Proc. Natl. Acad. Sci. USAVol. 77, No. 12, pp. 7199-7203, December 1980Biochemistry

Isolation and characterization of a Pseudomonas aeruginosa mutantproducing a nontoxic, immunologically crossreactive toxin A protein

(bacterial toxins/ADP-ribose-transferase)

STANLEY J. CRYZ, JR., RICHARD L. FRIEDMAN, AND BARBARA H. IGLEWSKIDepartment of Microbiology and Immunology, University of Oregon Health Sciences Center, Portland, Oregon 97201

Communicated by Kay D. Owen, September 12, 1980

ABSTRACT Nitrosoguanidine mutagenesis of Pseudomonasaeru inosa strain PAW- yielded a mutant strain, PAO-PR1,which produced a protein that was immunologically indistin-guishable from native toxin A and was nontoxic for culturedChinese hamster ovary cells. In contrast to native toxin, thecell-associated and extracellular crossreactive material (CRM),designated "CRM protein," possessed no adenosine diphos-phate-ribosylating activity. This CRM protein comigrated withnative toxin A on sodium dodecyl sulfate/polyacrylamide gels,could be immunoprecipitated with antitoxin from culture su-pernatants of strain PAO-PR1, and gave a reaction of identityin immunological assays. Equivalent amounts of toxin A antigenand CRM protein antigen were produced in liquid culture bytheir respective strains as quantitated in a radioimmunoassayfor toxin A. These data suggest that mutant strain PAO-PR1possesses one or more missense mutations within the structuralgene for toxin A that adversely affect enzymatic activity, therebyrendering the molecule nontoxic.

Pseudomonas aeruginosa is a leading cause of infections incompromised hosts (1, 2). P. aeruginosa synthesizes a numberof extracellular products that are believed to be involved in thepathogenesis of these infections (3, 4). The most toxic of theseproducts, a protein toxin termed "toxin A," is a potent inhibitorof eukaryotic protein synthesis (5, 6). Toxin A has been shownto act in a manner similar, if not identical, to that of diphtheriatoxin. Both toxins catalyze the transfer of the adenosine di-phosphate-ribosyl (ADP-Rib) moiety of NAD+ onto eukaryoticelongation factor 2, thereby rendering it nonfunctional (6-9).

Although the enzymology of toxin A has been studied in-tensely (8-11), little is known about either the immunochem-istry or structure-functional "domains" of the toxin molecule.Several reports have presented indirect evidence that suggeststoxin A (Mr 70,000) may possess two domains-one that con-tains the enzymatic active site and the other that functions inbinding the toxin to sensitive eukaryotic cell membranes.Culture supernatants of P. aeruginosa strain PA-103 have beenshown to contain an ADP-Rib-transferase of Mr 26,000-30,000that demonstrates partial immunological crossreactivity withnative toxin (9). The spontaneous appearance of this enzymein culture supernatants is believed to be caused by the cleavageof intact toxin by Pseudomonas proteases (9). Lory and Collier(12) have reported the generation of an enzymatically activepolypeptide (Mr 26,000) from intact toxin by partial digestionwith chymotrypsin in the presence of NAD. Purified toxin Asubjected to repeated freeze/thaw cycles dissociates into twopolypeptides (13). One polypeptide (Mr 27,000) possessedADP-Rib-transferase activity. It was postulated that the otherpeptide (Mr 45,000), which was enzymatically inactive, con-tained the membrane-binding region of the toxin molecule(13).

The publication costs of this article were defrayed in part by pagecharge payment. This article must therefore be hereby marked "ad-vertisement" in accordance with 18 U. S. C. §1734 solely to indicatethis fact.

7199

The isolation of a series of mutant j phages that code foraltered diphtheria toxin molecules that crossreact serologicallywith native toxin (crossreactive materials, CRMs) have provedto be extremely valuable in studying the biochemistry andimmunochemistry of the diphtheria toxin molecule (14-17).In an effort to study further the various structure-functionaspects of toxin A, we have attempted to isolate a similar classof mutant strains of P. aerugmnosa that synthesize altered toxinmolecules. The isolation and characterization of one such mu-tant is described in this report.

