Cloned diphtheria toxin within the periplasm of Escherichija ... - PNAS

Proc. Nati. Acad. Sci. USA Vol. 86, pp. 343-346, January 1989 Microbiology

Cloned diphtheria toxin within the periplasm of Escherichija coli causes lethal membrane damage at low pH (membrane insertion)

DONALD 0. O'KEEFE AND R. JOHN COLLIER* Department of Microbiology and Molecular Genetics, and The Shipley Institute of Medicine, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115

Communicated by Bernard D. Davis, September 30, 1988

ABSTRACT Acidic pH within endosomal vesicles of sensitive animal cells triggers a conformational change in diphtheria toxin (DT) that is believed to cause the B chain to insert into the vesicular membrane and the enzymic A chain to be released into the cytosol. In artificial lipid bilayers, DT forms ion-conductive channels under mildly acidic conditions (pH -5). Here we report a related phenomenon in Escherchia coli strains that secrete certain cloned DT-related proteins into their periplasm: the cells are rapidly killed at pH 5 but remain unharmed at pH 7. Expression of full-length DT (an active-site mutant, to comply with the National Institutes of Health recombinant DNA guidelines) causes acid-sensitivity, whereas expression of the A chain alone does not. The killed cells are not lysed, but inner-membrane functions are impaired (membrane potential, active transport, and ion impermeability). We propose that acidification of DT within the periplasm induces its insertion into the inner membrane, lethally damaging the permeability barrier. This discovery provides a potentially important selection procedure for mutations affecting the membrane insertion function of DT. Similar approaches may be useful in studying other proteins that undergo conditiondependent interaction with membranes.

inactivating the factor, blocking protein synthesis, and killing the cell. Various gene fragments encoding nontoxic or hypotoxic segments of DT have been cloned and expressed in Escherichia coli (11-13). Fragments containing the signal peptide are secreted to the periplasmic space, with the signal peptide being removed in the process leaving soluble biologically active proteins in this compartment (11, 14). Although the intact structural gene for DT has not yet been cloned, owing to constraints of the National Institutes of Health Guidelines, an intact DT gene containing a 3-base-pair mutation in an active-site residue (E148S) has been reconstructed and expressed (14). This mutation diminished ADP-ribosyltransferase activity and cytotoxicity by several hundred-fold. DT-E148S was secreted to the periplasmic space of E. coli, where it was processed into a stable and apparently correctly folded protein (14). The cloning and expression of a full-length enzymatically inactive form of DT allows us to investigate structural aspects of the toxin having participatory roles in receptor binding and membrane penetration. In this paper we demonstrate that when E. coli containing DT-E148S in the periplasm are exposed to acidic pH, the cells die. Several tests for innermembrane function suggest that DT-E148S inserts into the membrane and disrupts the permeability barrier. The lethal effect creates a positive genetic selection for mutant DT molecules incapable of membrane insertion. Such mutant toxins may be useful in elucidating the molecular mechanisms involved in DT translocation across mammalian cell membranes.

Diphtheria toxin (DT; Mr, 58,342) is representative of a class of toxic proteins that are believed to undergo membrane insertion and/or penetration under the mildly acidic conditions prevailing within certain intracellular membrane-bound vesicles. This class also includes Pseudomonas aeruginosa exotoxin A (1, 2) and modeccin (3, 4), and possibly anthrax toxin (5, 6), tetanus toxin (7, 8), and botulinum toxin (8, 9). Membrane insertion/penetration steps are not well understood for any toxin. This paper describes a method for studying the problem in bacteria. DT is synthesized by Corynebacterium diphtheriae as a single-chain protein containing a signal peptide that is removed during secretion from the bacterium yielding the 535-residue mature toxin. Before or soon after the toxin attaches to sensitive mammalian cells, it is cleaved proteolytically within one of its two disulfide loops; this generates an amino-terminal A fragment of DT (DTA, 193 residues) and a carboxyl-terminal B fragment (DTB, 342 residues), linked by a disulfide bond. After binding to its receptor the toxin undergoes receptor-mediated endocytosis and is conveyed to an endosome (10). Acidification of the endosomal lumen triggers a conformational change in the toxin, leading to membrane insertion by way of hydrophobic segments within DTB. This insertion somehow mediates transfer of DTA to the cytoplasmic face of the membrane, where it is believed to be released after reduction of the disulfide bond linking it to DTB. DTA then catalyzes transfer of the adenosine diphosphate ribose moiety of NAD to elongation factor 2, thereby

