Kristin J. Pederson

The N-terminal Domain of Pseudomonas aeruginosaExoenzyme 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.

The amino-terminal domain of Pseudomonas aeruginosa ExoS disrupts actin filaments via small-molecular-weight GTP-binding proteins.

Pseudomonas aeruginosa delivers exoenzyme S (ExoS) into the intracellular compartment of eukaryotic cells via a type III secretion pathway. Intracellular delivery of ExoS is cytotoxic for eukaryotic cells and has been shown to ADP‐ribosylate Ras in vivo and uncouple a Ras‐mediated signal transduction pathway. Functional mapping has localized the FAS‐dependent ADP‐ribosyltransferase domain to the carboxyl‐terminus of ExoS. A transient transfection system was used to examine cellular responses to the amino‐terminal 234 amino acids of ExoS (ΔC234). Intracellular expression of ΔC234 elicited the rounding of Chinese hamster ovary (CHO) cells and the disruption of actin filaments in a dose‐dependent manner. Expression of ΔC234 did not inhibit the expression of two independent reporter proteins, GFP and luciferase, or induce trypan blue uptake, which indicated that expression of ΔC234 was not cytotoxic to CHO cells. Carboxyl‐terminal deletion proteins of ΔC234 were less efficient in the elicitation of CHO cell rounding than ΔC234. Cytoskeleton rearrangement elicited by ΔC234 was blocked and reversed by the addition of cytotoxic necrotizing factor 1 (CNF‐1). CNF‐1 catalyses the deamidation of Gln‐63 of members of the Rho subfamily of small‐molecular‐weight GTP‐binding proteins, resulting in protein activation. This implies a role for small‐molecular‐weight GTP‐binding proteins in the disruption of actin by ΔC234. Together, these data identify ExoS as a cytotoxin that possesses two functional domains. Intracellular expression of the amino‐terminal domain of ExoS elicits the disruption of actin, while expression of the carboxyl‐terminal domain of ExoS possesses FAS‐dependent ADP‐ribosyltransferase activity and is cytotoxic to eukaryotic 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 ∼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.

In Vivo Rho GTPase-Activating Protein Activity of Pseudomonas aeruginosa Cytotoxin ExoS

ABSTRACT ExoS is a bifunctional type III cytotoxin secreted by Pseudomonas aeruginosa, which comprises a C-terminal ADP ribosyltransferase domain and an N-terminal Rho GTPase-activating protein (GAP) domain. In vitro, ExoS is a Rho GAP for Rho, Rac, and Cdc42; however, the in vivo modulation of Rho GTPases has not been addressed. Using a transient transfection system and delivery by P. aeruginosa, interactions were examined between the Rho GAP domain of ExoS and Rho GTPases in CHO cells. Rho GTPases were expressed as green fluorescent protein (GFP) fusion proteins to facilitate quantitation. GFP fusions of wild-type and dominant active Rho, Rac, and Cdc42 localized to discrete regions of CHO cells and appeared functional based upon their modulation of the actin cytoskeleton. Coexpression of the Rho GAP domain of ExoS changed the intracellular distribution of GFP-Rac and GFP-Cdc42 from a predominately membrane location to a cytosolic location. Coexpression of the Rho GAP domain of ExoS did not change the distribution of GFP-Rho, which was primarily in the cytosol. Coexpression of dominant active Rac (DARac) and DACdc42 inhibited actin reorganization by the Rho GAP domain but did not maintain the formation of actin stress fibers, which indicated that Rho had been inactivated. Similar results were observed when ExoS was delivered into CHO cells by P. aeruginosa. These data indicate that in vivo the Rho GAP activity of ExoS stimulates the reorganization of the actin cytoskeleton by inhibition of Rac and Cdc42 and stimulates actin stress fiber formation by inhibition of Rho.

A chromosomally encoded type III secretion pathway in Yersinia enterocolitica is important in virulence: Yersinia enterocolitica chromosomal type III secretion

