The Clostridium perfringens alpha-toxin

The gene encoding the alpha-(cpa) is present in all strains of Clostridium perfringens, and the purified alpha-toxin has been shown to be a zinc-containing phospholipase C enzyme, which is preferentially active towards phosphatidylcholine and sphingomyelin. The alpha-toxin is haemolytic as a result if its ability to hydrolyse cell membrane phospholipids and this activity distinguishes it from many other related zinc-metallophospholipases C. Recent studies have shown that the alpha-toxin is the major virulence determinant in cases of gas gangrene, and the toxin might play a role in several other diseases of animals and man as diverse as necrotic enteritis in chickens and Crohn's disease in man. In gas gangrene the toxin appears to have three major roles in the pathogenesis of disease. First, it is able to cause mistrafficking of neutrophils, such that they do not enter infected tissues. Second, the toxin is able to cause vasoconstriction and platelet aggregation which might reduce the blood supply to infected tissues. Finally, the toxin is able to detrimentally modulate host cell metabolism by activating the arachidonic acid cascade and protein kinase C. The molecular structure of the alpha-toxin reveals a two domain protein. The amino-terminal domain contains the phospholipase C active site which contains zinc ions. The carboxyterminal domain is a paralogue of lipid binding domains found in eukaryotes and appears to bind phospholipids in a calcium-dependent manner. Immunisation with the non-toxic carboxyterminal domain induces protection against the alpha-toxin and gas gangrene and this polypeptide might be exploited as a vaccine. Other workers have exploited the entire toxin as the basis of an anti-tumour system.     

Heat stability and species range of purified staphylococcal alpha-toxin

Cooper, Louis Z. (New England Medical Center Hospital, Boston, Mass.), Morton A. Madoff, and Louis Weinstein. Heat stability and species range of purified staphylococcal alpha-toxin. J. Bacteriol. 91:1686-1692. 1966.-Heating of high-titer purified staphylococcal alpha-toxin at 60 and 80 C resulted in a double-sloped curve of inactivation of the hemolytic effect on rabbit erythrocytes. Early inactivation was less at the lower temperature, but activity persisted for a longer time at 80 C. Toxin inactivated at 60 C showed renewed activity when heated briefly at 80 C. A precipitate which formed during heating of alpha-toxin at 60 or 80 C yielded hemolytic activity when resuspended and heated at 80 but not at 60 C. Supernatant fluid of heat-precipitated toxin was heat-labile and did not regain activity when heated at 80 C. The results indicate that the "paradoxical effect" of heating of staphylococcal alpha-toxin is not due to a thermolabile inhibitor, but results from alteration of the toxin molecule to a heat-stable active form. Demonstration of renewed activity by 80 C heating of purified toxin requires potent toxin preparations and brief heating periods. Hemolysis of erythrocytes of several animal species by purified alpha-toxin was generally similar to that produced by impure toxin. Rabbit cells were most susceptible. Human and horse erythrocytes hemolyzed to less than 0.1% of the extent of rabbit cells. Blood cells of other species were intermediate in their response to the lytic effect of alpha-toxin.     

Lethal Toxin of Bacillus cereus I. Relationships and Nature of Toxin, Hemolysin, and Phospholipase

Bacillus cereus phospholipase was characterized as a phospholipase C by the analysis of lecithin degradation products by thin-layer and paper chromatography. Methanol in the growth menstruum inhibited completely the synthesis of phospholipase C, whereas the synthesis of lethal toxin and hemolysin were only partially inhibited. Dialysis of preformed B. cereus products against ethyl alcohol and methanol did not inactivate hemolytic, phospholipase C, or lethal activity. The hemolytic and lethal activities of culture filtrates were completely abolished by trypsin, but phospholipase C activity was resistant to inactivation. Lethal and phospholipase C properties of culture filtrates were resistant to inactivation at 45 C, whereas the hemolytic activity was completely destroyed. Lethal, hemolytic, and phospholipase C activities appeared simultaneously in a complex growth menstruum, but the kinetics of synthesis were different in all cases. Resolution of B. cereus filtrates on columns of Sephadex showed that the phospholipase C, hemolysin, and lethal toxin are distinct proteins. Evidence is also presented which suggests a correlation between the synthesis of B. cereus toxin and the period of transition from vegetative growth to sporulation. The activity of each B. cereus product was cation-independent, as opposed to cation-dependency of the phospholipase C and lethal activities of Clostridium perfringens alpha-toxin. Immunological cross-reactivity between the B. cereus products and C. perfringens alpha-toxin was not apparent; indeed, they were shown to be antigenically distinct.     

