Hemodynamic and symptomatic effects of acute interventions on tilt in patients with postural tachycardia syndrome.

A variety of approaches have been used to alleviate symptoms in postural tachycardia syndrome (POTS). Drugs reported to be of benefit include midodrine, propranolol, clonidine, and phenobarbital. Other measures used include volume expansion and physical countermaneuvers. These treatments may influence pathophysiologic mechanisms of POTS such as alpha-receptor dysfunction, beta-receptor supersensitivity, venous pooling, and brainstem center dysfunction. The authors prospectively studied hemodynamic indices and symptom scores in patients with POTS who were acutely treated with a variety of interventions. Twenty-one subjects who met the criteria for POTS were studied (20 women, 1 man; mean age, 28.7 +/- 6.8 y; age range, 14-39 y). Patients were studied with a 5-minute head-up tilt protocol, ECG monitoring, and noninvasive beat-to-beat blood pressure monitoring, all before and after the administration of an intervention (intravenous saline, midodrine, propranolol, clonidine, or phenobarbital). The hemodynamic indices studied were heart rate (ECG) and systolic, mean, and diastolic blood pressure. Patients used a balanced verbal scale to record any change in their symptoms between the tilts. Symptom scores improved significantly after the patients received midodrine and saline. Midodrine and propranolol reduced the resting heart rate response to tilt (p <0.005) and the immediate and 5-minute heart rate responses to tilt (p <0.002). Clonidine accentuated the immediate decrease in blood pressure on tilt up (p <0.05). It was concluded that midodrine and intravenous saline are effective in decreasing symptoms on tilt in patients with POTS when given acutely. Effects of treatments on heart rate and blood pressure responses generally reflected the known pharmacologic mechanisms of the agents.

Clostridium septicum alpha toxin uses glycosylphosphatidylinositol-anchored protein receptors.

The alpha toxin produced by Clostridium septicum is a channel-forming protein that is an important contributor to the virulence of the organism. Chinese hamster ovary (CHO) cells are sensitive to low concentrations of the toxin, indicating that they contain toxin receptors. Using retroviral mutagenesis, a mutant CHO line (BAG15) was generated that is resistant to alpha toxin. FACS analysis showed that the mutant cells have lost the ability to bind the toxin, indicating that they lack an alpha toxin receptor. The mutant cells are also resistant to aerolysin, a channel-forming protein secreted by Aeromonas spp., which is structurally and functionally related to alpha toxin and which is known to bind to glycosylphosphatidylinositol (GPI)-anchored proteins, such as Thy-1. We obtained evidence that the BAG15 cells lack N-acetylglucosaminyl-phosphatidylinositol deacetylase-L, needed for the second step in GPI anchor biosynthesis. Several lymphocyte cell lines lacking GPI-anchored proteins were also shown to be less sensitive to alpha toxin. On the other hand, the sensitivity of CHO cells to alpha toxin was increased when the cells were transfected with the GPI-anchored folate receptor. We conclude that alpha toxin, like aerolysin, binds to GPI-anchored protein receptors. Evidence is also presented that the two toxins bind to different subsets of GPI-anchored proteins.

Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor.

Anthrax lethal toxin, produced by the bacterium Bacillus anthracis, is the major cause of death in animals infected with anthrax. One component of this toxin, lethal factor (LF), is suspected to be a metalloprotease, but no physiological substrates have been identified. Here it is shown that LF is a protease that cleaves the amino terminus of mitogen-activated protein kinase kinases 1 and 2 (MAPKK1 and MAPKK2) and that this cleavage inactivates MAPKK1 and inhibits the MAPK signal transduction pathway. The identification of a cleavage site for LF may facilitate the development of LF inhibitors.

Clostridium septicum alpha-toxin is proteolytically activated by furin.

