuberculosis pathogenesis in a mouse infection assay. Transfection of HeLa cells with a vector that produces a constitutively active form of RhoA (RhoA-V14) prevented the disruption of actin filaments and cell rounding by YopE. Production of an activated form of Rac1 (Rac1-V12), but not RhoA-V14, in HeLa cells interfered with YopE antiphagocytic activity. These results demonstrate that YopE functions as a RhoGAP to downregulate multiple Rho GTPases, leading to the disruption of actin filaments and inhibition of bacterial uptake into host cells.     

How the Pseudomonas aeruginosa ExoS toxin downregulates Rac

Pseudomonas aeruginosa is an opportunistic bacterial pathogen. One of its major toxins, ExoS, is translocated into eukaryotic cells by a type III secretion pathway. ExoS is a dual function enzyme that affects two different Ras-related GTP binding proteins. The C-terminus inactivates Ras through ADP ribosylation, while the N-terminus inactivates Rho proteins through its GTPase activating protein (GAP) activity. Here we have determined the three-dimensional structure of a complex between Rac and the GAP domain of ExoS in the presence of GDP and AlF3. Composed of approximately 130 residues, this ExoS domain is the smallest GAP hitherto described. The GAP domain of ExoS is an all-helical protein with no obvious structural homology, and thus no recognizable evolutionary relationship, with the eukaryotic RhoGAP or RasGAP fold. Similar to other GAPs, ExoS downregulates Rac using an arginine finger to stabilize the transition state of the GTPase reaction, but the details of the ExoS-Rac interaction are unique. Considering the intrinsic resistance of P. aeruginosa to antibiotics, this might open up a new avenue towards blocking its pathogenicity.     

A nonphosphorylated 14-3-3 binding motif on exoenzyme S that is functional in vivo.

14-3-3 proteins play an important role in a multitude of signalling pathways. The interactions between 14-3-3 and other signalling proteins, such as Raf and KSR (kinase suppressor of Ras), occur in a phospho-specific manner. Recently, a phosphorylation-independent interaction has been reported to occur between 14-3-3 and several proteins, for example 5-phosphatase, p75NTR-associated cell death executor (NADE) and the bacterial toxin Exoenzyme S (ExoS), an ADP-ribosyltransferase from Pseudomonas aeruginosa. In this study we have identified the amino acid residues on ExoS, which are responsible for its specific interaction with 14-3-3. Furthermore, we show that a peptide derived from ExoS, containing the 14-3-3 interaction site, effectively competes out the interaction between ExoS and 14-3-3. In addition, competition with this peptide blocks ExoS modification of Ras in our Ras modification assay. We show that the ExoS protein interacts with all isoforms of the 14-3-3 family tested. Moreover, in vivo an ExoS protein lacking the 14-3-3 binding site has a reduced capacity to ADP ribosylate cytoplasmic proteins, e.g. Ras, and shows a reduced capacity to change the morphology of infected cells.     

Auto-ADP-ribosylation of Pseudomonas aeruginosa ExoS

Pseudomonas aeruginosa Exoenzyme S (ExoS) is a bifunctional type-III cytotoxin. The N terminus possesses a Rho GTPase-activating protein (GAP) activity, whereas the C terminus comprises an ADP-ribosyltransferase domain. We investigated whether the ADP-ribosyltransferase activity of ExoS influences its GAP activity. Although the ADP-ribosyltransferase activity of ExoS is dependent upon FAS, a 14-3-3 family protein, factor-activating ExoS (FAS) had no influence on the activity of the GAP domain of ExoS (ExoS-GAP). In the presence of NAD and FAS, the GAP activity of full-length ExoS was reduced about 10-fold, whereas NAD and FAS did not affect the activity of the ExoS-GAP fragment. Using [(32)P]NAD, ExoS-GAP was identified as a substrate of the ADP-ribosyltransferase activity of ExoS. Site-directed mutagenesis revealed that auto-ADP-ribosylation of Arg-146 of ExoS was crucial for inhibition of GAP activity in vitro. To reveal the auto-ADP-ribosylation of ExoS in intact cells, tetanolysin was used to produce pores in the plasma membrane of Chinese hamster ovary (CHO) cells to allow the intracellular entry of [(32)P]NAD, the substrate for ADP-ribosylation. After a 3-h infection of CHO cells with Pseudomonas aeruginosa, proteins of 50 and 25 kDa were preferentially ADP-ribosylated. The 50-kDa protein was determined to be auto-ADP-ribosylated ExoS, whereas the 25-kDa protein appeared to represent a group of proteins that included Ras.     