and contain coding sequences downstream from the tac promoter, a hybrid promoter inducible with isopropyl 18D-thiogalactopyranoside and containing sequences from both the trp and lacUVS promoters of E. coli (16). pDO1 contains sequences encoding the full-length DT-E148S molecule. pDO3 was derived from pDO1 and encodes DTA-E148S plus 7 amino acids of the amino terminus of DTB, plus 2 amino acids added during cloning. ptacF2-E148S encodes F2E148S, a fragment of DT containing all of DTA-E148S and 55% of DTB. Plasmids used in this study are summarized in Table 1. E. coli JM103 was used in all experiments (15). Unless otherwise noted, all experiments were performed on uninduced cultures. Membrane Potential. Cells grown in L broth to an OD595 of 1.0 were resuspended in 2 mM Tris'HCI/50 mM NaCI, pH 7.0, to 3 x 109 cells per ml. For each experimental point, the cells were diluted 1:10 into 5 mM buffer at various pH values

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Abbreviations: DT, diphtheria toxin; DTA, fragment A of DT; DTB, fragment B of DT. *To whom reprint requests should be addressed.

MATERIALS AND METHODS Plasmids. All plasmids are derivatives of pCDptac2 (15)

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Table 1. Plasmids used in this study Encoded protein Plasmid DT-E148S, a full-length DT mutated at the active pDO1 site DTA-E148S, a DTA mutated at the active site pDO3 plus 7 amino acids of the amino terminus of DTB plus the dipeptide Lys-Ser F2, an enzymically active protein containing ptacF2 wild-type DTA and 55% of the amino terminus of DTB ptacF2-E148S F2-E148S, an F2 mutated at the active site

containing 50 mM NaCl. From this point, the cells were kept at 37°C in a thermoregulated magnetically stirred cuvette. The following buffers were used: Pipes (pH 7.0 and 6.5), Mes (pH 6.0), and sodium citrate/sodium succinate (pH 5.5 and 5.0). The cells were incubated for 1 min and then brought to 100 mM Tris-HCl (pH 7.5). One minute later 3,3'-dipropylthiadicarbocyanine iodide (Molecular Probes, Eugene, OR) was added to a final concentration of 1 ,ug/ml. The fluorescence, measured when the signal stabilized, was divided by the fluorescence at pH 7.0 to obtain relative fluorescence. Fluorescence was measured in a SLM AMINCO (Urbana, IL) SPF-500C spectrofluorometer with excitation at 645 nm and emission at 668 nm. Both slits were set at 5 nm. Proline Transport. Cells grown overnight in potassium-free M9 medium (sodium phosphate was substituted for potassium phosphate) were diluted 1:100 in the same medium. After growing for 5 hr, the cells were resuspended in growth medium to an OD595 of 1.0. Cells (0.1 ml) were then added to 1.9 ml of 20 mM buffer at various pH values containing 4.8 ,uCi (1 Ci = 37 GBq) of L-[2,3,4,5-3H]proline. After 10 min at 37°C, the cells were filtered through Millipore HA filters and washed with 5 ml of potassium-free M9 salts. The filters were dried and dissolved in OCS (Amersham), and the radioactivity was measured in a scintillation counter. Buffers were identical to those used in the membrane potential experiments.

86Rb EMux. Cells were grown as for the transport assays. After resuspending to an OD595 of 1.0, the cells were incubated in the presence of 86RbCl (20 ,Ci/ml) for 1 hr at 37°C. Cells (0.1 ml) were then added to 1.9 ml of 20 mM buffer at various pH values and incubated for 10 min at 37°C. The cells were then filtered through Millipore HA filters presoaked in wash buffer. The filters were washed with 5 ml of M9 salts supplemented with 10 mM RbCl and dried, and radioactivity was determined in a y counter. Buffers were identical to those used in the membrane potential experiments.