Clostridium septicum alpha-toxin is secreted as an inactive 46,450-Da protoxin. The protoxin is activated by proteolytic cleavage near the C terminus, which eventually causes the release of a 45-amino-acid fragment. Proteoytic activation and loss of the propeptide allow alpha-toxin to oligomerize and form pores on the plasma membrane, which results in colloidal-osmotic lysis. Activation may be accomplished in vitro by cleavage with trypsin at Arg367 (J. Ballard, Y. Sokolov, W. L. Yuan, B. L. Kagan, and R. K. Tweten, Mol. Microbiol. 10:627-634, 1993), which is located within the sequence KKRRGKR367S. A conspicuous feature of this site is a recognition site (RGKR) for the eukaryotic protease furin. Pro-alpha-toxin (AT[pro]) that was digested with trypsin or recombinant soluble furin yielded the 41,327-Da active form (AT[act]). A mutated alpha-toxin in which the furin consensus site was altered to KKRSGSRS at the cleavage site (AT[SGSR]) was cleaved and activated by trypsin but not by furin. In cytotoxicity assays, wild-type Chinese hamster ovary (CHO) and furin-deficient CHO (FD11) cells were killed by AT(pro) but not by AT(SGSR). Both cell types were killed by AT(SGSR) that was preactivated with trypsin. Propidium iodide uptake assays revealed that FD11 cells were approximately 22% less sensitive to AT(pro) than were CHO cells. AT(pro)-induced cell lysis of FD11 cells, assessed by propidium iodide uptake, was partially prevented by leupeptin (5 mM) and completely prevented by antipain (2.5 mM). The inhibition by antipain suggested the presence of cysteine or serine proteases that could also activate AT(pro). These findings demonstrate that furin is involved in the activation of C. septicum alpha-toxin on the cell surface but that alternate eukaryotic proteases can also activate the toxin. Regardless of the activating protease, the furin consensus site appears to be essential for the activation of alpha-toxin on the cell surface.

A role for PACE4 in the proteolytic activation of anthrax toxin protective antigen.

Several bacterial protein toxins require activation by eukaryotic proteases. Previous studies have shown that anthrax toxin protective antigen (PA), Pseudomonas exotoxin A (PE), and diphtheria toxin (DT) are cleaved by furin C-terminal to the sequences RKKR, RQPR, and RVRR, respectively. Because furin-deficient cells retain some sensitivity to PA and DT, it is evident that other cellular proteases can activate these toxins. Whereas furin has been shown to require arginine residues at positions -1 and -4 for substrate recognition, another protease with an activity which could substitute for furin in toxin activation, the furin-related protease PACE4, requires basic residues in the -1, -2, and -4 positions of the substrate sequence. To examine the relative roles of furin and PACE4 in toxin activation, we used furin-deficient CHO cells (FD11 cells) transfected with either the furin (FD11/furin cells) or PACE4 (FD11/PACE4 cells) gene. Mutant PA proteins containing the cleavage sequence RAAR or KR were cytotoxic toward cells expressing only PACE4. In vitro cleavage data demonstrated that PACE4 can recognize RAAR and, to a much lesser extent, KR and RR. When extracts from PACE4-transfected cells were used as a source of proteases, PACE4 had minimal activity, indicating that it had been partially inactivated or did not remain associated with the cell membranes. Cleavage of iodinated PA containing the sequence RKKR or RAAR was detected on the surface of all cell types tested, but cleavage of a dibasic sequence was detected only intracellularly and only in cells that expressed furin or PACE4. The data provide evidence that PACE4 is present at the exterior of cells, that it plays a role in the proteolytic activation of anthrax toxin PA, and that PACE4 can activate substrates at the sequence RAAR or KR.

Furin regulates both the activation of Pseudomonas exotoxin A and the Quantity of the toxin receptor expressed on target cells.

Pseudomonas exotoxin A (PE) binds and enters mammalian cells via the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP). The toxin then requires proteolytic cleavage to generate an enzymatically active fragment with translocates to the cell cytosol and inhibits protein synthesis. To assess the role of furin in determining toxin susceptibility, CHO cells were transfected with a mouse furin gene (CHO+fur cells) and maintained under neomycin selection. Cells expressing the transfected gene were about two- to threefold more sensitive to PE than were cells expressing only a neomycin resistance gene (CHO+neo cells). Possible reasons for the increased toxin sensitivity include the cleavage of a greater number of PE molecules and/or the conversion of more single-chain LRP to the processed, two-chain form. Processing of LRP appears to be necessary to allow the surface display of this receptor. Results of ligand binding studies indicated that the CHO+fur cells displayed about twofold more surface-expressed LRP than did CHO+neo cells. In addition, the in vitro cleavage of PE by recombinant furin enhanced toxin potency about threefold for CHO+neo cells but enhanced it very little for CHO+fur cells. This suggested that CHO+fur cells were processing PE at close to the maximum usable rate. Together these findings suggest that furin is involved in at least two separate protein processing pathways that each contribute to the sensitivity of cells to PE.