ADP-ribosylation of oncogenic Ras proteins by Pseudomonas aeruginosa exoenzyme S in vivo

The exoenzyme S (ExoS)-producing Pseudomonas aeruginosa strain, 388, and corresponding ExoS knock-out strain, 388Δ exoS , were used in a bacterial and mammalian co-culture system as a model for the contact-dependent delivery of ExoS into host cells. Examination of DNA synthesis and Ras ADP-ribosylation in tumour cell lines expressing normal and mutant Ras revealed a decrease in DNA synthesis concomitant with ADP-ribosylation of Ras proteins after exposure to ExoS-producing bacteria, but not after exposure to non-ExoS-producing bacteria. Examination of normal H-Ras, K-Ras and N-Ras by two-dimensional electrophoresis after exposure to bacteria revealed differences in the degree of ADP-ribosylation by ExoS, with H-Ras being modified most extensively. ADP-ribosylation of oncogenic forms of Ras was examined in vivo using cancer lines expressing mutant forms of H-, N- or K-Ras. The mutant Ras proteins were modified in a manner qualitatively similar to their normal counterparts. Using Ras/Raf-1 co-immunoprecipitation after co-culture, it was found that exposure to ExoS-producing bacteria caused a decrease in the amount of Raf-1 associated with EGF-activated Ras and oncogenic Ras. The results from this study indicate that ExoS ADP-ribosylates both normal and mutant Ras proteins in vivo and inhibits signalling through Ras.     

Modulation of host cell endocytosis by the type III cytotoxin, Pseudomonas ExoS

Pseudomonas aeruginosa ExoS is a bifunctional type III cytotoxin that possesses Rho GTPase-activating protein (RhoGAP) and ADP-ribosyltransferase (ADPr) activities. In the current study, the RhoGAP and ADPr activities of ExoS were tested for the ability to disrupt mammalian epithelial cell physiology. RhoGAP, but not ADPr, inhibited internalization/phagocytosis of bacteria, while ADPr, but not RhoGAP, inhibited vesicle trafficking, both general fluid-phase uptake and EGF-activated EGF receptor (EGFR) degradation. In ADPr-intoxicated cells, upon EGF activation, EGFR co-localized with clathrin-coated vesicles (CCV), which did not mature into Rab5-positive early endosomes. Constitutively, active Rab5 recruited EGFR from CCV to early endosomes. Consistent with the inhibition of Rab5 function by ADPr, several Rab proteins including Rab5 and 9, but not Rab4, were ADP ribosylated by ExoS. Thus, the two enzymatic activities of ExoS have different effects on epithelial cells with RhoGAP inhibiting bacterial internalization and ADPr interfering with CCV maturation. The ability ADPr to inhibit mammalian vesicle trafficking provides a new mechanism for bacterial toxin-mediated virulence.     

ADP-ribosylation and functional effects of Pseudomonas exoenzyme S on cellular RalA