Platelet Damaging Factor, a Fifth Activity of Staphylococcal α-Toxin

Crude and purified staphylococcal alpha-toxin were used to demonstrate that the platelet-damaging effect of crude alpha-toxin represents a fifth activity of the alpha-toxin molecule. The homogeneity of the purified toxin employed was demonstrated by ultracentrifugation, Ouchterlony, and immunoelectrophoretic methods. Continuous-flow electrophoretic migration studies demonstrated under a variety of conditions that the platelet-damaging and the alpha-hemolytic activities migrated as a unit. Fractionation studies with the use of Sephadex G-100, carboxymethyl cellulose, and diethylaminoethyl cellulose failed to separate these two activities. Further, when alpha-toxin of demonstrated purity and crude toxin were adjusted to the same hemolytic activity, they possessed the same platelet-damaging activity. In addition, heat-reactivation studies with crude alpha-toxin revealed that the platelet-damaging effect was inactivated and reactivated in parallel with alpha-hemolytic activity. Comparable studies with purified alpha-toxin showed parallel inactivation of both activities at 60 C. Additional heating at 100 C failed to reactivate either activity. Electron micrographs revealed that purified alpha-toxin produced distinct degenerative changes in rabbit platelets. These studies also provided definite evidence that purified alpha-toxin has a damaging effect on human platelets. Monovalent alpha-antisera prevented platelet damage.     

Heat reactivation of the alpha-hemolytic, dermonecrotic, lethal activities of crude and purified staphylococcal alpha-toxin

Manohar, M. (University of Minnesota, St. Paul), S. Kumar, and R. K. Lindorfer. Heat reactivation of the alpha-hemolytic, dermonecrotic, and lethal activities of crude and purified staphylococcal alpha-toxin. J. Bacteriol. 91:1681-1685. 1966.-Crude staphylococcal toxin loses its alpha-hemolytic activity more rapidly at 60 than at 100 C. This paradoxical behavior has been postulated to be due to the presence of a thermolabile inhibitor in crude toxin. This work provides experimental evidence for the presence of a thermolabile "protective inhibitor." This substance(s) protects the alpha-toxin against destruction at 60 C, yet simultaneously inhibits the hemolytic activity of alpha-toxin under the same conditions. Of greater importance, this work also demonstrates that the dermonecrotic and lethal activities of crude toxin are inactivated and reactivated in parallel with the alpha-hemolytic activity. Crude staphylococcal toxin possessing a high alpha-hemolytic titer when heated to 60 C for 30 min lost its alpha-hemolytic, dermonecrotic, and lethal activity. However, when this same toxin was immediately exposed to 100 C, a remarkable simultaneous reactivation of all three of these activities occurred. Contrariwise, electrophoretically purified alpha-hemolysin, which also possessed dermonecrotic and lethal activity, showed no reactivation under these conditions, thus demonstrating that reactivation is due to a substance(s) distinct from the alpha-toxin. The fact that alpha-hemolytic, dermonecrotic, and lethal activities were inactivated at 60 C and simultaneously reactivated at 100 C provides additional proof that these activities are all associated with one toxic component. The probability is remote that three separate entities would exhibit the same rate of inactivation and the same strange reactivation.     