Vaccination with genetically modified Shiga-like toxin IIe prevents edema disease in swine.

Escherichia coli strains producing Shiga-like toxin II variant (SLT-IIe, formerly called SLT-IIv) cause edema disease in weaned pigs. Vaccination of pigs with a genetically modified form of Shiga-like toxin IIe, SLT-IIe(E167Q), has been previously shown to be nontoxic and to induce antibodies to SLT-IIe (V.M. Gordon. S.C. Whipp, H.W. Moon, A.D. O'Brien, and J.E. Samuel, Infect, Immun. 60:485-502, 1992). Fifty micrograms of SLT-IIe(E167Q) toxin was used to vaccinate suckling pigs at 1 and 2 weeks of age. Both vaccinated and nonvaccinated pigs were orally inoculated with an SLT-IIe-producing strain of E. coli after weaning (3 to 4 weeks of age). Pigs fed a low-protein diet that were not vaccinated with SLT-IIe(E167Q) developed subclinical edema disease, histologically evident as vascular necrosis. Pigs fed a high-protein diet that were not vaccinated with SLT-IIe(E167Q) developed clinical edema disease manifested as vascular necrosis, reduced weight gain, ataxia, palpebral edema, lateral recumbency, and death. Pigs vaccinated with SLT-IIe(E167Q) had a reduction in the incidence of subclinical edema disease and never developed clinical edema disease. These data demonstrate that vaccination with a genetically modified form of SLT-IIe prevents edema disease and are consistent with the notion that diet influences susceptibility to edema disease.

Proteolytic activation of bacterial toxins by eukaryotic cells is performed by furin and by additional cellular proteases.

Before intoxication can occur, anthrax toxin protective antigen (PA), Pseudomonas exotoxin A (PE), and diphtheria toxin (DT) must be activated by proteolytic cleavage at specific amino acid sequences. Previously, it was shown that PA and DT can be activated by furin. In Chinese hamster ovary (CHO) cells, wild-type (RKKR) and cleavage site mutants of PA, each administered with a modified form of anthrax toxin lethal factor (the N terminus of lethal factor fused to PE domain III), had the following potencies: RKKR (wild type) (concentration causing 50% cell death [EC50] = 12 ng/ml) > or = RAAR (EC50 = 18 ng/ml) > FTKR (EC50 = 24 ng/ml) > STRR (EC50 = 49 ng/ml). In vitro cleavage of PA and cleavage site mutants of PA by furin demonstrated that native PA (RKKR) and PA with the cleavage sequence RAAR are substrates for furin. To characterize eukaryotic proteases that play a role in activating bacterial toxins, furin-deficient CHO cells were selected after chemical mutagenesis. Furin-deficient cells were resistant to PE, whose cleavage site, RQPR, constitutes a furin recognition site and to all PA cleavage site mutants, but were sensitive to DT (EC50 = 2.9 ng/ml) and PA (EC50 = 23 ng/ml), whose respective cleavage sites, RKKR and RVRR, contain additional basic residues. Furin-deficient cells that were transfected with the furin gene regained sensitivity to PE and PA cleavage site mutants. These studies provide evidence that furin can activate the three toxins and that one or more additional proteases contribute to the activation of DT and PA.

In vitro processing of anthrax toxin protective antigen by recombinant PC1 (SPC3) and bovine intermediate lobe secretory vesicle membranes.