Exoenzyme S (ExoS) is a bifunctional virulence factor directly translocated into eukaryotic cells by the type III secretory process of Pseudomonas aeruginosa. Bacterial translocation of ExoS into epithelial cells is associated with diverse effects on cell function, including inhibition of growth, alterations in cell morphology, and effects on adherence processes. Preferred substrates of the ADP-ribosyltransferase (ADPRT) portion of ExoS include low molecular weight G-proteins (LMWG-proteins) in the Ras family. In examining the ADP-ribosylation and functional effects of ExoS on RalA, ExoS was found to ADP-ribosylate endogenous RalA and recombinant RalADeltaCAAX at multiple sites, with Arg52 identified as the preferred site of ADP-ribosylation. The binding of RalA to the Ral binding domain (RBD) of its downstream effector, RalBP1, was inhibited by bacterially translocated ExoS, indicating an effect of ExoS on cellular RalA function. In vitro analyses confirmed that ADP-ribosylation of RalA directly interfered with its ability to bind to the RBD of RalBP1. The studies support the fact that RalA is a cellular substrate of bacterially translocated ExoS and that ADP-ribosylation by ExoS affects RalA interaction with its downstream effector, RalBP1.     

Ras (CXXX) and Rab (CC/CXC) prenylation signal sequences are unique and functionally distinct.

Rab proteins typically lack the consensus carboxyl-terminal CXXX motif that signals isoprenoid modification of Ras and other isoprenylated proteins and, instead, terminate in either CC or CXC sequences (C = cysteine, X = any amino acid). To compare the functional relationship between the Ras CXXX and the Rab CC/CXC motifs, we have generated chimeric Ras proteins terminating in Rab carboxyl-terminal CC or CXC sequences. These mutant Ras proteins were not isoprenylated in vitro or in vivo, demonstrating that the CC and CXC sequences alone are not sufficient to replace a CXXX sequence to signal Ras isoprenoid modification. Surprisingly, chimeric Ras/Rab proteins terminating in significant lengths of carboxyl-terminal sequences from Rab1b (7-139 residues), Rab2 (5-151 residues), or Rab3a (12 residues) were also not isoprenylated. These results demonstrate that the sequence requirements for isoprenoid modification of Rab proteins are more complex than the simple tetrapeptide CXXX sequence for isoprenoid modification of Ras proteins and suggest that the Rab geranylgeranyl transferase(s) requires recognition of protein conformation to signal the addition of geranylgeranyl groups. Finally, competition studies demonstrate that a common geranylgeranyl transferase activity is responsible for the modification of Rab proteins terminating in CC or CXC motifs.     

Exoenzyme S binds its cofactor 14-3-3 through a non-phosphorylated motif

14-3-3 proteins belong to a family of conserved molecules, which play a regulatory role and participate in signal transduction and checkpoint control pathways. 14-3-3 proteins bind phosphoserine-phosphorylated ligands, such as the Raf-1 kinase and Bad, through recognition of the phosphorylated consensus motif, RSXpSXP (where pS is phosphoserine). Recently, a phosphorylation-independent interaction has been reported to occur between 14-3-3 and a small number of proteins, for example the 43 kDa inositol polyphosphate 5-phosphatase, glycoprotein Ib, p75NTR-associated cell-death executor (NADE) and the bacterial ADP-ribosyltransferase toxin exoenzyme S (ExoS). It has been suggested that specific residues of 14-3-3 proteins are required for activation of the bacterial toxin ExoS. An unphosphorylated peptide derived from a phage display library, known as the R18 peptide, and a synthetic peptide derived from ExoS inhibit the interaction between ExoS and 14-3-3. In this report we identify the amino acid sequence on ExoS which is responsible for its specific interaction with 14-3-3, both in vitro and in vivo. In addition, we believe that this interaction is critical for the ADP-ribosylation of an endogenous target, Ras, by ExoS both in vitro and in vivo. Loss of the 14-3-3-binding site on ExoS results in an ExoS molecule that is unable to efficiently inactivate Ras and shows a reduced capacity to change the morphology of infected cells, together with reduced killing activity.     

