Vol. 13, No. 5 Printed in U.S.A.

INFECTION AND IMMUNITY, May 1976, p. 1467-1472 Copyright X) 1976 American Society for Microbiology

Temperature-Dependent Inactivating Factor ofPseudomonas aeruginosa Exotoxin A MICHAEL L. VASIL,* PINGHUI V. LIU, AND BARBARA H. IGLEWSKI Department of Microbiology and Immunology, University of Oregon Health Sciences Center, Portland, Oregon 97201,* and Department of Microbiology and Immunology, University ofLouisville Health Sciences Center, Louisville, Kentucky 40201 Received for publication 31 December 1975

The adenosine diphosphate ribosyl transferase activity of Pseudomonas aeruginosa exotoxin A (PA toxin) was found to be rapidly destroyed by heating at 45 to 60 C but not by heating at 70 to 90 C (for at least 30 min). This phenomenon has been previously described for other bacterial toxins (staphylococcal alpha-toxin and Vibrio parahaemolyticus hemolysin) and is termed an Arrhenius effect. In contrast, the Arrhenius effect was not seen when the PA toxin was heat-treated as above and tested for cell toxicity or mouse lethality. Although the PA toxin treated at 70 C for 30 min retained a significant proportion (>70%) of its adenosine diphosphate ribosyl transferase activity, the cell toxicity and mouse lethality of the toxin were virtually abolished. A temperature-dependent inactivating factor that has proteolytic activity and is co-purified with the PA toxin was shown to be responsible for the Arrhenius effect. PA toxin separated from the factor by conventional disc gel electrophoresis or PA toxin preparations lacking the factor did not show the Arrhenius effect.

The role of extracellular substances of Pseudomonas aeruginosa in the pathogenesis of Pseudomonas infections in humans is not well understood. However, exotoxin A (PA toxin), originally described by Liu et al. (12, 13), has the potential to be an important virulence factor of P. aeruginosa (4, 5, 9, 18). Recently, Iglewski and Kabat (9) reported that the PA toxin inhibits mammalian protein synthesis by the same mechanism as diphtheria toxin. Both toxins catalyze transfer of the adenosine 5'-diphosphate-ribosyl moiety of NAD+ (nicotinamide adenine dinucleotide) onto the same amino acid of elongation factor 2 in a stereochemically identical fashion (B. H. Iglewski, L. P. Elwell, P. V. Liu, and D. Kabat, in S. Shaltiel [ed. ], Proceedings of the 4th International Symposium on the Metabolic Interconversion of Enzymes, in press). However, the two toxins do not cross-react immunologically and differ in some of their structural properties. Unlike diphtheria toxin, which is a proenzyme that must be cleaved and reduced to produce an enzymatically active fragment (A fragment) (6), the intact PA toxin (molecular weight, 66,000) appears to be an active adenosine 5'diphosphate ribosyl transferase (ADPR-transferase) (9; Iglewski et al., in press). During the course of our study to further characterize the structure-function relationship of the PA toxin, we found that the ADPR-

transferase activity of PA toxin, at various stages during purification, was rapidly destroyed by heating at 45 to 60 C but not by heating at 70 C to 90 C for at least 30 min. This phenomenon, previously demonstrated with Vibrio parahaemolyticus hemolysin (17, 19, 20) and with staphylococcal alpha-toxin (2, 3, 8), has been termed an "Arrhenius effect." The Arrhenius effect was not observed when the PA toxin was tested for mouse lethality or cell toxicity because both of these activities were destroyed when the toxin was heated at 70 C. In the present study, the characteristics of the Arrhenius effect on the enzyme activity of PA toxin are described and data are presented suggesting that this effect is a result of a temperature-dependent inactivating factor with proteolytic activity, which co-purifies with the PA toxin.