Protective antigen (PA), an 83-kDa protein produced by Bacillus anthracis, requires proteolytic activation at a tetrabasic site (RKKR167) before it can combine with either edema factor or lethal factor on the cell surface. The complex is then endocytosed and the target cell intoxicated. Previous work has demonstrated that furin, a ubiquitously distributed, subtilisin-like protease, can perform this cleavage. In this study, another member of the furin family, PC1 (SPC3), was tested as a putative processing enzyme for PA. Recombinant PC1, partially purified from the medium of stably transfected L-cells, cleaved PA to a 63-kDa fragment (PA63) and a 20-kDa fragment (PA20). Amino-terminal sequence analysis of the 63 kDa product demonstrated that cleavage occurred between Arg167 and Ser168. The pH optimum for in vitro PA cleavage was 6.0 and the enzymatic activity was calcium-dependent. Medium from untransfected L-cells did not cleave PA. Site-directed mutagenesis of the tetrabasic cleavage site revealed that PC1 preferred to cleave sequences containing basic residues at positions -1 and -4 relative to the wild-type cleavage site, demonstrating that PC1 can cleave substrates at a monobasic residue site in vitro. Substrates having basic residues at the -1 and -2 positions were cleaved with approximately twofold less efficiency than wild-type PA. Mutants of PA containing basic residues in positions -1 and either -2 or -4 of the cleavage site were predicted to be substrates for PC1 and were more toxic to L-cells expressing PC1 than to untransfected L-cells. These results demonstrate that PA is cleaved by PC1 in vivo. Membranes from bovine intermediate lobe secretory vesicles which contain both prohormone convertases, PC1 and PC2, also cleaved PA to PA63 with a pH optimum of 5.5. Immunodepletion studies using antisera against PC1 and PC2 showed that these are the enzymes primarily responsible for the cleavage of PA in the membrane preparation. Thus, both recombinant PC1 and a membrane preparation containing endogenous PC1 can activate PA.

Evidence that proteolytic separation of Shiga-like toxin type IIv A subunit into A1 and A2 subunits is not required for toxin activity.

The role for proteolytic activation of Shiga-like toxin type II variant (SLT-IIv) A subunit was examined using site-directed mutagenesis. Processing of the enzymatically active A subunit by trypsin results in cleavage at an arginine residue(s) (Arg247 and/or Arg250) located between two cysteines. After reduction of the disulfide bond, the processed A subunit separates into an enzymatically active A1 and an A2 peptide. Substitution mutations were created in SLT-IIv that replaced each or both of the two arginines with either glutamic acid (R247E, R250E, or R247E/R250E) or histidine (R247H, R250H, or R247H/R250H). The products of all glutamic acid substitution mutations were immunoreactive but were not cytotoxic due to an inability to assemble into holotoxin. The products of all histidine substitution mutations had cytotoxic activities, enzymatic activities, and a lethal dose for mice similar to that of native toxin. R247H and R250H were susceptible to proteolytic cleavage while R247H/R250H was resistant to processing by exogenously added trypsin. Native toxin incubated with Vero cells was completely cleaved while only a fraction of R247H/R250H was cleaved. These results demonstrate that cleavage of SLT-IIv can be mediated by proteases with different specificities and suggest efficient cleavage is not required for toxicity.

Comparison of the relative toxicities of Shiga-like toxins type I and type II for mice.

In earlier studies using a streptomycin-treated mouse model of infection caused by enterohemorrhagic Escherichia coli (EHEC), animals fed Shiga-like toxin type II (SLT-II)-producing strains developed acute renal cortical necrosis and died, while mice fed Shiga-like toxin type I (SLT-I)-producing clones did not die (E. A. Wadolkowski, L. M. Sung, J. A. Burris, J. E. Samuel, and A. D. O'Brien, Infect. Immun. 58:3959-3965, 1990). To examine the bases for the differences we noted between the two toxins in the murine infection model, we injected mice with purified toxins and carried out histopathological examinations. Despite the genetic and structural similarities between the two toxins, SLT-II had a 50% lethal dose (LD50) which was approximately 400 times lower than that of SLT-I when injected intravenously or intraperitoneally into mice. Histopathologic examination of toxin-injected mice revealed that detectable damage was limited to renal cortical tubule epithelial cells. Passive administration of anti-SLT-II antibodies protected mice from SLT-II-mediated kidney damage and death. Immunofluorescence staining of normal murine kidney sections incubated with purified SLT-I or SLT-II demonstrated that both toxins bound to cortical tubule and medullary duct epithelial cells. Compared with SLT-I, SLT-II was more heat and pH stable, suggesting that SLT-II is a relatively more stable macromolecule. Although both toxins bound to globotriaosylceramide, SLT-I bound with a higher affinity in a solid-phase binding assay. Differences in enzymatic activity between the two toxins were not detected. These data suggest that structural/functional differences between the two toxins, possibly involving holotoxin stability and/or receptor affinity, may contribute to the differential LD50s in mice.

