162

Bioehimiea et Biophysica Acta, 1138(1992) 162-166 © 1992 Elsevier Science Publishers B.V. All rights reserved 1)925-4439/92/$115.l}11

BBADIS 61116

Conformational integrity of a recombinant toxoid of Pseudomonas aeruginosa exotoxin A containing a deletion of glutamic acid-553 Kevin P. Killeen * a n d R. J o h n Collier Department of Microbiology and Moh'eular Genetics, Hart,ard Medical School and Shipley Institute of Medicine, Boston, MA (U.S.A.)

(Received 27 August 1991)

Key words: Recombinant toxoid: Deletion: Conformation A mutant form of Pseudomonas aeruginosa exotoxin A (ETA) carrying a deletion of glutamic acid-553, an important active-site residue, was expressed in an ETA-negative strain of P. aeruginosa and shown to be exported from the cells as efficiently as wild-type ETA. The mutant protein, purified from the culture medium, was devoid of ADP-ribosyltransferase activity. Protein conformation was barely perturbed by the deletion, as determined by a number of measures, including affinity for substrate NAD, proteinase sensitivity, absorbance and fluorescence spectroscopy, and differential scanning calorimetry. The conformationai integrity and stability of the mutant toxin are consistent with potential use of the protein in vaccines or as a carrier in preparing conjugate vaccines.

Exotoxin A (613 residues) is the most toxic of several virulence factors of Pseudomonas aeruginosa [1]. Reports that passive immunization against E T A confers significant protection to burned mice [2,3] and that high serum antitoxin levels are correlated with a greater chance of survival in humans with P. aeruginosa infections [4,5] have spurred interest in developing a vaccine against this toxin. Earlier work has demonstrated that wild-type E T A can be inactivated by chemical modification with glutaraldehyde, formalin or formalin-lysine [6,7], or by photoaffinity inactivation [8]. In each case, however, the product retained slight activity, reverted to a partially active state under certain conditions or was altered antigenically. In an earlier study from this laboratory, we reported a mutant form of E T A (ETA-E553A) in which glutamic acid-553 (Glu-553 or E553) had been deleted [9]. This 'recombinant toxoid', which was synthesized in

Escherichia coli, showed no detectable ADP-ribosyltransferase, NAD-glycohydrolase or cytotoxic activity; but antiserum raised against the protein inhibited ADP-ribosyltransferase and cytotoxic activities and immunization with ETA-E553A protected mice from lethal doses of wild-type ETA [9]. In the current study, we first transformed the cloned mutant and wild-type forms of E T A into a toxin-minus (tox-) strain of Pseudornonas aeruginosa PAl03, with the goal of facilitating isolation and purification of the proteins. P. aeruginosa is known to export E T A to the culture medium, unlike E. coli, in which the toxin is secreted into the periplasmic compartment [10]. The mutant and wild-type proteins were exported equally well from P. aeruginosa and were isolated from the culture medium, purified and characterized. The results demonstrate that the drastic reduction in enzymic and cytotoxic activities due to the deletion of Glu-553 is accompanied only by slight perturbation of protein structure. This suggests that the approach of deleting an active-site residue may be generally useful in generating toxoids by recombinant D N A technology.

* Present address: Virus Research Institute, 61 Moulton Street, Cambridge, MA 02139, U.S.A. Abbreviation: ETA, exotoxin A.

Materials and Methods

Correspondence: R.J. Collier, Department of Microbiology and Molecular Genetics, Harvard Medical School and Shipley Institute of Medicine, 260 Longwood Avenue, Boston, MA 02115, U.S.A.

Bacterial strains and plasmids. An ETA-negative strain of P. aeruginosa PAl03 constructed by Stephen Lory, University of Washington, (WA, U.S.A.) by inser-