MATERIALS AND METHODS Preparation and heat treatment of PA toxin. P. aeruginosa (PA 103) was used throughout this study for toxin production (12). PA toxin was purified by precipitation with zinc acetate (stage I) and (NH4)2SO4 (stage II) and chromatographed on columns of diethylaminoethyl-cellulose (stage III) and Sephadex G-200 (stage IV) (13). Stage IVa and IVb toxins were prepared from different batches of culture supernatant of P. aeruginosa (13). PA toxin preparations (0.1 ml) were heat-treated in a Haake water bath (Polyscience Corp., Evans1467



ton, Ill.) as described in the tables. Concentrations of the heat-treated toxin preparations were as follows: stage IV, 100 jig/ml of total protein; stage I and II, at a concentration where the ADPR-transferase activity as determined by titration with known amounts of stage IV toxin was equivalent to that of the stage IV preparation above. Assay for ADPR-transferase activity. Aminoacyltransferase-containing enzymes were prepared from crude extracts of rabbit reticulocytes as described by Allen and Schweet (1) and modified by Collier and Kandel (7). ADPR-transferase activity was measured according to the procedure of Collier and Kandel (7). The assay mixture contained 25 Mul of buffer [5 mM tris(hydroxymethyl)aminomethane-hydrochloride, pH 8.2, 0.1 mM ethylenediaminetetraacetic acid, 40 mM dithiothreitol (Sigma)], 25 gl of reticulocyte enzymes, 5 Ml (0.367 ,uM) of nicotinamide [U-'4C]NAD (136 Ci/mol, Amersham/Searle) and 10 gl of toxin sample. After 5 min of incubation at 25 C, 65 MlI of 10% trichloroacetic acid was added, and the precipitates were collected, washed, and analyzed in a Nuclear-Chicago low-background gasflow counter as described previously (9; Iglewski et al., in press). Inhibition of protein synthesis in mouse L cells. Inhibition of protein synthesis in mouse L cells by PA toxin was determined by measuring inhibition of [3H]amino acid incorporation as modified from a method described previously (10). Hanks minimal essential medium was modified to contain one-tenth the normal concentration of essential and nonessential amino acids, 2% undialyzed or dialyzed (against Hanks balanced salt solution) fetal calf serum and 1% glutamine. Hanks modified minimum essential medium was used to wash and maintain cells during assay and as a diluent. Breifly, washed cells (106) seeded 18 h previously in 35-mm tissue culture dishes (Corning) were incubated for 4 h at 37 C in 0.9 ml of assay medium with various concentrations of preparations of stage IV toxin or controls without toxin. Then 1 MCi of [3H]amino acid mixture (New England Nuclear Corp., NET-250) was added to a final volume of 1 ml, and the cells were incubated for an additional 1 h at 37 C. Plates were harvested by washing twice with phosphate-buffered saline, treating with 0.25% trypsin (5 min, 37 C) and lysing the cells with 1 ml of distilled water. Trichloroacetic acid was added at a final concentration of 10%, and samples were incubated at 90 C for 15 min. The precipitates were processed for counting of radioactivity in a Beckman LS200 spectrometer (Iglewski et al., in press). Percent inhibition of incorporation was determined from the mean of triplicate samples exposed to PA toxin as compared with triplicate samples from control cultures without toxin incubated under identical conditions. Control cultures without toxin showed linear incorporation over the entire 5-h period with the concentration of amino acids described above, regardless of whether dialyzed or undialyzed serum was used. Mouse lethality. The lethality of PA toxin preparations for Swiss-Webster mice was determined as previously described (9, 12). Analytical disc gel electrophoresis. Formulations

INFECT. IMMUN. for sample gels and spacer gels employing a high-pH discontinuous buffer system were as described by Maizel (15). Electrophoresis was performed in a Buchler bath assembly, and the length of the sample gels was 5.5 cm. Samples of 100 Mu contained 20 Mug of stage IVa toxin or 15 Mug of stage IVb toxin. The buffer was precooled to 4 C, and electrophoresis was carried out at 4 C with 5 mA/gel for approximately 1.5 h. Duplicate gels of each toxin preparation were run. One gel was immediately fixed and stained with 0.5% aniline blue black in 7.0% acetic acid and then decolorized with 10% acetic acid in 20% methanol. The duplicate gel was immediately sliced (approximately 1.5 mm/fraction), and each fraction was crushed and eluted at 4 C for 12 h with 1 ml of phosphate buffer (0.2 M sodium phosphate, 1 mM dithiothreitol, 0.1 mM ethylenediaminetetraacetic acid, pH 7.0). The ADPR-transferase activity of 10 MlI of the eluate from each gel fraction was assayed as above. Liquification of gelatin. To test for protease activity, 20 Mul of stage IVa or IVb toxin (250 Ag/ml) or water was added to 0.2 ml of 12% gelatin (Difco) in tubes (10 by 75 mm). Toxin and gelatin or controls with water and gelatin were heated at 55 C for 30 min and then cooled in a 25 C water bath for 1 h. A positive reaction is scored if the gelatin remains liquid after removal from the water bath.