MATERIALS AND METHODSBacterial Strains and Growth Conditions. P. aeruginosa

strain PAO-1 has been described in detail (18). This strain hasbeen shown to produce toxin A, alkaline protease, and elastase,and it is virulent in a variety of animal models (unpublisheddata). Cultures were routinely grown in the trypticase soy brothdialysate (TSBD) medium of Liu (19), which was treated withChelex 100 chelating resin (minus 400 mesh, Bio-Rad) to re-move iron as described (20). Culture conditions for toxin pro-duction were as follows: 0.1 ml of an overnight culture (18 hr,32°C) was innoculated into 10 ml of low-iron TSBD mediumper 250-ml erlenmeyer flask. Cultures were grown for 20 hr at32°C on a reciprocating water bath (Lab-Line) to give maximalaeration. Cultures were harvested by centrifuging at 12,000 Xg for 15 min at 4°C and were sterilized by membrane filtration(0.45 Am, Millipore).Chemical Mutagenesis. Midlogarithmic-phase cells (A540

of 10 ml = 0.25) were harvested by centrifugation at 12,000 Xg for 15 min. Cells were washed in 10 ml of buffer (50 mMTris/50 mM maleic acid, pH 6.0) and resuspended in 5 ml ofbuffer. N-Methyl-N'-nitro-N-nitrosoguanidine (Sigma) wasadded to a final concentration of 50 ug/ml. Cultures were in-cubated for 60 min at 320C, washed twice in low-iron TSBDmedium, resuspended in 10 ml of low-iron TSBD medium, andincubated for 20 hr at 320C to allow mutant clones to segregate.The above mutagenesis procedure resulted in a decrease ofapproximately 50% in culture viability.

Cytotoxicity Assay. The Chinese hamster ovary (CHO) cellcytotoxicity assay was performed as described (21). The mini-mal cytotoxic dose for 2 X 104 CHO cells was approximately0.01 ng of purified toxin A.Assay for the Identification of Nontoxic Mutant Clones.

To facilitate the screening of a large number of mutagen-treated clones for a nontoxinogenic phenotype, techniquessimilar to those of Murphy et al. (22) were used. To each wellof a conical-bottomed 96-well polystyrene microtiter plate(Linbro) was added 200 Al of low-iron TSBD medium. Single

Abbreviations: CRM, crossreacting material; RIA, radioimmunoassay;ADP-Rib, adenosine diphosphate ribose; CHO, Chinese hamster ovary;TSBD, trypticase soy broth dialysate.

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colonies from mutagen-treated cultures pregrown on nutrientagar (Difco) plates were placed into each well, and the plateswere incubated for 20 hr at 320C. Bacterial cells were thenpelleted by centrifugation of the microtiter plates for 20 minat 2500 X g. Approximately 2 Al of supernatant culture wastransferred from each microtiter well to a corresponding flat-bottomed microtiter well freshly seeded with 2 X 104 CHOcells. Plates were incubated for 72 hr at 370C in the presenceof 5% C02/95% air and scored colorimetrically as described(21). Plates containing bacterial cultures were immediatelyfrozen at -20'C after removal and transfer of the supernatant.Control experiments showed that 2 Ml of strain PAO-1 culturesupernatant was toxic for CHO cells under the above conditions.Furthermore, this toxicity was completely neutralized by theinclusion of specific antitoxin. Control experiments furtherdemonstrated that when cultures of PAO-1 were grown formaximal toxin production (in 10 ml of low-iron TSBD mediumper 250-ml erlenmeyer flask for 20 hr at 320C with maximumaeration), 0.5 Al of culture supernatant was the largest quantitythat could be tested in which cytotoxicity could be neutralizedby antitoxin. In contrast, the culture supernatant from themutant strain PAO-T1, which produced no detectable extra-cellular toxin (23), was found to be nontoxic at the abovelevels.

Antisera. Antitoxin was prepared against highly purifiedtoxin A as described (13). IgG from hyperimmune antitoxin(antitoxin IgG) was collected by repeated precipitation of crudeantiserum with ammonium sulfate as described (21).