Interaction of Staphylococcal α-Toxin with Artificial and Natural Membranes

Comparison of hemolytic activity and chromate-releasing activity of partially purified preparations of staphylococcal alpha-toxin indicated the presence of a lytic factor other than alpha-toxin. This lytic release factor (RF) was isolated from the preparations and was shown to be active against both lipid spherules and erythrocytes. Heat-purified alpha-toxin (HP alpha-toxin) disrupted spherules, with the formation of fragments which always showed the presence of ring structures similar in dimensions (ca. 90 A) to pure alpha 12S-toxin. The interaction of HP alpha-toxin with spherules was accompanied by loss of hemolytic activity and adsorption of toxic protein. The alpha 12S-toxin, although only weakly hemolytic, was shown to be lytic for spherules. An alpha 12S-free toxin rapidly disrupted spherules, with formation of fragments with attached rings similar in dimensions to the alpha 12S molecule. Lipid monolayer experiments showed that HP alpha-toxin could penetrate lipid monolayers by virtue of a hydrophobic interaction. Effects of HP alpha-toxin on rabbit and human erythrocyte ghosts were similar to its effects on spherules, in that rings appeared on membrane fragments. Toxin-lysed rabbit erythrocytes showed similar rings on the resulting membrane fragments. However, rings were not seen on toxin-treated rabbit erythrocytes in the prelytic lag phase; this result and the fact that human erythrocytes are largely insensitive to alpha-toxin were interpreted as evidence against a lytic mechanism involving ring formation as the primary event. Rings were interpreted as toxin polymers similar to alpha 12S molecules, formed from specifically orientated active toxin molecules at the surface of lipid structures. Possible mechanisms for toxin lysis of spherules and erythrocytes are discussed.     

Purification and characterization of alpha-toxin of Clostridium oedematiens type A

The alpha-toxin of Clostridium oedematiens type A was purified from culture filtrate by two steps of column chromatography and repeated gel filtration. The purified alpha-toxin proved homogeneous in polyacrylamide gel electrophoresis and agar gel double diffusion. The molecular weight of the alpha-toxin was estimated at 280,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and at 260,000 by gel filtration on a Sephadex G-200 column. The isoelectric point determined by isoelectric focusing polyacrylamide gel electrophoresis was 6.1. No dissociation of the purified alpha-toxin into subunits was demonstrated in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 50% lethal and edematizing doses per mg protein of the purified alpha-toxin were 5.9 X 10(4) and 5.9 X 10(5), respectively. The L +/50 doses per mg protein of the toxin was 4.6 X 10(3). The purified alpha-toxin, when injected intradermally into the rabbit skin, induced increased vascular permeability. The toxin contained little or no hemolytic or lecithinase activity. These results attest that the lethal, edematizing and vascular permeability-enhancing activities elicited by C. oedematiens type A culture reside on the same protein molecule.     

Staphylococcus aureus alpha-toxin activates phospholipases and induces a Ca2+ influx in PC12 cells

Staphylococcal alpha-toxin at subcytotoxic concentrations stimulated phosphatidylinositol turnover and arachidonic acid release in undifferentiated cultures of pheochromocytoma PC12 cells. Stimulation of phospholipase A2 but not C was dependent on extracellular calcium. Addition of staphylococcal alpha-toxin to PC12 cells caused a dose-dependent, biphasic increase in intracellular calcium measured by fura-2 fluorescence technique. Elevation of intracellular Ca2+ content occurred with a time course similar to those observed for stimulation of phospholipase A2. Alteration of membrane structure and formation of staphylococcal alpha-toxin pores facilitating an influx of Ca2+, represent the probable mechanisms by which phospholipases C and A2 are activated, respectively. These results suggest a possible involvement of Ca2+, phosphoinositides and arachidonic acid metabolites in the pathogenic action of staphylococcus alpha-toxin and caution against the general usage of this toxin as a permeabilizing agent to study stimulus-secretion coupling in secretory cells.     

NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens

For over 30 years a phospholipase C enzyme called alpha-toxin was thought to be the key virulence factor in necrotic enteritis caused by Clostridium perfringens. However, using a gene knockout mutant we have recently shown that alpha-toxin is not essential for pathogenesis. We have now discovered a key virulence determinant. A novel toxin (NetB) was identified in a C. perfringens strain isolated from a chicken suffering from necrotic enteritis (NE). The toxin displayed limited amino acid sequence similarity to several pore forming toxins including beta-toxin from C. perfringens (38% identity) and alpha-toxin from Staphylococcus aureus (31% identity). NetB was only identified in C. perfringens type A strains isolated from chickens suffering NE. Both purified native NetB and recombinant NetB displayed cytotoxic activity against the chicken leghorn male hepatoma cell line LMH; inducing cell rounding and lysis. To determine the role of NetB in NE a netB mutant of a virulent C. perfringens chicken isolate was constructed by homologous recombination, and its virulence assessed in a chicken disease model. The netB mutant was unable to cause disease whereas the wild-type parent strain and the netB mutant complemented with a wild-type netB gene caused significant levels of NE. These data show unequivocally that in this isolate a functional NetB toxin is critical for the ability of C. perfringens to cause NE in chickens. This novel toxin is the first definitive virulence factor to be identified in avian C. perfringens strains capable of causing NE. Furthermore, the netB mutant is the first rationally attenuated strain obtained in an NE-causing isolate of C. perfringens; as such it has considerable vaccine potential.     