The N-terminal domain of Pseudomonas aeruginosa exoenzyme S is a GTPase-activating protein for Rho GTPases

Pseudomonas aeruginosa exoenzyme S (ExoS) is a bifunctional cytotoxin. The ADP-ribosyltransferase domain is located within the C terminus part of ExoS. Recent studies showed that the N terminus part of ExoS (amino acid residues 1-234, ExoS(1-234)), which does not possess ADP-ribosyltransferase activity, stimulates cell rounding when transfected or microinjected into eukaryotic cells. Here we studied the effects of ExoS(1-234) on nucleotide binding and hydrolysis by Rho GTPases. ExoS(1-234) (100-500 nM) did not influence nucleotide exchange of Rho, Rac, and Cdc42 but increased GTP hydrolysis. A similar increase in GTPase activity was stimulated by full-length ExoS. Half-maximal stimulation of GTP hydrolysis by Rho, Rac, and Cdc42 was observed at 10-11 nM ExoS(1-234), respectively. We identified arginine 146 of ExoS to be essential for the stimulation of GTPase activity of Rho proteins. These data identify ExoS as a GTPase-activating protein for Rho GTPases.     

Intracellular trafficking of Pseudomonas ExoS, a type III cytotoxin

Pseudomonas aeruginosa ExoS is a bifunctional type III cytotoxin that disrupts Ras- and Rho-signaling pathways in mammalian cells. A hydrophobic region (residues 51-77, termed the membrane localization domain) targets ExoS to the plasma membrane (PM) and late endosomes of host cells. In the current study, metabolic inhibitors and dominant-negative proteins that disrupt known vesicle-trafficking pathways were used to define the intracellular trafficking of ExoS. Release of ExoS from PM was independent of dynamin and ADP ribosylation factor 6 but inhibited by methyl-beta-cyclodextrin, a cholesterol-depleting reagent, and perinuclear localization of ExoS was disrupted by nocodazole. p50 dynamitin, a dynein inhibitor partially disrupted perinuclear localization of ExoS. Methyl-beta-cyclodextrin and nocodazole inhibited the ability of type-III-delivered ExoS to ADP-ribosylated Golgi/endoplasmic reticulum-resident Ras. Methyl-beta-cyclodextrin also relocated ExoS from the perinuclear region to the PM, indicating that ExoS can cycle through anterograde as well as through retrograde trafficking pathways. These findings show that ExoS endocytosis is cholesterol dependent, and it utilizes host microtubules, for intracellular trafficking. Understanding how type III cytotoxins enter and traffic within mammalian cells may identify new targets for therapeutic intervention of gram-negative bacterial pathogens.     

Pseudomonas aeruginosa exoenzyme S, a double ADP-ribosyltransferase, resembles vertebrate mono-ADP-ribosyltransferases

Previous data indicated that Pseudomonas aeruginosa exoenzyme S (ExoS) ADP-ribosylated Ras at multiple sites. One site appeared to be Arg41, but the second site could not be localized. In this study, the sites of ADP-ribosylation of c-Ha-Ras by ExoS were directly determined. Under saturating conditions, ExoS ADP-ribosylated Ras to a stoichiometry of 2 mol of ADP-ribose incorporated per mol of Ras. Nucleotide occupancy did not influence the stoichiometry or velocity of ADP-ribosylation of Ras by ExoS. Edman degradation and mass spectrometry of V8 protease generated peptides of ADP-ribosylated Ras identified the sites of ADP-ribosylation to be Arg41 and Arg128. ExoS ADP-ribosylated the double mutant, RasR41K,R128K, to a stoichiometry of 1 mol of ADP-ribose incorporated per mol of Ras, which indicated that Ras possessed an alternative site of ADP-ribosylation. The alternative site of ADP-ribosylation on Ras was identified as Arg135, which was on the same alpha-helix as Arg128. Arg41 and Arg128 are located within two different secondary structure motifs, beta-sheet and alpha-helix, respectively, and are spatially separated within the three-dimensional structure of Ras. The fact that ExoS could ADP-ribosylate a target protein at multiple sites, along with earlier observations that ExoS could ADP-ribosylate numerous target proteins, were properties that have been attributed to several vertebrate ADP-ribosyltransferases. This prompted a detailed alignment study which showed that the catalytic domain of ExoS possessed considerably more primary amino acid homology with the vertebrate mono-ADP-ribosyltransferases than the bacterial ADP-ribosyltransferases. These data are consistent with the hypothesis that ExoS may represent an evolutionary link between bacterial and vertebrate mono-ADP-ribosyltransferases.     