An enzymatic mutant of Shiga-like toxin II variant is a vaccine candidate for edema disease of swine.

Edema disease (ED) of weanling pigs is caused by an infection with Escherichia coli that produces Shiga-like toxin II variant (SLT-IIv). Pathology identical to that caused by ED can be duplicated in pigs that are injected with less than 10 ng of purified SLT-IIv per kg of body weight. Therefore, SLT-IIv was mutated to create an immunoreactive form of the toxin that was significantly reduced in enzymatic activity. Initially, purified SLT-IIv was treated with formaldehyde which abrogated cytotoxic activity. Pigs were vaccinated with the toxoid (100 micrograms) to determine whether a toxoid was a viable vaccine candidate and whether young pigs were capable of mounting an immune response. Although the pigs developed a neutralizing antibody titer (1:128 to 1:512) 28 days postinjection, they also lost weight and developed ED lesions. The deleterious effect of the toxoid appeared to result from residual enzymatic activity or a reversion to a toxic form. An alternative method, site-directed mutagenesis, was employed to consistently reduce the enzymatic activity of SLT-IIv. Glutamate at position 167 of the mature A subunit was replaced by aspartate (E167D), and arginine at position 170 was replaced by lysine (R170K). These mutations reduced cytotoxic activity 10(4)-fold and 10-fold, respectively, while the enzymatic activities were decreased 400-fold and 5-fold, respectively. The activity of a toxin that contained both mutations (SLT-IIvE167D/R170K) closely resembled that of SLT-IIvE167D. When position 167 was replaced by glutamine (E167Q), the cytotoxic activity decreased 10(6)-fold and the enzymatic activity decreased approximately 1,500-fold. Pigs that were vaccinated with purified, mutant toxin designated SLT-IIvE167Q developed a neutralizing antibody titer of 1:512 21 days postinjection, and their tissues were free of ED lesions. These data suggest that SLT-IIvE167Q may represent an effective vaccine against ED.

Adenylate cyclase toxin from Bordetella pertussis. Conformational change associated with toxin activity.

Adenylate cyclase (AC) toxin from Bordetella pertussis interacts with and enters eukaryotic cells to catalyze the production of supraphysiologic levels of cyclic AMP. Although the calmodulin-activated enzymatic activity (ability to convert ATP to cyclic AMP in a cell-free assay) of this molecule is calcium independent, its toxin activity (ability to increase cyclic AMP levels in intact target cells) requires extracellular calcium. Toxin activity as a function of calcium concentration is biphasic, with no intoxication occurring in the absence of calcium, low level intoxication (200-300 pmol of cyclic AMP/mg of Jurkat cell protein) occurring with free calcium concentrations between 100 nM and 100 microM and a 10-fold increase in AC toxin activity at free calcium concentrations above 300 microM. The molecule exhibits a conformational change when free calcium concentrations exceed 100 microM as demonstrated by shift in intrinsic tryptophan fluorescence, an alteration in binding of one anti-AC monoclonal antibody, protection of a fragment from trypsin-mediated proteolysis, and a structural modification as illustrated by electron microscopy. Thus, it appears that an increase in the ambient calcium concentration to a critical point and the ensuing interaction of the toxin with calcium induces a conformational change which is necessary for its insertion into the target cell and for delivery of its catalytic domain to the cell interior.

Hemolytic activity of adenylate cyclase toxin from Bordetella pertussis.

Adenylate cyclase (AC) toxin from B. pertussis enters eukaryotic cells where it produces supraphysiologic levels of cAMP. Purification of AC toxin activity [(1989) J. Biol. Chem. 264, 19279] results in increasing potency of hemolytic activity and electroelution of the 216-kDa holotoxin yields a single protein with AC enzymatic, toxin and hemolytic activities. AC toxin and E. coli hemolysin, which have DNA sequence homology [(1988) EMBO J. 7, 3997] are immunologically cross-reactive. The time courses of hemolysis elicited by the two molecules are strikingly different, however, with AC toxin eliciting cAMP accumulation with rapid onset, but hemolysis with a lag of greater than or equal to 45 min. Finally, osmotic protection experiments indicate that the size of the putative pore produced by AC toxin is 3-5-fold smaller than that of E. coli hemolysin.