Crystal structure of the C. perfringens alpha-toxin with the active site closed by a flexible loop region

Clostridium perfringens biotype A strains are the causative agents of gas-gangrene in man and are also implicated as etiological agents in sudden death syndrome in young domestic livestock. The main virulence factor produced by these strains is a zinc-dependent, phosphatidylcholine-preferring phospholipase C (alpha-toxin). The crystal structure of alpha-toxin, at pH 7.5, with the active site open and therefore accessible to substrate has previously been reported, as has calcium-binding to the C-terminal domain of the enzyme at pH 4.7. Here we focus on conformation changes in the N-terminal domain of alpha-toxin in crystals grown at acidic pH. These changes result in both the obscuring of the toxin active site and the loss of one of three zinc ions from it. Additionally, this "closed" form contains a small alpha helix, not present in the open structure, which hydrogen bonds to both the N and C-terminal domains. In conjunction with the previously reported findings that alpha-toxin can exist in active and inactive forms and that Thr74Ile and Phe69Cys substitutions markedly reduced the haemolytic activity of the enzyme, our work suggests that these loop conformations play a critical role in the activity of the toxin.     

Biochemical and immunological properties of the C-terminal domain of the alpha-toxin of Clostridium perfringens

Abstract The C-terminal domain of the alpha-toxin (cpa_247–370) of Clostridium perfringens has been expressed in Escherichia coli and purified. Antiserum raised against cpa_247–370 reacted in an identical manner to anti-alpha-toxin serum when used to map epitopes in the C-terminal domain, suggesting that cpa_247–370 was immunologically and structurally identical to this region in the alpha-toxin. The isolated cpa_247–370 was devoid of sphingomyelinase activity or haemolytic activity and was not cytotoxic for mouse lymphocytes. Haemolytic activity was detected when cpa_247–370 was tested with the N-terminal domain of the alpha-toxin (cpa_1–249), confirming that cpa_247–370 confers haemolytic properties on the phospholipase C activity of the alpha-toxin. Haemolytic activity was not detected if cpa_247–370 was tested with the Bacillus cereus phosphatidylcholine phospholipase C, nor if cpa_1–249 and cpa_247–370 were incubated sequentially with erythrocytes.     

Role of tryptophan-1 in hemolytic and phospholipase C activities of Clostridium perfringens alpha-toxin

Replacement of the Trp-1 in Clostridium perfringens alpha-toxin with tyrosine caused no effect on hemolytic and phospholipase C (PLC) activities or on binding to the zinc ion, but that of the residue with alanine, glycine and histidine led to drastic decreases in these activities and a significant reduction in binding to the zinc ion. The hemolytic and PLC activities of W1H and W1A were significantly increased by the preincubation of these variant toxins with zinc ions, but the preincubation of W1G with the metal ion caused little effect on these activities. Gly-Ile-alpha-toxin, which contained an additional Gly-Ile linked to the N-terminal amino acid of alpha-toxin, did not show hemolytic activity, but showed about 6% PLC activity of the wild-type toxin. A mutant toxin, which contained an additional Gly-Ile linked to the N-terminus of a protein lacking 4 N-terminal residues of alpha-toxin, showed about 1 and 6% hemolytic and PLC activities of the wild-type toxin, respectively. Incubation of the mutant toxin with zinc ions caused a significant increase in PLC activity. These observations suggested that Trp-1 is not essential for toxin activity, but plays a role in binding to zinc ions.     

Isolation and characterization of a type II restriction endonuclease from Streptococcus thermophilus

The alpha-toxin (phospholipase C) of Clostridium perfringens has been reported to contain catalytically essential zinc ions. We report here that histidine residues are essential for the co-ordination of these ion(s). Incubation of alpha toxin with diethylpyrocarbonate, a histidine modifying reagent, did not result in the loss of phospholipase C activity unless the protein was first incubated with EDTA, suggesting that zinc ions normally protect the susceptible histidine residues. When the amino acid sequences of three phospholipase C's were aligned, essential zinc binding histidine residues in the non-toxic B. cereus phospholipase C were found in similar positions in the toxic C. perfringens enzyme and the weakly toxic C. bifermentans phospholipase C.     