Rab GTPases containing a CAAX motif are processed post-geranylgeranylation by proteolysis and methylation

Post-translational modification by protein prenylation is required for membrane targeting and biological function of monomeric GTPases. Ras and Rho proteins possess a C-terminal CAAX motif (C is cysteine, A is usually an aliphatic residue, and X is any amino acid), in which the cysteine is prenylated, followed by proteolytic cleavage of the AAX peptide and carboxyl methylation by the Rce1 CAAX protease and Icmt methyltransferase, respectively. Rab GTPases usually undergo double geranylgeranylation within CC or CXC motifs. However, very little is known about processing and membrane targeting of Rabs that naturally contain a CAAX motif. We show here that a variety of Rab-CAAX proteins undergo carboxyl methylation, both in vitro and in vivo, with one exception. Rab38(CAKS) is not methylated in vivo, presumably because of the inhibitory action of the lysine residue within the AAX motif for cleavage by Rce1. Unlike farnesylated Ras proteins, we observed no targeting defects of overexpressed Rab-CAAX proteins in cells deficient in Rce1 or Icmt, as reported for geranylgeranylated Rho proteins. However, endogenous geranylgeranylated non-methylated Rab-CAAX and Rab-CXC proteins were significantly redistributed to the cytosol at steady-state levels and redistribution correlates with higher affinity of RabGDI for non-methylated Rabs in Icmt-deficient cells. Our data suggest a role for methylation in Rab function by regulating the cycle of Rab membrane recruitment and retrieval. Our findings also imply that those Rabs that undergo post-prenylation processing follow an indirect targeting pathway requiring initial endoplasmic reticulum membrane association prior to specific organelle targeting.     

Intracellular localization modulates targeting of ExoS,a type III cytotoxin,to eukaryotic signalling proteins

ExoS is a bifunctional type III cytotoxin produced by Pseudomonas aeruginosa. Residues 96-232 comprise the Rho GTPase activating protein (Rho GAP) domain, whereas residues 233-453 comprise the 14-3-3-dependent ADP-ribosyltransferase domain. Earlier studies showed that the N-terminus targeted ExoS to intracellular membranes within eukaryotic cells. This N-terminal targeting region is now characterized for cellular and biological contributions to intoxications by ExoS. An ExoS(1-107)-green fluorescent protein (GFP) fusion protein co-localized with alpha-mannosidase, which indicated that the fusion protein localized near the Golgi. Residues 51-72 of ExoS (termed the membrane localization domain, MLD) were necessary and sufficient for membrane localization within eukaryotic cells. Deletion of the MLD did not inhibit type III secretion of ExoS from P. aeruginosa or type III delivery of ExoS into eukaryotic cells. Type III-delivered ExoS(DeltaMLD) localized within the cytosol of eukaryotic cells, whereas type III-delivered ExoS was membrane associated. Although type III-delivered ExoS(DeltaMLD) stimulated the reorganization of the actin cytoskeleton (a Rho GAP activity), it did not ADP-ribosylate Ras. Type III-delivered ExoS(DeltaMLD) and ExoS showed similar capacities for eliciting a cytotoxic response in CHO cells, which uncoupled the ADP-ribosylation of Ras from the cytotoxicity elicited by ExoS.     