Immunological Assays. The agar-well assay for the detectionof toxin A production by actively growing bacterial cells wasperformed as described (24). A liquid-phase radioimmunoassay(RIA) for toxin A was developed and is described here. Car-rier-free Na'25I [1 mCi (1 Ci = 3.7 X 1010 becquerels); Amer-sham] was added to 50 ,ug of toxin and 10 ,ug of lactoperoxidasein 1 ml of phosphate-buffered saline (pH 7.2). The enzymaticiodination reaction was initiated by the addition of a 25-M41 al-iquot of 0.03% H202 (25). After a 5-min incubation at 220C,an additional 25-Ml aliquot of 0.03% H202 was added, and thereaction continued for 5 min. The labeled preparation was di-alyzed for 18 hr at 4°C against 1 liter of saline. The dialyzedsample was applied at 4°C to a 2.2 X 30 cm Sephadex G-100column (88-ml bed volume) equilibrated with phosphate-buffered saline containing 1 mg of bovine serum albumin perml and was eluted with the same buffer. Fractions containingthe radiolabeled toxin were pooled. Sodium azide was addedto a final concentration of 0.02%, and the samples were storedat 40C. Samples had specific activities of at least 2 ,Ci/,ug oftoxin protein and were greater than 85% immunoprecipitablein the presence of excess antitoxin (see below).IgG Sorb (Enzyme Center, Boston, MA), a formalin-fixed

preparation of a protein A-bearing strain of Staphylococcusaureus, was used as a particulate immunoadsorbent for immunecomplexes containing IgG (26). Lyophilized samples were re-constituted to 10% (vol/vol) in 150 mM NaCl/5 mM EDTA/50mM Tris, pH 7.4 (NET buffer), and were stored at 4°C. Im-mediately prior to use in RIAs, cells were pelleted by centrif-ugation at 4000 X g for 15 min at 4°C. Cells were resuspendedto 10% (vol/vol) in NET buffer containing 0.5% Nonidet P-40(Particle Data Laboratories, Elmhurst, IL) and incubated for10 min at 22°C. The cells were then washed once with NETbuffer containing 0.05% Nonidet P-40 and finally were resus-pended to 10% (vol/vol) in assay buffer (NET buffer containing0.05% Nonidet P-40 and 1 mg of bovine serum albumin perml).

RIAs were carried out in 0.5 ml (final volume) in 10 X 75mmpolystyrene test tubes. Assay buffer was used as diluent for all

reagents. Reagents were added in the following sequence: (i)assay buffer, (ii) '25I-labeled toxin (approximately 10,000 cpmin 10 ,l), (iii) nonradioactive antigen (either purified toxin orculture supernatants sterilized by membrane filtration; 5-100,Ml per assay), and (iv) antitoxin (0.01 ,l, sufficient to immu-noprecipitate 50-60% of the total radioactivity in the absenceof competing antigen). Assay mixtures were incubated for 15min at 220C, and then 50 ,l of prepared IgG Sorb was added.The samples were incubated an additional 10 min at 22"C.Immune complexes were collected by centrifugation at 4000X g for 15 min at 40C and were washed twice with 1 ml of assaybuffer. The final pellets were assayed for radioactivity in aBeckman Biogamma counter.

ADP-Rib-Transferase Assay. The ability of culture super-natants and purified toxin A to transfer ADP-Rib to wheat germEF-2 was determined as described (21). Culture supernatants(10 ,l per assay) were tested both before and after activationwith urea/dithiothreitol (10). Biologically active toxin A wasquantitated by comparing the enzyme activity per volume ofculture supernatant to a standard curve generated by usingpurified toxin A as described (21).

Extraction of Cell-Associated Toxin. Cells (20 ml) grownunder conditions for maximal toxin production as describedabove were washed twice in 50 mM Tris buffer (pH 7.0),resuspended in half the original volume of the same buffer, anddisrupted by sonication with eight 30-sec bursts (50% maximaloutput) using a Biosonik IV (Bronwill) sonicator. After eachburst, cells were placed on ice for 30 sec. Samples examined bylight microscopy showed almost total cell lysis. Cellular debriswas removed by centrifugation at 15,000 X g for 20 min at 40C.Sonicates were concentrated 10-fold in an Amicon microsoluteconcentrator (Amicon, Lexington, MA) and then were dialyzedagainst 500 vol of Tris buffer for 18 hr.NaDodSO4/Polyacrylamide Gel Electrophoresis. Na-