Clostridium perfringens alpha-toxin: characterization and mode of action

Clostridium perfringens type A strains that produce alpha-toxin cause gas gangrene, which is a life-threatening infection with fever, pain, edema, myonecrosis and gas production. Intramuscular injection of the toxin or Bacillus subtilis carrying the alpha-toxin gene causes myonecrosis and produces histopathological features of the disease. Immunization of mice with alpha-toxin or fragments of the toxin prevents gas gangrene caused by C. perfringens. The toxin possesses phospholipase C (PLC), sphingomyelinase (SMase) and biological activities causing hemolysis, lethality and dermonecrosis. These biological activities are closely related to PLC and/or SMase activities. However, there is yet some uncertainty about the biological activities induced by the PLC and SMase activities of alpha-toxin. Based on the isolation and characterization of the gene for alpha-toxin and a comparison of the toxin with enzymes of the PLC family, significant progress has been made in determining the function-structure of alpha-toxin and the mode of action of the toxin. To provide a better understanding of the role of alpha-toxin in tissue damage in gas gangrene, this article summarizes current knowledge of the characteristics and mode of action of alpha-toxin.     

Threonine-74 Is a Key Site for the Activity of Clostridium perfringens Alpha-Toxin

A mutant toxin (MT) that abolished almost 99% of the hemolytic activity of alpha-toxin was isolated by random polymerase chain reaction (PCR) mutagenesis of the gene for Clostridium perfringens alpha-toxin. In the mutant toxin, the amino acids at Tyr (Y)-62, Thr (T)-74 and Ile (I)-345 were substituted with His, Ile and Met, respectively. Replacement of T-74 with Ile by site-directed mutagenesis resulted in the loss of hemolytic, phospholipase C and sphingomyelinase activities by 1/250-fold of that of the wild-type. The replacement of Y-62 with Ile or I-345 with Met alone did not affect the activities of the toxin. T74I mutant bound to sheep erythrocyte membranes and specifically bound [⁶⁵Zn]²⁺ in Tris-buffered saline, in the same manner as the wild-type, and contained 2 mol of zinc ions per mol of protein. These results suggest that the T-74 residue plays a key role in these biological activities of C. perfringens alpha-toxin.     

Clostridium perfringens Alpha-Toxin Induces Gm1a Clustering and Trka Phosphorylation in the Host Cell Membrane

Clostridium perfringens alpha-toxin elicits various immune responses such as the release of cytokines, chemokines, and superoxide via the GM1a/TrkA complex. Alpha-toxin possesses phospholipase C (PLC) hydrolytic activity that contributes to signal transduction in the pathogenesis of gas gangrene. Little is known about the relationship between lipid metabolism and TrkA activation by alpha-toxin. Using live-cell fluorescence microscopy, we monitored transbilayer movement of diacylglycerol (DAG) with the yellow fluorescent protein-tagged C1AB domain of protein kinase C-γ (EYFP-C1AB). DAG accumulated at the marginal region of the plasma membrane in alpha toxin-treated A549 cells, which also exhibited GM1a clustering and TrkA phosphorylation. Annexin V binding assays showed that alpha-toxin induced the exposure of phosphatidylserine on the outer leaflet of the plasma membrane. However, H148G, a variant toxin which binds cell membrane and has no enzymatic activity, did not induce DAG translocation, GM1a clustering, or TrkA phosphorylation. Alpha-toxin also specifically activated endogenous phospholipase Cγ-1 (PLCγ-1), a TrkA adaptor protein, via phosphorylation. U73122, an endogenous PLC inhibitor, and siRNA for PLCγ-1 inhibited the formation of DAG and release of IL-8. GM1a accumulation and TrkA phosphorylation in A549 cells treated with alpha-toxin were also inhibited by U73122. These results suggest that the flip-flop motion of hydrophobic lipids such as DAG leads to the accumulation of GM1a and TrkA. We conclude that the formation of DAG by alpha-toxin itself (first step) and activation of endogenous PLCγ-1 (second step) leads to alterations in membrane dynamics, followed by strong phosphorylation of TrkA.     

