Acute myocardial ischemia associated with ingestion of bupropion and pseudoephedrine in a 21-year-old man.

A 21-year-old man presented to the emergency department with atypical chest pain, diaphoresis and shortness of breath. His electrocardiogram revealed ST segment elevation in leads II, III, aVF, V5 and V6, elevated creatine kinase-MB subunit levels and positive troponin I. He denied the use of cocaine, and smoking was his only risk factor for coronary artery disease. The patient was diagnosed with an acute myocardial infarction, yet an emergency coronary angiogram revealed normal coronary arteries. His medication history revealed recent commencement of bupropion for smoking cessation and pseudoephedrine as a nonprescription influenza remedy. It was postulated that this patient experienced acute coronary vasospasm in the presence of these two known sympathomimetic agents. The present case is the first report linking bupropion to an acute coronary syndrome, and one of a few cases associated with pseudoephedrine.

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 chromosomally encoded type III secretion pathway in Yersinia enterocolitica is important in virulence.

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 degrees 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 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.

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.

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 (DeltaC234). Intracellular expression of DeltaC234 elicited the rounding of Chinese hamster ovary (CHO) cells and the disruption of actin filaments in a dose-dependent manner. Expression of DeltaC234 did not inhibit the expression of two independent reporter proteins, GFP and luciferase, or induce trypan blue uptake, which indicated that expression of DeltaC234 was not cytotoxic to CHO cells. Carboxyl-terminal deletion proteins of DeltaC234 were less efficient in the elicitation of CHO cell rounding than DeltaC234. Cytoskeleton rearrangement elicited by DeltaC234 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 DeltaC234. 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.

Interaction of 14-3-3 with a nonphosphorylated protein ligand, exoenzyme S of Pseudomonas aeruginosa.

The 14-3-3 proteins are a family of conserved, dimeric proteins that interact with a diverse set of ligands, including molecules involved in cell cycle regulation and apoptosis. It is well-established that 14-3-3 binds to many ligands through phosphoserine motifs. Here we characterize the interaction of 14-3-3 with a nonphosphorylated protein ligand, the ADP-ribosyltransferase Exoenzyme S (ExoS) from Pseudomonas aeruginosa. By using affinity chromatography and surface plasmon resonance, we show that the zeta isoform of 14-3-3 (14-3-3zeta) can directly bind a catalytically active fragment of ExoS in vitro. The interaction between ExoS and 14-3-3zeta is of high affinity, with an equilibrium dissociation constant of 7 nM. ExoS lacks any known 14-3-3 binding motif, but to address the possibility that 14-3-3 binds a noncanonical phosphoserine site, we assayed ExoS for protein-bound phosphate by using mass spectrometry. No detectable phosphoproteins were found. A phosphopeptide ligand of 14-3-3, pS-Raf-259, was capable of inhibiting the binding of 14-3-3 to ExoS, suggesting that phosphorylated and nonphosphorylated ligands may share a common binding site, the conserved amphipathic groove. It is conceivable that 14-3-3 proteins may bind both phosphoserine and nonphosphoserine ligands in cells, possibly allowing kinase-dependent as well as kinase-independent regulation of 14-3-3 binding.

Pseudomonas aeruginosa exoenzyme S is a biglutamic acid ADP-ribosyltransferase.

Kinetic analysis of two mutations within Pseudomonas aeruginosa exoenzyme S (ExoS) showed that a E379D mutation inhibited expression of ADP-ribosyltransferase activity but had little effect on the expression of NAD glycohydrolase activity while a E381D mutation inhibited expression of both activities. These data identify ExoS as a biglutamic acid ADP-ribosyltransferase, where E381 is the catalytic residue and E379 contributes to the transfer of ADP-ribose to the target protein.

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 deltaN222), and a point mutant, deltaN222-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 deltaN222 expressed ADP-ribosyltransferase activity. Co-transfection of deltaN222, but not deltaN222-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 deltaN222 resulted in a greater percentage of cells staining with trypan blue than when cells were transfected with either deltaN222-E381A or control plasmid. Together, these data indicate that expression of the ADP-ribosyltransferase domain of exoenzyme S is cytotoxic to eukaryotic cells.

The ClpP protein, a subunit of the Clp protease, modulates ail gene expression in Yersinia enterocolitica.

Yersinia enterocolitica is a gastrointestinal pathogen of humans and animals. Ail is a 17kDa cell-surface protein that confers on Y. enterocolitica resistance to serum killing and the ability to attach to and invade cells in vitro. The ail gene of Y. enterocolitica is regulated by temperature and growth phase. In stationary phase, ail transcript is only detected when bacteria are grown at the host temperature of 37 degrees C. Our laboratory previously described a group of mini-Tn10 mutants, which expressed ail in stationary phase at 28 degrees C. In one of these mutants, DP5102::mini-Tn10 3-2, the mini-Tn 10 inserted into a gene encoding a protein with 90.3% identity to the ClpP protease subunit from Escherichia coli. Expression of ail in stationary phase at 28 degrees C was also derepressed in a directed Y. enterocolitica clpP mutant. Analysis of ail transcripts in the wild-type and clpP mutant strains indicated that there is a single start site of transcription of ail and that the effect of the clpP mutation was on the initiation of transcription at this site. Similar to E. coli, a clpX homologue was identified downstream of clpP. The Y. enterocolitica clpP gene complemented the clpP mutant phenotype, repressing the expression of both ail transcript levels and cell surface-expressed Ail protein. Thus, ClpP has a role in the modulation of ail transcription in Y. enterocolitica.

Ail expression in Yersinia enterocolitica is affected by oxygen tension.

We investigated several environmental factors for their abilities to regulate ail gene expression and found that ail transcript levels are regulated by oxygen tension. Bacteria growing under anaerobiosis at 37 degrees C repress ail mRNA and Ail expression, resulting in a loss of Ail-mediated serum resistance and cell invasion.