Introduction

163 tion of the tetracycline resistance gene within the coding sequence of the exotoxin A gene, was generously provided to us. Plasmid pRC357, kindly provided by Rockford Draper, University of Texas, (TX, U.S.A.) is a bifunctional vector, capable of replication in E. coli and P. aeruginosa [11]. pCDPT2, encoding wild-type ETA from P. aeruginosa PAK [10], and pCDPT2E553A [12] were constructed as described, pRCCD or pRCCDE553A was constructed by inserting a 2.1 kb EcoR1 fragment from pCDPT2 or pCDPT2E553A, respectively, into a unique EcoR1 site of pRC357. All restriction enzyme digestions were performed as described [13], with enzymes from New England Biolabs. Protein purification. E T A from P. aeruginosa PAl03 was purified as described [14]. Toxin-negative PA 103 (pRCCD or pRCCDE553A) was grown overnight in L-broth supplemented with 20 p~g/ml tetracycline and 300 p~g/ml carbenicillin. The culture was diluted to an initial A550 of 0.1 in 8 1 of the same medium supplemented with 1.9 g/1 nitrilotriacetic acid and grown at 32°C for 1 h, when isopropyl /3-D-thiogalactopyranoside (IPTG, Sigma) was added to a final concentration of 1 mM. Cultures were grown to a final A55o of 5.0, and culture supernates were concentrated to 100 mi with a mini-tangential flow apparatus (Millipore) and dialyzed against several exchanges of 10 mM Tris-HCl, 1 mM E D T A (pH 8.0) at 4°C. Chromatography on immunoaffinity and FPLC anion-exchange columns (Mono-Q, Pharmacia) was performed as described by Madshus and Collier [15]. Immunodetection of protein was performed as described [15]. Polyacrylarnide gel electrophoresis. Proteins were boiled for 10 rain in the presence of sodium dodecyl sulfate (SDS) and separated on a 11.25% polyacrylamide gel, as described by Laemmli [16]. Samples were analyzed for total protein by Coomassie blue staining [17] or for ETA-related material by immunoblot analysis [18]. Western blots were performed as described by Douglas et al. [10]. Proteinase digestion. Native E T A or ETA-E553A (200 ~ g / m l , in 50 mM Tris-HCl, pH 8.0) was digested with 5, 10 or 50 p~g/ml chymotrypsin or trypsin at 25°C for 30 min. Activated E T A or ETA-E553A in the presence of 2 ~ M NAD was digested with 2.5, 10, or 25 ~ g / m l thermolysin at 25°C for 30 rain. Activation and assay of ETA and ETA-E553A. Purified wild-type ETA and ETA-E553A were activated in the presence of 6 M urea and 2 mM DT-F, and carboxymethylated, by the procedure of Carroll and Collier [19]. ADP-ribosyltransferase activity of native or carboxymethylated ETA or ETA-E553A was measured as described [9]. Differential scanning calorimetry. Thermal properties of ETA or ETA-E553A were determined on a Microcal MC-2D differential scanning calorimeter. ETA or ETA-E553A (1.5 m g / m l in 50 mM sodium phosphate,

pH 8.0) was scanned from 20 to 80°C at a rate of 90°C/h with filter constant maintained at 15 s. The calorimeter was interfaced to an IBM PC microcomputer using a converter board (Data Translation-DT2801) for automatic data collection and interpretation. Data were deconvoluted with DA-2 software (Microcad to determine enthalpy ( A H ) values. Results

Wild-type ETA and ETA-E553A were subcloned into a bifunctional vector, pRC357 [11], and the resulting plasmids were transformed into a tox- strain of P. aeruginosa PAl03 (pRCCD or pRCCDE553A), induced for expression and tested for secretion. The two proteins were exported equally well, to a level of approx. 1.7 mg/I, and in each case constituted the most abundant protein in the culture medium (Fig. 1). The culture supernates were concentrated and the proteins purified, initially on an anti-ETA-E553A immunoaffinity column (30-fold purification) and finally on a Pharmacia Mono-Q column. Each product gave a single band on SDS-polyacrylamide gels. The full-length proq~

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2

3

4

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Fig. 1. Purity of ETA-E553A after each step in purification as determined by electrophoretic gel band patterns. Secreted proteins from exotoxin A - PAl03 (pRCCD A553) were isolated and ETAE553A was purified as described under Materials and Methods. Samples removed after each step were separated on an 11.25% SDS-polyacrylamide gel and stained with Coomassie blue. Lanes: 1, M r standards; 2, wild-type ETA; 3, total secreted fraction; 4, following immunoaffinity chromatography; and 5, following anion-exchange chromatography (Mono-Q Sepharose).

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NAB (uM) Fig. 2. NAD-dependent quenching of intrinsic fluorescene of native or activated ETA wild-type and ETA-E553J. Native ETA, A; ETAE553A, i ; activated ETA, ©; or ETA-E553zl, e, 150 nM, dissolved in TE 10, was excited at 290 nm (1 nm bandpass) and emission at 335 nm (10 nm bandpass) was monitored as a function of the concentration of NAD. Corrections for beam attenuation were made by titrating a solution of tryptophan with NAD in parallel. Fluorescence measurements were on a SPF-500C Spectrofluorometer (SLM Aminco).

teins were the only toxin-related species detectable by Western blot analysis throughout the purification, implying resistance of both proteins to attack by contaminating proteinases (data not shown). ETA-E553A prepared from the tox- P. aeruginosa PAl03 possessed no detectable ADP-ribosyltransferase activity ( < 5 • 10 -5 that of wild-type toxin; data not shown), a finding consistent with results obtained with impure ETAE553A from E. coil [9]. Affinity for a ligand may be used as a measure of the conformational integrity of a protein, and we used quenching of intrinsic protein fluorescence to measure affinity of NAD for wild-type or mutant E T A (Fig. 2). Native ETA is a proenzyme, which may be activated by

partial unfolding and reduction. We therefore first treated wild-type and mutant ETA with urea and dithothreitol, followed by carboxymethylation, to maximize both the enzymic activity and the potential to bind NAD. NAD quenched intrinsic fluorescence in the activated wild-type or mutant ETA and EadieHofstee analysis of the data yielded similar dissociation constants for the two proteins (K