Numerous Gram‐negative bacteria use a type III, or contact dependent, secretion system to deliver proteins into the cytosol of host cells. All of these systems identified to date have been shown to have a role in pathogenesis. We have identified 13 genes on the Yersinia enterocolitica chromosome that encode a type III secretion apparatus plus two associated putative regulatory genes. In order to determine the function of this chromosomally‐encoded secretion apparatus, we created an in frame deletion of a gene that has homology to the hypothesized inner membrane pore, ysaV. The ysaV mutant strain failed to secrete eight proteins, called Ysps, normally secreted by the parental strain when grown at 28°C in Luria–Bertani (LB) broth supplemented with 0.4 M NaCl. Disruption of the ysaV gene had no effect on motility or phospholipase activity, suggesting this chromosomally encoded type III secretion pathway is distinct from the flagella secretion pathway of Y. enterocolitica. Deletion of the ysaV gene in a virulence plasmid positive strain had no effect on in vitro secretion of Yops by the plasmid‐encoded type III secretion apparatus. Secretion of the Ysps was unaffected by the presence or absence of the virulence plasmid, suggesting the chromosomally encoded and plasmid‐encoded type III secretion pathways act independently. Y. enterocolitica thus has three type III secretion pathways that appear to act independently. The ysaV mutant strain was somewhat attenuated in virulence compared with the wild type in the mouse oral model of infection (an approximately 0.9 log difference in LD50). The ysaV mutant strain was nearly as virulent as the wild type when inoculated intraperitoneally in the mouse model. A ysaV probe hybridized to sequences in other Yersinia spp. and homologues were found in the incomplete Y. pestis genome sequence, indicating a possible role for this system throughout the genus.

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 α‐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(ΔMLD) localized within the cytosol of eukaryotic cells, whereas type III‐delivered ExoS was membrane associated. Although type III‐delivered ExoS(ΔMLD) stimulated the reorganization of the actin cytoskeleton (a Rho GAP activity), it did not ADP‐ribosylate Ras. Type III‐delivered ExoS(ΔMLD) 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.

Intracellular expression of the ADP-ribosyltransferase domain of Pseudomonas exoenzyme S is cytotoxic to eukaryotic cells

Exoenzyme S of Pseudomonas aeruginosa is an ADP‐ribosyltransferase, which is secreted via a type III‐dependent secretion mechanism and has been demonstrated to exert cytotoxic effects on eukaryotic cells. Alignment studies predict that the amino‐terminus of exoenzyme S has limited primary amino acid homology with the YopE cytotoxin of Yersinia, while biochemical studies have localized the FAS‐dependent ADP‐ribosyltransferase activity to the carboxyl‐terminus. Thus, exoenzyme S could interfere with host cell physiology via several independent mechanisms. The goal of this study was to define the role of the ADP‐ribosyltransferase domain in the modulation of eukaryotic cell physiology. The carboxyl‐terminal 222 amino acids of exoenzyme S, which represent the FAS‐dependent ADP‐ribosyltransferase domain (termed ΔN222), and a point mutant, ΔN222‐E381A, which possesses a 2000‐fold reduction in the capacity to ADP‐ribosylate, were transiently expressed in eukaryotic cells under the control of the immediate early CMV promoter. Lysates from cells transfected with ΔN222 expressed ADP‐ribosyltransferase activity. Co‐transfection of ΔN222, but not ΔN222‐E381A, resulted in a decrease in the steady‐state levels of two reporter proteins, green fluorescent protein and luciferase, in both CHO and Vero cells. In addition, transfection with ΔN222 resulted in a greater percentage of cells staining with trypan blue than when cells were transfected with either ΔN222‐E381A or control plasmid. Together, these data indicate that expression of the ADP‐ribosyltransferase domain of exoenzyme S is cytotoxic to eukaryotic cells.

Intracellular localization and processing of Pseudomonas aeruginosa ExoS in eukaryotic cells

ExoS is a type III cytotoxin of Pseudomonas aeruginosa, which modulates two eukaryotic signalling pathways. The N‐terminus (residues 1–234) is a GTPase activating protein (GAP) for RhoGTPases, while the C‐terminus (residues 232–453) encodes an ADP‐ribosyltransferase. Utilizing a series of N‐terminal deletion peptides of ExoS and an epitope‐tagged full‐length ExoS, two independent domains have been identified within the N‐terminus of ExoS that are involved in intracellular localization and expression of GAP activity. N‐terminal peptides of ExoS localized to the perinuclear region of CHO cells, and a membrane localization domain was localized between residues 36 and 78 of ExoS. The capacity to elicit CHO cell rounding and express GAP activity resided within residues 90–234 of ExoS, which showed that membrane localization was not required to elicit actin reorganization. ExoS was present in CHO cells as a full‐length form, which fractionated with membranes, and as an N‐terminally processed fragment, which localized to the cytosol. Thus, ExoS localizes in eukaryotic cells to the perinuclear region and is processed to a soluble fragment, which possesses both the GAP and ADP‐ribosyltransferase activities.