RESULTS Effect of heat treatment on ADPR-transferase activity of PA toxin. Aliquots (0.1 ml) of PA toxin at various stages of purification were heated, and the ADPR-transferase activity of each was assayed. For all stages of toxin tested (I, II, and IV) the ADPR-transferase activities of those preparations heated at the lower temperatures (55 C) were significantly reduced in relation to the enzyme activities of the corresponding untreated preparations or those heated at 70 C for 30 min (Table 1). However, such an "Arrhenius effect" was not observed with all preparations of stage IV toxin. The ADPR-transferase activity of stage IVb did not show an Arrhenius effect and was relatively stable at 55 C, which may be a reflection of its degree of purity. The differences between stages IVa and IVb will be discussed further. Figure 1 is a plot of the thermal inactivation of the ADPR-transferase activity of stage IVa toxin that demonstrates an Arrhenius effect. These data indicate that the optimum range of inactivation is between 50 and 60 C and that the ADPR-transferase activity of PA toxin is in fact quite thermostable at 90 C for 30 min. In addition, there is significant inactivation of the enzyme activity at temperatures as low as 35 to 40 C. Effect of heat-treated PA toxin on cell toxicity and mouse lethality. It has been previously reported that PA toxin inhibits protein


VOL. 13, 1976

synthesis in cultured mammalian cells (18). Therefore, in light of the observed Arrhenius effect on the ADPR-transferase activity, it was also of interest to determine whether such an effect would be seen when heated preparations of stage IVa toxin were tested for inhibition of protein synthesis in mouse L cells and for TABLE 1. Effect of heat treatment on the ADPRtransferase activity of various toxin preparations % Relative

Heat treatment


Stage I (zinc acetate precipitate) 55 C, 30 min 70 C, 30 min

8.9 45.0

Stage II (NH4)2SO4 precipitate 55 C, 30 min 70 C, 30 min

6.5 72.6

Stage IVa (Sephadex G-200 fraction) 55 C, 30 min 70 C, 30 min

mouse lethality. The data given in Table 2 show that there was no Arrhenius effect and that the toxicity of PA toxin to mouse L cells was significantly reduced at 55 C and virtually destroyed at 70 C for 30 min. The data pertaining to the effect of heat treatment on the mouse lethality of PA toxin are presented in Table 3. An Arrhenius effect was not observed, and in fact the mouse lethality of stage IVa PA toxin was completely destroyed by treatment at 55 C for 30 min. These data are consistent with the thermolability of PA toxin (assayed by mouse lethality) previously reported by Callahan (55 C, 10 min) (5) and Liu (70 C, 30 min) (12). It is of interest, TABLE 2. Effect of heat treatment on toxicity of stage IV toxin to mouse L cellsa % Inhibitiona


0 76

(jAg) Untreated

Stage IVb (Sephadex G-200 fraction) 82 55 C, 30 min 52 70 C, 30 min " Percent activities are based on the acid-insoluble radioactivity counts per minute from the ADPR-transferase assay where the counts per minute from the assay of the untreated toxin preparations are equivalent to 100% activity. Treated and untreated samples from the same toxin preparation were diluted identically to obtain a maximum of 2,500 counts/min for the untreated sample. (Five-tenths microgram of protein of stage IVb toxin gives 100% activity in the ADPR-transferase assay.) 100 C

1 1469

55 C, 30 min

70 C, 30 m

2.2 80.3 68 0 0 58.3 ND NDb ND ND ND ND Toxicity is expressed in terms of percent inhibition of [3H]amino acid incorporation based on 6,891 counts/min incorporated for controls without toxin that give 0% inhibition.

2 1 1.5 0.1 0.05 0.01


97.5 97.6 97.8 88 84 60

ND, Not done.