DodSO4/polyacrylamide slab gel electrophoresis was per-formed as described (27). Gels composed of 10% (wt/vol) re-

solving gel and a 4.5% (wt/vol) stacking gel were cast at a

thickness of 0.75 mm. Crude culture supernatants were steril-ized by membrane filtration (0.45 Mlm; Millipore) and were

concentrated 10-fold by filtration through an Amicon PM-10membrane in a nitrogen atmosphere. Concentrates were di-alyzed against 500 vol of 50 mM Tris (pH 7.8) at 4°C. Immu-noprecipitation of toxin and mutant protein was accomplishedby the addition of 10 Ml of antitoxin IgG to 2 ml of concentratedsupernatant. The mixture was incubated for 15 min at 220C.Then 200 Ml of a 10% IgG Sorb solution was added and gentlymixed for 15 min at 22°C. Immune complexes were removedby centrifugation and washed in 2 ml of 50mM Tris (pH 7.0).Adsorbed antibody-antigen complexes were removed from thefixed staphylococcal cells by heating at 100°C for 5 min in thepresence of 5% (vol/vol) 2-mercaptoethanol and 1.25% (wt/vol)NaDodSO4. The staphylococci were removed by centrifugation,and an appropriate amount of the supernatant was applied ontothe gel. Gels were electrophoresed at room temperature at a

constant power of 1.2 W/gel in the buffer system as described(26). Gels were stained with 0.05% Coomassie brilliant blueR-250 in 25% (vol/vol) isopropanol and 10% (vol/vol) aceticacid and were destained in 10% acetic acid.

RESULTS

Approximately 22,000 mutagen-treated clones were screenedby the CHO cell assay, resulting in the identification of fourindependently derived mutant strains that displayed a nontoxicphenotype. Mutant and parental strains were recloned andgrown in liquid culture under optimal conditions for toxin Aproduction. The culture supernatants were assayed for toxin-

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induced CHO cell toxicity, enzymatic activity, and toxin an-.tigen concentration (Table 1). All mutant strains were foundto be negative for toxin-induced CHO cell cytotoxicity. Neitherenzyme activity nor toxin antigen could be detected in culturesupernatants from mutant strains PAO-A2, PAO-B16, andPAO-C15. The culture supernatant from mutant PAO-PR1 alsowas found to possess no enzyme activity either before or aftertreatment with urea/dithiothreitol. The PAO-PR1 supernatant,however, contained parental levels of toxin antigen (Table 1).These preliminary results suggested that mutant strains PAO-A2, PAO-B16, and PAO-C15 were regulatory mutants whosenontoxic phenotype was because of the absence of productionor the hypoproduction of toxin A. Mutant strain PAO-PR1appeared to be producing a CRM protein, with its nontoxicphenotype caused by a mutation that adversely affected theenzymatic activity of toxin A.The phenotypic stability of strain PAO-PR1 was tested in the

following manner. A single colony was inoculated into 10 mlof low-iron TSBD medium and was grown for 18 hr at 320C.The culture was diluted and plated for single colonies. A singlecolony was then used to inoculate fresh medium. This cycle wasrepeated five times. Ten colonies from the final cycle wereselected and grown under conditions for optimal toxin pro-duction. Culture supernatants from all of these cultures con-tained toxin antigen but no enzyme activity.The CRM protein produced by strain PAO-PR1 was im-

munologically indistinguishable from native toxin (Figs. 1 and2). In a liquid phase RIA for toxin A, increasing quantities ofculture supernatant from strains PAO-1 and PAO-PR1, whichcontained nearly identical toxin A antigen concentrations (1.96and 2.10 yg/ml, respectively), gave superimposable lineardisplacement curves (Fig. 1). In addition, when tested in theagar-well immunodiffusion assay against antitoxin, a band ofidentity and equal intensity was formed between the mutant(PAO-PR1) and parental (PAO-1) strains (Fig. 2). In contrast,mutant strains PAO-A2, PAO-B15, and PAO-C16 gave noprecipitin band with antitoxin, and culture supernatants fromthese strains gave no displacement of labeled antigen in the RIAfor toxin A (data not shown).