ADP-ribosylation of cyclophilin A by Pseudomonas aeruginosa exoenzyme S

The virulence of the opportunistic pathogen Pseudomonas aeruginosa (Pa) is in part mediated by the type III secretion (TTS) of bacterial proteins into eukaryotic hosts. Exoenzyme S (ExoS) is a bifunctional Pa TTS effector protein, with GTPase-activating (GAP) and ADP-ribosyltransferase (ADPRT) activities. Known cellular substrates of TTS-translocated ExoS (TTS-ExoS) ADPRT activity include proteins in the Ras superfamily and ERM family proteins. This study describes the ADP-ribosylation of a non-G-protein substrate of TTS-ExoS, cyclophilin A (CpA), a peptidyl-prolyl isomerase (PPIase). Four novel 17 kDa proteins (pI 6.5-6.8) were recognized in a proteomic screen of lysates of human epithelial cells that had been exposed to ExoS-producing Pa, but not an isogenic non-ExoS producing strain. The proteins were identified as isoforms of CpA using MALDI-TOF mass spectrometry and confirmed by Western blotting. Mutagenesis analysis identified arginine 55 and 69 of CpA as sites of ExoS ADP-ribosylation. Examination of the effect of ExoS ADP-ribosylation on CpA function found a moderate (19%) decrease in prolyl isomerization of a Xaa-Pro containing peptides. In comparison, GST-CpA co-immunoprecipitation studies found ExoS ADP-ribosylation of CpA to efficiently inhibit CpA binding to calcineurin/PP2B phosphatase. Our results support that ExoS ADP-ribosylates and affects the function of the cytosolic protein, CpA, with the predominant functional effect relating to interference of CpA-cellular protein interactions.     

The Legionella pneumophila effector protein DrrA is a Rab1 guanine nucleotide-exchange factor

The intracellular pathogen Legionella pneumophila avoids fusion with lysosomes and subverts membrane transport from the endoplasmic reticulum to create an organelle that supports bacterial replication. Transport of endoplasmic reticulum-derived vesicles to the Legionella-containing vacuole (LCV) requires bacterial proteins that are translocated into host cells by a type IV secretion apparatus called Dot/Icm. Recent observations have revealed recruitment of the host GTPase Rab1 to the LCV by a process requiring the Dot/Icm system. Here, a visual screen was used to identify L. pneumophila mutants with defects in Rab1 recruitment. One of the factors identified in this screen was DrrA, a new Dot/Icm substrate protein translocated into host cells. We show that DrrA is a potent and highly specific Rab1 guanine nucleotide-exchange factor (GEF). DrrA can disrupt Rab1-mediated secretory transport to the Golgi apparatus by competing with endogenous exchange factors to recruit and activate Rab1 on plasma membrane-derived organelles. These data establish that intracellular pathogens have the capacity to directly modulate the activation state of a specific member of the Rab family of GTPases and thus further our understanding of the mechanisms used by bacterial pathogens to manipulate host vesicular transport.     

Molecular heterogeneity of a type III cytotoxin, Pseudomonas aeruginosa exoenzyme S

Pseudomonas aeruginosa ExoS is a bifunctional type III cytotoxin. The N-terminus (residues 1-232) is a Rho GTPase activating protein (GAP) domain, while the C-terminus (residues 233-453) is a FAS-dependent ADP-ribosyltransferase domain that targets Ras and Ras-like GTPases. A membrane localization domain (residues 51-72) localizes ExoS to a perinuclear region within eukaryotic cells. Recent studies observed that ExoS is auto-ADP-ribosylated upon delivery into eukaryotic cells. Auto-ADP-ribosylated ExoS analyzed from eukaryotic cells displayed pI heterogeneity and prompted an analysis of this heterogeneity. Bacterial-associated ExoS and ExoS that had been secreted by P. aeruginosa also showed pI heterogeneity with five charge forms ranging in pI from 5.1 to 5.9. The pI heterogeneity of ExoS was independent of a mass change and thus represented molecular charge conformers. Urea was not required to observe the pI conformers of ExoS; it enhanced the resolution and formation of pI conformers during the focusing component of the analysis. ExoS(E381D), a mutant deficient in ADP-ribosyltransferase activity, isolated from cultured cells showed charge forms that migrated to a more acidic pI than type III secreted ExoS but more basic than auto-ADP-ribosylated ExoS. Incubation of cell lysates with Mn(2+) shifted the pI of ExoS(E381D) to a pI identical to secreted ExoS. This indicates that within the mammalian cells ExoS undergoes a negatively charged modification, in addition to auto-ADP-ribosylation observed for wild-type ExoS. ExoT, ExoU, and YopE also focus into multiple pI forms, suggesting that this is a common property of type III cytotoxins.