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markedly inhibited after acid treatment, whereas cells synthesizing the corresponding mutant DTA (DTA-E148S) or containing the vector alone were unaffected (data not shown). Next we plated E. coli containing plasmids encoding various forms of DT-E148S on solid media with various pH values and determined cell viability by colony counts. As seen in Fig. 1, viability of E. coli synthesizing full-length DT-E148S decreased by -50 times at pH 6.0; by 105 at pH 5.5; and by almost 107 at pH 5.0. Death at acidic pH was rapid. A 1-min exposure of cells to pH 5.0, followed by plating at pH 8.0, yielded maximal levels of cell killing (data not shown). By contrast, cells producing DTA-E148S, or cells harboring the plasmid vector lacking DT sequences, showed only a modest (-2 times) decrease in viability at pH 5.0 and were essentially unaffected at higher pH values. When cells were induced with 1 mM isopropyl P-D-thiogalactopyranoside at pH 6.0 viability of DT-E148S-producing cells decreased by -105 compared to cells induced at pH 7.5, whereas cells producing DTA-E148S were unaffected (data not shown). This suggests that the lethality observed is a function of both the pH and the concentration of DT-E148S in the periplasm. Uninduced strains producing DT-E148S and DTA-E148S contained stoichiometrically equivalent amounts of DT-related protein in the periplasm (-5000 molecules per cell). A DT fragment (F2-E148S) comprising all of DTA-E148S plus 55% of DTB caused a similar, although somewhat lesser, decline in viability at pH 5.0, but little loss of viability at higher pH values (Fig. 1). A strain expressing wild-type F2 gave identical results (data not shown). The F2 fragment is 6 residues longer than CRM45 (23, 24), a chain-termination fragment that contains a substantial fraction of the hydrophobic region within DTB (25). CRM45 has been shown to form ion-conductive channels under acidic pH conditions (26). These results implicate hydrophobic regions of DTB in the pH-dependent killing of E. coli. The fact that both the F2 and F2-E148S fragments were produced in substantially lower amounts than DT-E148S, as estimated by ELISA, may account for their reduced effect on viability. 100 o

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RESULTS When exposed to acidic pH, DT and certain truncated forms of DT undergo a conformational change that exposes latent hydrophobic regions within the B chain (17-19). If the pH is reduced in the presence of artificial planar lipid bilayers, the toxin undergoes membrane insertion and forms ion-conductive channels (20). Similarly, if toxin that is bound to the mammalian cell surface is exposed to acidic conditions, it is able to translocate the DTA moiety across the membrane into the cytosol (21, 22). The behavior of DT under acidic conditions suggested that membrane-insertion-competent forms of the toxin residing in the periplasm of E. coli might insert into the plasma membrane when the bacteria were exposed to acid pH. Initially we monitored cell growth spectrophotometrically. Cultures were adjusted to pH 5.0, incubated for 10 min, and neutralized; and 2 hr later the OD595 was measured. The results indicated that growth of cells synthesizing DT-E148S was

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pH FIG. 1. Viability of cells harboring plasmids encoding all or part of DT-E148S when plated on media of various pH values. E. coli JM103, containing plasmids, were grown to midlogarithmic phase and plated on L agar containing either 100 mM Pipes (pH 7.5-6.5) or 50 mM sodium citrate/50 mM sodium succinate (pH 6.0-5.0). One day later viability was assessed and scored relative to cells plated at pH 7.5. Identical results were seen at pH 5.5 when 100 mM sodium acetate was used instead of sodium citrate/sodium succinate. Plasmids are as follows: o, pDO1 (DT-E148S); A, pDO3 (DTA-E148S); o, ptacF2-E148S (F2-E148S).


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FIG. 3. Proline transport (A) and 86Rb efflux (B) in E. coli JM103 containing various plasmids after acid pH treatment. Cell-associated radioactivity is expressed relative to samples incubated at pH 7.0. o, pDO1 (DT-E148S); A, pDO3 (DTA-E148S); and a, pCDptac2 (vector).

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The acid-induced death seen in cells synthesizing DTE148S or F2-E148S might result from the formation of ion-conducting channels in the inner membrane, similar to those formed when cells are treated with certain colicins (27). In vitro, colicin El forms channels in artificial lipid membranes at low pH (28). In vivo, it depolarizes the inner membrane of E. coli, impairing active transport, and there is an accompanying loss of intracellular ions (27). To determine if there were effects of acidic pH on inner membrane function in cells expressing DT-E148S, we measured membrane potential, active transport, and Rb efflux. To measure membrane potential we used the fluorescent dye 3,3'-dipropylthiadicarbocyanine iodide, which undergoes membrane potential-sensitive concentration and quenching within cells (29, 30). Fig. 2 shows that at pH 6.0 or lower the potential in cells producing DT-E148S was dissipated. A minimal effect was observed in cells containing either the DTA-E148S plasmid or the vector alone. When cells producing DTA-E148S were treated at pH 5.0 in the presence of externally added DT at 0.1 mg/ml, no effect was observed (data not shown). This demonstrates that dissipation of the membrane potential is due to cell-associated toxin and not to toxin released from spontaneously lysing cells. Fig. 3A shows that the acid-induced loss of membrane potential in cells producing DT-E148S is accompanied by a loss in active transport as measured by proline uptake, and Fig. 3B shows that the toxin-producing cells are unable to retain 86Rb at pH 6.0 or below. For each of these assays little or no effect of acid pH was found on cells containing the plasmid vector alone or those synthesizing only DTA-E148S. Microscopic examination of cells containing the whole toxin treated at pH 5.0 showed that cellular integrity was maintained. Furthermore, we found that glucose-6-phosphate dehydrogenase, a cytoplasmic enzyme, remained cellassociated after treatment of DT-E148S-producing cells at low pH.