Native toxin and CRM protein were isolated from culturesupernatants of strains PAO-1 and PAO-PR1 by immunopre-cipitation with antitoxin IgG and fixed staphylococci. Culturesupernatant from the toxin-deficient strain PAO-T1 and un-inoculated growth medium were treated in a similar manner

Table 1. Characteristics of mutant strainsADP-Rib-transferase

Toxin-induced activitytBacterial CHO cell Unacti- Acti- Antigen,strain cytotoxicity* vated vated ysg/mlt

PAO-1 (parental) + 21 344 1.02PAO-T1 - 0 0 0PAO-A2 - 0 0 0PAO-C15 - 0 0 0PAO-B16 - 0 0 0PAO-PR1 - 0 0 1.16

Cultures (10 ml) were grown for 20 hr at 32°C, the supernatantsharvested and sterilized by membrane filtration. Three independentexperiments gave similar results.* Based upon the ability of 0.5 ul of culture supernatant to preventa color change. This is the largest quantity of culture supernatantin which cytotoxicity can be completely neutralized by antitoxin.

t Expressed as cpm/10 ,ul of sample assayed for 30 min at 25°C. Thelevel of detectability for this assay is 0.1 ng of toxin A per ml.

t Quantitated by RIA in which the level of detectability is 0.5 ng oftoxin A per ml.

10

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6

5

4

3

1

0 25 50Culture supernatant, gg

Toxin, ngFIG. 1. RIA for toxin A: competition for antitoxin binding by

culture supernatants of P. aeruginosa PAO-1 and PAO-PR1. 1/B,Fraction of radiolabeled toxin bound; *, purified toxin; A, PAO-1culture supernatant; 0, PAO-PR1 culture supernatant.

and were used as controls. The immunoprecipitates weresubjected to NaDodSO4/polyacrylimide slab gel electropho-resis, and the resulting profiles are shown in Fig. 3. A proteinthat comigrated with purified toxin A was immunoprecipitatedfrom both PAO-1 and PAO-PR1 culture supernatants. A cor-responding band was not seen with PAO-T1 immunoprecipi-tates. The other bands seen on the gels were identified as arisingfrom the staphylococcal cell-IgG proteins, which were solu-bilized during the dissociation procedure. Other than thepresence or absence of the toxin band, the gel profiles of theimmunoprecipitates from the three strains were essentiallyidentical to what was seen when inoculated growth mediumwas immunoprecipitated (data not shown). These results showthat the CRM protein produced by strain PAO-PR1 has a mo-lecular weight similar to that of native toxin A.

These results indicate that strain PAO-PR1 produced a CRMprotein that is nontoxic because of a loss of enzymatic activity.The production of such an altered protein conceivably couldbe caused by either (i) a mutation within the toxin structuralgene or (ii) improper processing of the synthesized toxin mol-

FIG. 2. Agar-well assay of strains PAO-1 and PAO-PR1. Cultureswere grown for 24 hr at 320C. Antitoxin IgG (4 Ml) was added to thecenter well and plates were reincubated for an additional 24 hr at320C.

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1 2 3 4 5 6

.

a2oad e tAedS lo-

n

cocci and the mixture was heated at 1001C for 5min. Twentyul of this

mixture was applied onto the gel. Toxin (2 jMg) and molecular weightstandards (5 M~g) were also heated at 100cC for 5 min in a Na-

DodSO4S2-mercaptoethanol solution before application on the gel.Well 1, toxin A; well 2, immunoprecipitate of strain PAO-1; well 3,

immunoprecipitate of strain PAO-T1 culture supernatant; well 4,

immunoprecipitate of strain PAO-PR1 culture supernatant; well 5,

toxin A; well 6, standards in daltons (from top to' bottom): phos-

phorylase b (92,000), bovine serum albumin (68,000), pyruvate kinase

(57,000), ovalbumin (45,000), lactate dehydrogenase (36,000), soybean

trypsin inhibitor (21,000), and lysozyme (14,000).

ecule within the bacterial cell. In an attempt to differentiate

between the two possibilities, cell-associated toxins from strains

PAO-PR1 and PAO-1 were isolated and compared. Ten-fold

concentrates of culture supernatants from both strains contained

similar amounts of toxin antigen, but again enzymatic activitycould only be detected in the PAO-1 concentrates (Table 2). The

enzymatic activity of extracellular toxin A increases followingpreincubation with urea/dithiothreitol (10, 11, 13). In contrast,

cell-associated toxin from strain PAO-L was found to be in the

activated form, with enzymatic activity decreasing after

urea/dithiothreitol treatment. Similar findings have been re-

ported for P. aeruginosa strain PA-10S (28). In contrast, cell-

associated toxin derived from strain PAO-PR1 was enzymati-

cally inactive before and after activation with urea/di-thiothreitol. The enzymatic activity of PAO-1 cell-associated

toxin was completely neutralized with antitoxin IgG (data not

shown). Surprisingly, we were unable to detect cell-associatedtoxin antigen by RIA in sonicates from either strain PAO-1 or