Adenylate cyclase toxin from Bordetella pertussis. Identification and purification of the holotoxin molecule.

Bordetella pertussis adenylate cyclase (AC) toxin is a calmodulin-activated adenylate cyclase enzyme which has the capacity to enter eukaryotic target cells and catalyze the conversion of endogenous ATP into cyclic AMP. In this work, the AC holotoxin molecule is identified and isolated. It is a single polypeptide of apparent 216 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Monoclonal antibodies which immunoprecipitate AC activity from extracts of wild type B. pertussis (BP338) react with this 216-kDa band on Western blots, and it is absent from a transposon Tn5 mutant (BP348) specifically lacking AC toxin. Isolation of the 216-kDa protein to greater than 85% purity by hydrophobic chromatography, preparative sucrose gradient centrifugation, and affinity chromatography using either calmodulin-Sepharose or monoclonal antibody coupled to Sepharose 4B yields stepwise increases in AC toxin potency, to a maximum of 88.3 mumol of cAMP/mg of target cell protein/mg of toxin. Electroelution of the 216-kDa band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis yields a preparation with both AC enzyme and toxin activities. These data indicate that this protein represents the AC holotoxin molecule.

Adenylate cyclase toxins from Bacillus anthracis and Bordetella pertussis. Different processes for interaction with and entry into target cells.

Adenylate cyclase (AC) toxins produced by Bacillus anthracis and Bordetella pertussis were compared for their ability to interact with and intoxicate Chinese hamster ovary cells. At 30 degrees C, anthrax AC toxin exhibited a lag of 10 min for measurable cAMP accumulation that was not seen with pertussis AC toxin. This finding is consistent with previous data showing inhibition of anthrax AC toxin but not pertussis AC toxin entry by inhibitors of receptor-mediated endocytosis (Gordon, V. M., Leppla, S. H., and Hewlett, E. L. (1988) Infect. Immun. 56, 1066-1069). Treatment of target Chinese hamster ovary cells with trypsin or cycloheximide reduced anthrax AC toxin-induced cAMP accumulation by greater than 90%, but was without effect on pertussis AC toxin. In contrast, incubation of the AC toxins with gangliosides prior to addition to target cells inhibited cAMP accumulation by pertussis AC toxin, but not anthrax AC toxin. To evaluate the role of lipids in the interaction of pertussis AC toxin with membranes, multicompartmental liposomes were loaded with a fluorescent marker and exposed to toxin. Pertussis AC toxin elicited marker release in a time- and concentration-dependent manner and required a minimal calcium concentration of 0.2 mM. These data demonstrate that the requirements for intoxication by the AC toxins from B. anthracis and B. pertussis are fundamentally different and provide a perspective for new approaches to study the entry processes.

Inhibitors of receptor-mediated endocytosis block the entry of Bacillus anthracis adenylate cyclase toxin but not that of Bordetella pertussis adenylate cyclase toxin.

Bordetella pertussis and Bacillus anthracis produce extracytoplasmic adenylate cyclase toxins (AC toxins) with shared features including activation by calmodulin and the ability to enter target cells and catalyze intracellular cyclic AMP (cAMP) production from host ATP. The two AC toxins were evaluated for sensitivities to a series of inhibitors of known uptake mechanisms. Cytochalasin D, an inhibitor of microfilament function, abrogated the cAMP response to B. anthracis AC toxin (93%) but not the cAMP response elicited by B. pertussis AC toxin. B. anthracis-mediated intoxication of CHO cells was completely inhibited by ammonium chloride (30 mM) and chloroquine (0.1 mM), whereas the cAMP accumulation produced by B. pertussis AC toxin remained unchanged. The block of target cell intoxication by cytochalasin D could be bypassed when cells were first treated with anthrax AC toxin and then exposed to an acidic medium. These data indicate that despite enzymatic similarities, these two AC toxins intoxicate target cells by different mechanisms, with anthrax AC toxin entering by means of receptor-mediated endocytosis into acidic compartments and B. pertussis AC toxin using a separate, and as yet undefined, mechanism.