X 80 80t .Ss





a 20\

Temperature (°C)

FIG. 1. Effect of heat treatment on the ADPR-transferase activity of stage IVa PA toxin. were treated at the various temperatures for 10 (A), 20 (0) or 30 min (0).

Samples, 0.1 ml,




however, that even though there is minimal inhibition of protein synthesis and essentially no mouse lethality by PA toxin heated at 70 C for 30 min, the toxin still retains a significant proportion of its ADPR-transferase activity when treated under those conditions. Isolation of a temperature-dependent inactivating factor of PA toxin by polyacrylamide disc gel electrophoresis. When the stage IVa and IVb preparations of PA toxin were subjected to conventional disc gel electrophoresis and stained for protein, stage IVa had two bands, whereas stage IVb showed only one (Fig. 2). Duplicates of the gels in Fig. 2 subjected to electrophoresis at the same time were sliced, and fractions were eluted and assayed for ADPR-transferase activity. Only those fracTABLE 3. Effect of heat treatment on stage IV toxin on mouse lethality Mortality

(no. dead/ no. in-



10 LD500

None 55 C, 30 min 70 C, 30 min

4/4 0/4 0/4


None 55 C, 30 min 70 C, 30 min

4/4 0/4 0/4

2 LD50

None 55 C, 30 min 70 C, 30 min

4/4 0/4 0/4



1 LD5,(

None 55 C, 30 min 70 C, 30 min aOne LD,0 (mean lethal dose) = 0.75 IVa total protein.

TABLE 4. Effect of heat treatment on the ADPRtransferase activity ofproteins P1 and P2 (PA toxin) isolated by conventional disc gel electrophoresis % Activitya

Toxin prepn

Protein Untreated

55 C, 70 C, 30 30 min min

Stage IVa

2/4 0/4 0/4 ,gg of stage

P1 0 0 0 P2 100 90 58 P1 + P2b 100 10 62 P1 Stage IVb NP" P2 100 91 43 Percent activities are based on the acid-insoluble radioactivity (counts per minute) from the ADPR-transferase assay where the counts per minute of the untreated protein P2 (PA toxin) is equivalent to 100% activity. b Protein P1 and protein P2 were mixed at a ratio of 1:5. Protein 1 not present in this toxin preparation. "


I Va P1


tions corresponding by distance to the stained band P2 from both stages IVa and IVb showed enzyme activity. In addition, the P2 protein eluted from the stage IVa gel killed three of four mice, whereas the P1 protein was not lethal when injected into four mice, thus indicating that the P2 protein was PA toxin. The P2 protein of stages IVa and IVb isolated from the acrylamide gels did not show an Arrhenius effect. When the P2 protein was assayed for ADPR-transferase activity after the usual heat treatment, its thermostability resembled that of stage IVb (Table 4). In contrast, after the P1 and P2 proteins were mixed at a ratio of 1:5 (P1/P2), a distinct Arrhenius effect was seen when the mixture was heat treated and assayed for enzyme activity. The effect was very similar to that seen with stage I, II, or IVa toxin preparations. This strongly suggests that



FIG. 2. Conventional disc gel electrophoresis of stage IVa and IVb preparations of PA toxin. P1 is the temperature-dependent inactivating factor, and P2 is the PA toxin.


VOL. 13, 1976

P1 protein is a temperature-dependent inactivating factor and is responsible for the Arrhenius effect of the ADPR-transferase activity of the PA toxin. Proteolytic activity of the temperature-dependent inactivating factor. When a stage IVa toxin preparation heated at 55 C for 30 min was subjected to conventional disc gel electrophoresis, the P2 protein (PA toxin) band was significantly diminished, and no other protein bands were seen that could account for an altered P2 protein (Fig. 3). The P1 band did not appear to be reduced, and its mobility on the gel was not altered (Fig. 3). Stage IVa and IVb toxin preparations were also tested for protease activity in gelatin. Only the stage IVa toxin preparation, which contains the P1 protein, gave a positive reaction. The stage IVb toxin preparation, which lacks the P1 protein, and the water control did not liquify gelatin by the method described. DISCUSSION The mechanism of the Arrhenius effect has been studied by several investigators since the effect was first shown with staphylococcal alpha-toxin in 1907 (2, 3, 8, 16). Landsteiner and his collaborators (11) proposed that the effect was due to the existence of some substance that could interact with and inactivate alpha-toxin at 60 C but not at higher temperatures. Data presented by Manohar et al. (16) supported this hypothesis. On the other hand, Arbuthnott et al. (2) and Cooper et al. (8) reported that the Arrhenius effect of alpha-toxin resulted from the formation of insoluble, inactive aggregates at 60 C that became soluble and biologically active at higher temperatures. More recently, crude hemolysin of V. parahaemolyticus was reported to show an Arrhenius