Table 2. Enzymatic activity of cell-associated and extracellulartoxin from strains PAO-1 and PAO-PR1

Enzymatic activitytToxin Unacti- Acti- Antigen,

Strait source* vated vated ,ug/mlt

PAO-1 Cell-associated 400 33 NRExtracellular 319 1153 4.32

PAO-PR1 Cell-associated 0 0 NRExtracellular 0 0 3.88

* Cell-associated toxin was derived from sonicated cells. For extra-cellular toxin, crude culture supernatants were concentrated 10-foldand dialyzed before being assayed.

t Expressed as cpm/10 A1 of sample assayed for 30 min at 250C.Quantitated by RIA in which the level of detectability is 0.5 ng oftoxin A per ml; NR, nonreactive.

PAO-PR1, even though enzymatic activity was present inPAO-1 sonicates. Sonicates from strains PAO-1 and PAO-PR1were treated with Triton X-100 (final concentration, 0.5%) priorto testing in RIA to determine if cell-associated toxin might beheld in an immunologically unreactive complex. Control ex-periments showed that detergent treatment did not adverselyaffect the enzymatic activity of PAO-1 sonicates (data notshown). Cell-associated toxin antigen could not be detected byRIA in the detergent-treated sonicates from either strain.

DISCUSSIONAlthough intensely studied, many aspects of the structure-function and immunochemistry of the toxin A molecule remainunknown. Toxin A is known to be released from the bacterialcell predominantly in the proenzyme form (9-11). Expressionof enzymatic activity is dependent upon alterations in theconformation of the toxin molecule, which involves breakageof intramolecular disulfide bonds, thereby exposing the enzymeactive site (10, 11). An enzymatically active fragment thatimmunologically crossreacts with toxin has been isolated andcharacterized (9, 13). However, the relationship of this fragmentto intact toxin and the physical location of the enzyme activesite within the toxin molecule are unknown. Another fragmentderived from intact toxin that may possess the functional "do-main" responsible for binding to sensitive eukaryotic cells hasbeen identified (13). This fragment has not yet been isolatedin a biologically active form.

In an effort to answer various questions regarding thestructure-function and immunochemistry of toxin A, we areattempting to isolate mutant strains that synthesize alteredproteins serologically related to native toxin (CRMs). In thepresent study we report the isolation of one such mutant strain,PAO-PR1. The CRM protein produced by strain PAO-PR1 isnontoxic for CHO cells apparently because of a loss of enzy-matic activity that is essential for the toxicity associated withthe molecule. The alteration(s) within the CRM protein thatresult in loss of enzymatic activity do not detectably alter theantigenicity of the molecule as evidenced by (i) a band ofidentity with toxin A when tested by gel immunodiffusionagainst antitoxin (Fig. 2) and (ii) the ability of the CRM proteinto compete equally with toxin for antitoxin binding in RIA (Fig.1). The fact that the CRM protein and native toxin comigrateon NaDodSO4/polyacrylamide gel electrophoresis indicatesthat they have an identical, or near identical, molecular weight(Fig. 3). It was somewhat surprising that the sonicates of theparental strain PAO-1, which contained enzymatic activity thatcould be neutralized by antitoxin, did not compete in RIA. Thismay be caused by the conformation of cell-associated toxinbeing such that, although enzymatic activity is expressed, theportion of the toxin molecule against which the predominance

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of antibody is directed may be altered and, hence, unrecog-nizable. The fact that expression of enzymatic activity of cell-associated toxin is not dependent upon unfolding of the toxinmolecule by urea/dithiothreitol treatment, as is extracellulartoxin, indicates that intracellular and extracellular toxin A differin configuration. Given the lack of information concerning theprocesses involved in the synthesis and release of toxin A, wecannot rule out the possibility that the loss of activity of theCRM protein is because of improper processing of the toxinmolecule. However, the fact that there is no cell-associatedenzymatic activity in strain PAO-PR1 sonicates and the fact thatequivalent quantities of extracellular antigen are found in themutant and parental strains make this unlikely. The abovefindings are consistent with the hypothesis that strain PAO-PR1contains at least one missense mutation within the structuralgene for toxin A at site(s) that are essential for the expressionof enzymatic activity and toxicity.The CRM protein produced by strain PAO-PR1 (CRM 66)