Identification of residues critical for toxicity in Clostridium perfringens phospholipase C, the key toxin in gas gangrene.

Clostridium perfringens phospholipase C (PLC), also called alpha-toxin, is the major virulence factor in the pathogenesis of gas gangrene. The toxic activities of genetically engineered alpha-toxin variants harboring single amino-acid substitutions in three loops of its C-terminal domain were studied. The substitutions were made in aspartic acid residues which bind calcium, and tyrosine residues of the putative membrane-interacting region. The variants D269N and D336N had less than 20% of the hemolytic activity and displayed a cytotoxic potency 103-fold lower than that of the wild-type toxin. The variants in which Tyr275, Tyr307, and Tyr331 were substituted by Asn, Phe, or Leu had 11-73% of the hemolytic activity and exhibited a cytotoxic potency 102- to 105-fold lower than that of the wild-type toxin. The results demonstrated that the sphingomyelinase activity and the C-terminal domain are required for myotoxicity in vivo and that the variants D269N, D336N, Y275N, Y307F, and Y331L had less than 12% of the myotoxic activity displayed by the wild-type toxin. This work therefore identifies residues critical for the toxic activities of C. perfringens PLC and provides new insights toward understanding the mechanism of action of this toxin at a molecular level.     

A型产气荚膜梭菌α毒素基因表达及其免疫保护作用的初步研究

利用PCR技术,从A型产气荚膜梭菌标准株染色体DNA中扩增出α毒素基因,构建了含α毒素基因的重组菌株BL21(DE3)(pXETA02)。经酶切鉴定和序列测定证实,构建的表达质粒pXETA02含有α毒素基因序列。经SDS-PAGE、Western blot分析和ELISA检测,重组菌株表达的α毒素蛋白能够被α毒素单抗识别。表达优化结果表明,以IPTG为诱导剂诱导α毒素表达的优化条件是:培养基pH 7.5,培养温度37℃,IPTG浓度0.8mmol/L,菌体生长密度OD600达到0.8时加入IPTG,诱导时间5h,此时α毒素蛋白表达量为34.83%。以乳糖为诱导剂诱导α毒素表达的优化条件是:培养基pH7.5、培养温度37℃,乳糖浓度0.1g/L,菌体生长密度OD600达到0.8时加入乳糖,诱导时间5h,α毒素蛋白表达量为23.82%。动物实验结果表明,用重组菌株α毒素蛋白免疫的小鼠可以抵抗1MLD的A型产气荚膜梭菌标准株C57-1毒素攻击。     

Physical properties and function of phallolysin

Phallolysin, a mixture of two to three cytolytic proteins (all of Mr 34 000), has been isolated from Amanita phalloides mushrooms and purified to homogeneity (specific activity 24 000 hemolytic units/mg of protein). After separation by isoelectric focusing, the amino acid composition of two of these proteins has been determined. They are rich in water-soluble amino acids and contain one tryptophan residue each, but no cysteine or methionine. Mr was determined to be 34 000 in the native form as well as under denaturing conditions, indicating that the native proteins exist as monomers. Many of the physical properties of phallolysin are strikingly similar to those of staphylococcal alpha-toxin, e.g., molecular weight, existence of multiple forms, pI values, amino acid composition, and thermolability (60 degrees C). Pure phallolysin allowed us to prepare a radioactively labeled toxin. Labeling was achieved by reaction with formaldehyde, followed by reduction with sodium [3H]borohydride. With the labeled toxin (specific activity 7-14 Ci/mmol, ca. 60% biological activity), we investigated its binding to human A2 erythrocytes. We determined the number of receptors on these cells (2 X 10(4) per cell) as well as their affinity to the toxin (KD = 4 X 10(-9) M). In studies on the mechanism of cytolytic activity, we were able to distinguish at least three sequential events: binding of the toxin to human erythrocytes, K+ release, and membrane rupture (hemoglobulin release). These steps could be characterized by different kinetics as well as by different temperature dependencies. Again, the kinetic data for phallolysin are very closely related to those obtained for staphylococcal alpha-toxin.(ABSTRACT TRUNCATED AT 250 WORDS)