effect similar to that of the staphylococcal alpha-toxin (17). However, Takeda et al. (19, 20) subsequently showed that the Arrhenius effect of V. parahaemolyticus hemolysin is due to the presence of a temperature-dependent inactivating factor found in crude hemolysin preparations. This factor can be separated from the hemolysin by diethylaminoethyl-cellulose chromatography (19). This report described an Arrhenius effect of the ADPR-transferase activity of Pseudomonas exotoxin A, although such an effect was not seen when other biological properties (cell toxicity and mouse lethality) of PA toxin were examined (Tables 2 and 3). It has also been demonstrated that a temperature-dependent inactivating factor present in some PA toxin preparations is responsible for the Arrhenius effect. This inactivating factor was separated from the PA toxin by conventional polyacrylamide disc gel electrophoresis and appears to be identical to the protein designated P1 (Table 4 and Fig. 2). The data from polyacrylamide disc gel electrophoresis of stage IVa toxin heated at 55 C and the gelatinase activity of stage IVa toxin, but not IVb toxin, strongly suggest that the inactivating factor is a protease. Several implications of this study deserve further comment. Because the inactivating factor is co-purified with the PA toxin and its presence affects the thermostability of the PA toxin, the classification of thermostability or thermolability for bacterial toxins would best remain tentative until the toxin has been purified to homogeneity. This is also of interest in view of the fact that a heat-stable toxin (Z toxin) of P. aeruginosa has recently been described (14). The Z toxin produces cytopathic effects on cultured cells and has a molecular weight almost identical to that of exotoxin A.



it 55C 30min FIG. 3. Conventional disc gel electrophoresis of a stage IVa preparation of PA toxin (50 pg/ml) untreated (UT) and heated at 55 C for 30 min.




The partially purified Z toxin was differentiated from the A toxin on the basis of its thermostability at 70 C (14). Figure 1 shows that the inactivating factor can reduce the ADPR-transferase activity of the PA toxin at temperatures as low as 30 C in a comparatively short period of time (30 min). Thus, it would probably be best to perform all the stages of purification of PA toxin at temperatures where the inactivating factor is not likely to affect the PA toxin, perhaps at 4 C. Such considerations may lead to better yields of PA toxin. Data presented in Tables 1 to 3 indicate that, even though greater than 90% of the mouse lethality and cell -toxicity is lost when the toxin is treated at 70 C, the ADPR-transferase is still quite active (>70% of untreated toxin). These data suggest that a property of the PA toxin molecule is required for the sequence of events that precedes intracellular inhibition of protein synthesis via the ADPR-transferase activity of the toxin. This could be similar to diphtheria toxin: toxicity that requires fragment A and B is destroyed by heat, whereas the ADPR-transferase activity of fragment A is heat stable (6). Studies are currently in progress to elucidate the structure-function relationships of the PA toxin and ultimately to determine the role of this toxin in the pathogenesis of human Pseudomonas infections. ACKNOWLEDGMENTS This study was supported by Public Health Service grants IAI 11137 and AI 05283 from the National Institute of Allergy and Infectious Diseases and the Oregon Heart Association. We gratefully acknowledge the excellent technical assistance of Sophia Chung Fegan, David Oldenburg (in Portland), Dorothy Wilson, and Mary Jacob (in Louisville).