has properties which are very similar to those of CRM 197, a

nontoxic protein serologically related to diphtheria toxin codedfor by the mutant f3-phage lysogen C7 (fltox-197) (29). CRM197, which has the same molecular weight as native diphtheriatoxin, is enzymatically inactive and nontoxic due to a missensemutation within the fragment A moiety of the toxin molecule(14). The 17,000-dalton COOH-terminal region of the diph-theria toxin molecule, which functions in cell binding (30, 31),is unaltered in CRM 197, as evidenced by its ability to com-pletely block intoxication of cultured cells by native diphtheriatoxin. A single preliminary competitive blocking experimentshowed that the purified CRM 66 protein in a ratio of 500:1(CRM protein:toxin A protein) blocked intoxication of CHOcells by native Pseudomonas toxin A. This observation is sug-gestive, but additional experiments are required to determineif the CRM 66 protein actually binds to sensitive eukaryotic cellmembranes.

Given the immunological identity of the CRM 66 protein andnative toxin, if the purified CRM 66 protein is found to benontoxic in animals, it may have potential for use as a

toxoid.We wish to thank Dr. Lesley Hallick for her help in the analysis of

the data and in the preparation of this manuscript. Expert technicalassistance was provided by Jack Lile and John Bradley. This investi-gation was supported by U.S. Public Health Service Grant IAI-14671from the National Institute of Allergy and Infectious Diseases. R.L.F.was supported by a fellowship from the Cystic Fibrosis Foundation.1. Feigin, R. D. & Shearer, W. T. (1975) J. Pediatr. 87, 677-694.2. Reynolds, H. Y., Levine, A. S., Wood, R. E., Zierdt, C. H., Dale,

D. G. & Pennington, J. E. (1975) Ann. Int. Med. 82, 819-831.

3. Liu, P. V. (1974) J. Infect. Dis. 128,506-513.4. Homma, J. Y. (1977) Jpn. Med. Assoc. 78,275-293.5. Pavlovskis, 0. R. & Shackelford, A. H. (1974) Infect. Immun.

9,540-546.6. Iglewski, B., Elwell, L. P., Liu, P. V. & Kabat, D. (1975) in Pro-

ceedings of the Fourth International Symposium on the In-terconversions of Enzymes, ed. Shaltiel, S. (Springer, NewYork).

7. Iglewski, B. H. & Kabat, D. (1975) Proc. Natl. Acad. Sci. USA72,2284-2288.

8. Iglewski, B. H.., Liu, P. V. & Kabat, D. (1977) Infect. Immun.15, 183-184.

9. Chung, P. W. & Collier, R. J. (1977) Infect. Immun. 16, 832-841.

10. Leppla, S. H. (1976) Infect. Immun. 14, 1077-1086.11. Leppla, S. H., Martin, 0. C. & Muehl, L. A. (1978) Biochem.

Biophys. Res. Commun. 81, 532-538.12. Lory, S. & Collier, R. J. (1980) Infect. Immun. 28, 494-501.13. Vasil, M. L., Kabat, D. & Iglewski, B. H. (1977) Infect. Immun.

16,353-361.14. Uchida, T., Pappenheimer, A. M., Jr. & Greany, R. (1973) J. Biol.

Chem. 248, 3838-3844.15. Matsuda, M., Kanei, C. & Yoneda, M. (1972) Biochem. Biophys.

Res. Commun. 64,43-49.16. Laird, W. & Groman, N. B. (1976) J. Virol. 19, 208-219.17. Holmes, R. K. (1976) J. Virol. 19, 195-207.18. Holloway, B. W., Krishnapillai, V. & Morgan, A. F. (1979) Mi-

crobiol. Rev. 43,73-102.19. Liu, P. V. (1973) J. Infect. Dis. 128, 506-513.20. Bjorn, M. J., Iglewski, B. H., Ives, S. K., Sadoff, J. C. & Vasil, M.

L. (1978) Infect. Immun. 19,785-791.21. Iglewski, B. H. & Sadoff, J. C. (1979) Methods Enzymol. 60,

353-361.22. Murphy, J. R., Bacha, P. & Teng, M. (1978) J. Clin. Microbiol.

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uest

on

Oct

ober

8, 2

020