DISCUSSION The results presented are consistent with a model whereby an acidic environment, inducing a conformational change in the B moiety of periplasmic DT-E148S, enables the protein to insert into the inner membrane and disrupt membrane potential. Insertion of the toxin into a membrane devoid of the

toxin receptor found on sensitive mammalian cells is not surprising, since the toxin is known to insert into protein-free artificial lipid bilayers under acidic conditions. Loss of inner-membrane function, as demonstrated, could account for the lethal effects of acidic pH on toxin-containing cells and seems likely to be the primary effect of the toxin. We have not yet studied the possibility that outer-membrane integrity or function is altered. It may be that channels similar to those observed in artificial bilayer systems are formed in the inner membrane (diameter estimates are in the range of 18 A) (26), but we lack specific information about this. Preliminary results indicate that ultraviolet-absorbing material of low molecular mass is released when DT-E148S-producing cells are treated at low pH. Although acidification of the extracellular milieu of E. coli should cause an approximately parallel decrease in pH of the periplasm, it is unlikely that the periplasmic pH is precisely the same as that ofthe external medium. Stock et al. (31) have predicted that the periplasmic pH should be significantly below that in the medium, due to the Donnan potential across the outer membrane and the buffering capacity of ionic species within the periplasm. Regardless of the precise values of periplasmic pH, one would only expect an approximate correlation between pH profiles of cell killing with those of membrane insertion activity (20) or membrane translocation (21) in other systems. Membrane insertion in some systems represents initial rate measurements whereas in others it does not, and all the activities depend on variables besides pH (e.g., ionic strength) that can not be precisely controlled in the bacterial periplasm. Although our results do not entirely exclude the possibility that the DTA moiety of DT-E148S and F2-E148S might play some role in generating the acid-sensitivity phenotype, ADPribosyltransferase activity is almost certainly irrelevant. No macromolecular substrates for DTA have been found in E. coli, and DTA expression in the cytoplasm, as well as its secretion to the periplasm, is without apparent detrimental effect (32). More importantly, we have found that the pH-sensitivity profiles of strains synthesizing wild-type (E148) and mutant (E148S) forms of the F2 fragment are identical. Finally, it is noteworthy that peptides contained within DTB are sufficient for channel formation in vitro (33). An important implication of these studies is that they define an approach to isolating additional classes of mutants. For studies of DT structure and activity the system can be used as a positive genetic selection of toxin mutations affecting membrane insertion and/or formation of ionconductive channels. Directed and/or random mutagenesis

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of the cloned toxin gene, followed by transformation into E. coli and analysis of survivors plated onto acidic plates, may yield various classes of mutants useful in analyzing the poorly understood processes of membrane insertion and translocation. Membrane perturbations caused by DT may not be restricted to acid induction; other variables (e.g., ionic strength, specific ions, or osmolarity) might be used to induce lethality, and hence be used for selection. Furthermore, ifcell death is due to periplasmic DT-E148S and not to a minor population of toxin molecules left in the cytoplasm then this system might also be used to isolate conditional secretory mutants, either of the toxin or of bacterial constituents. The general approach described might also be adapted to the study of other proteins that normally interact with biological membranes at low pH or under other conditions. Besides the other toxic proteins that undergo pH-dependent membrane interactions, there are proteins present on certain enveloped viruses (e.g., the hemagglutinin of influenza) that mediate fusion between the viral envelope and target membranes when exposed to a low pH (34) and a number of other proteins that induce fusion of phospholipid vesicles at low pH (35). This work was supported by National Institutes of Health grants (AI-22021 and AI-22848). Partial support was also received from the Shipley Institute of Medicine. D.O.O. is the recipient of a National Research Service Award from the National Institute of Allergy and Infectious Diseases. 1. Morris, R. E. & Saelinger, C. B. (1986) Infect. Immun. 52,445453. 2. Farahbakhsh, Z. T., Baldwin, R. L. & Wisnieski, B. J. (1986)

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