LITERATURE CITED 1. Allen, E. S., and R. S. Schweet. 1962. Synthesis of hemoglobin in a cell-free system. J. Biol. Chem. 237:760-767. 2. Arbuthnott, J. P., J. H. Freer, and A. W. Burnheimer. 1967. Physical states of staphylococcal a-toxins. J. Bacteriol. 94:1170-1177. 3. Arrhenius, S. 1907. Immunochemie Anwendungen der physikalischem Chemie auf die Lehre von den physiologischen Antikoerpern. Akademische Verlagsgelsellschaft, Leipzig. 4. Atik, M., P. V. Liu, B. A. Hanson, S. Amini, and C. F.


6. 7.

8. 9.


11. 12.









Rosenberg. 1968. Pseudomonas exotoxin shock. J. Am. Med. Assoc. 205:134-140. Callahan, L. T. 1974. Purification and characterization of Pseudomonas aeruginosa exotoxin. Infect. Immun. 9:113-118. Collier, R. J. 1975. Diphtheria toxin: mode of action and structure. Bacteriol. Rev. 39:54-85. Collier, R. J., and J. Kandel. 1971. Structure and activity of diphtheria toxin. I. Thiol-dependent dissociation of a fraction of toxin into enzymatically active and inactive fragments. J. Biol. Chem. 246:14961503. Cooper, L. Z., M. A. Madoff, and L. Weinstein. 1966. Heat stability and species range of purified staphylococcal a-toxin. J. Bacteriol. 91:1686-1692. Iglewski, B. H., and D. Kabat. 1975. NAD-dependent inhibition of protein synthesis by Pseudomonas aeruginosa toxin. Proc. Natl. Acad. Sci. U.S.A. 72:2284-2288. Iglewski, B. H., M. B. Rittenberg, and W. J. Iglewski. 1975. Preferential inhibition of growth and protein synthesis in Rous sarcoma virus transformed cells by diphtheria toxin. Virology 65:272-275. Landsteiner, K., and R. von Rauchenbichler. 1909. Uber das Verhalten des Staphylolysins bein Erwarmen. Z. Immunitaetsforsch. 1:439-448. Liu, P. V. 1973. Exotoxins of Pseudomonas aeruginosa. I. Factors that influence the production of exotoxin A. J. Infect. Dis. 128:506-513. Liu, P. V., S. Yoshii, and H. Hsieh. 1973. Exotoxins of Pseudomonas aeruginosa. II. Concentration, purification, and characterization of exotoxin A. J. Infect. Dis. 128:506-513. Ludovici, P. P., F. A. Roinestad, and F. S. Wong. 1975. Column chromatography and cell culture assay of Pseudomonas aeruginosa toxin Z preparations. Appl. Microbiol. 30:293-297. Maizel, J. V. 1971. Polyacrylamide gel electrophoresis of viral proteins, p. 180. In K. Maramorosch and H. Koproski (ed.), Methods in virology, vol 5. Academic Press, New York. Monohar, M., S. Kumar, and R. K. Lindorfer. 1966. Heat reactivation of the a-hemolytic, dermonecratic, and lethal activities of crude and purified staphylococcal a-toxin. J. Bacteriol. 91:1681-1685. Miwatani, T., Y. Takeda, J. Sakurai, A. Yoshihara, and S. Tzga. 1972. Effect of heat (Arrhenius effect) on crude hemolysin of Vibrio parahaemolyticus. Infect. Immun. 6:1031-1033. Pavlovskis, 0. R., and F. B. Gordan. 1972. Pseudomonas aeruginosa exotoxin: effect on cell cultures. J. Infect. Dis. 125:631-636. Takeda, Y., Y. Hari, and T. Miwatani. 1974. Demonstration of a temperature-dependent inactivating factor of the thermostable direct hemolysin in Vibrio parahaemolyticus. Infect. Immun. 10:6-10. Takeda, Y., Y. Hari, S. Taga, J. Sakurai, and T. Miwatani. 1975. Characterization of the temperature-dependent inactivating factor of the thermostable direct hemolysin in Vibrio parahaemolyticus. Infect. Immun. 12:449-454.

Temperature-dependent inactivating factor of Pseudomonas aeruginosa exotoxin A.

Vol. 13, No. 5 Printed in U.S.A. INFECTION AND IMMUNITY, May 1976, p. 1467-1472 Copyright X) 1976 American Society for Microbiology Temperature-Depe...
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