Vol. 60, No. 2

INFECTION AND IMMUNITY, Feb. 1992, p. 510-517 0019-9567/92/020510-08$02.00/0 Copyright C 1992, American Society for Microbiology

Isolation and Characterization of Toxin A Excretion-Deficient Mutants of Pseudomonas aeruginosa PAO1 ABDUL N. HAMOOD,t* DENNIS E. OHMAN,t SUSAN E. WEST,§ AND BARBARA H. IGLEWSKI Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Box 672, Rochester, New York 14642 Received 13 August 1991/Accepted 14 November 1991

We have isolated and characterized four toxin A excretion-deficient mutants of Pseudomonas aeruginosa PAO1. Similar to previously described mutants (B. Wretlind and 0. R. Pavlovskis, J. Bacteriol. 158:801-808, 1984), the mutants appear to have a pleiotropic defect in the excretion of several extracellular products, including toxin A, elastase, alkaline phosphatase, and phospholipase C. However, the mutants are not defective in the excretion of either alkaline protease or exoenzyme S. We also examined the localization and processing of toxin A in these mutants by using pulse-labeling experiments. Mature toxin A was found to be localized to the membranes only. Our results suggest that toxin A is localized to the outer membrane but is not exposed to the extracellular surfaces of the outer membranes. The results also suggest that toxin A obtained from the excretion-deficient mutants has intact disulfide bonds.

group of toxin A excretion-deficient mutants. Localization of toxin A in different compartments of such mutants will enable us to determine the specific steps of toxin A excretion in P. aeruginosa. Excretion-deficient mutants have been used previously in protein excretion studies. The processing and excretion of aerolysin in Aeromonas hydrophila were examined by using aerolysin excretion-deficient mutants (19, 20). In this study, we describe the characterization of four of the toxin A excretion-deficient mutants. The mutants are defective in the excretion of several extracellular products. We also examined the localization and processing of toxin A in these mutants.

Toxin A is one of several virulence-related extracellular products produced by Pseudomonas aeruginosa (24). The toxin is synthesized as a 71-kDa precursor which is processed to a 68-kDa mature toxin upon excretion (10, 25). Pseudomonas toxin A causes ADP-ribosylation of elongation factor 2 in the eucaryotic cell, resulting in the inhibition of protein synthesis and cell death (21). Defining the mechanism through which these virulencerelated extracellular products are excreted is an important step in understanding the pathogenesis of infection of their bacterial host. DNA sequence analysis showed that these extracellular products are synthesized as larger precursors with leader peptides at their amino terminus ends (4, 10, 29, 40). The leader peptide, which is removed during the excretion of these proteins, has the same characteristic features as other leader peptides of other procaryotic secreted proteins (4, 10, 29). In addition, many of these products are translocated to the extracellular environment through the periplasm (17, 19, 22). However, available evidence suggests that toxin A bypasses the periplasmic space during its excretion in P. aeruginosa (27). Lory et al. (27) suggested that toxin A is directly transferred from the cytoplasm of P. aeruginosa to the extracellular environment through regions of inner-outer membrane fusion (Bayer junction). This unique mechanism of excretion could be the function of both the toxin A molecule and the P. aeruginosa excretion machinery. Toxin A leader peptide alone is less likely to be the only factor involved in toxin A excretion. A recent study suggests that besides the leader peptide, certain regions of the mature toxin are required for the excretion of toxin A in P. aeruginosa (14). To examine the role of other cellular components in toxin A excretion in P. aeruginosa, we have generated a

MATERIALS AND METHODS Bacterial strain and culture conditions. The mutants were derived from P. aeruginosa PAO1 (18). For toxin A and alkaline protease production, the cells were grown at 32°C in Trypticase soy broth dialysate that was treated with Chelex100 (TSB-DC) (34). The dialysate was supplemented with 0.05 M monosodium glutamate and 1% glycerol. TSB-DC plates were prepared by the addition of 1.5% Noble agar to the TSB-DC broth. For exoenzyme S production, the cells were grown in S-defined medium as previously described (32). Elastase production was detected in cells grown in peptone-Trypticase soy broth (5% peptone, 0.25% Trypticase soy broth, pH 7.4). Phosphate-deficient medium (28) was used for the production of alkaline phosphatase. Phospholipase C production was detected in cells grown in a specific phospholipase C medium (3). For pulse-labeling experiments, the cells were grown in minimum medium 1 x A

(31).

Corresponding author. t Present address: Department of Microbiology, Texas Tech University Health Sciences Center, Lubbock, TX 79430. t Present address: Department of Microbiology and Immunology, University of Tennessee, and Veterans Administration Medical Center, Memphis, TN 38163. § Present address: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, *

Isolation of mutants. Mutagenesis of PAO1 was done as previously described (34). PAO1 cells were grown to mid-log phase in nutrient broth with 0.5% yeast extract and treated with either 100 ,ug of N-methyl-N'-nitro-N-nitrosoguanidine (NTG) or 0.5% (vol/vol) ethylmethyl sulfonate for 60 min at 37°C. Cells were then diluted in nutrient broth with 0.5% yeast extract and incubated at 37°C overnight with shaking. Toxin A-deficient mutants were identified by the previously

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described agar-well assay (34) using toxin A-specific antiserum. Antisera against toxin A, elastase, and alkaline protease were prepared in rabbits as previously described (14, 35, 39). Antiserum against gel-denatured toxin A was also prepared in rabbits as previously described (35). Fractionation of P. aeruginosa was done according to the procedure described by Cheng et al (6). Intact cells and spheroplasts were lysed by passing them twice through a French pressure cell (SLM Instruments, Inc., American Instrument Co., Urbana, Ill.) at 10,000 lb/in2. In all fractionation experiments, the fractions were assayed for glucose 6-phosphate dehydrogenase and P-lactamase activities. Glucose 6-phosphate dehydrogenase is a cytoplasmic marker (28), and P-lactamase is a periplasmic marker (1). The inner and outer membranes of P. aeruginosa were separated on a 50 to 70% sucrose density step gradient as described previously (16). Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and immunoblotting were done as previously described (15). Pulse-labeling and immunoprecipitation. Cells grown in TSB-DC medium at 32°C (to maximize toxin A production) were subcultured into 1 xA medium and grown at the same temperature to an optical density at 540 nm (OD540) of about 2.0. A 1-ml volume of the cell suspension was centrifuged (15,000 x g for 10 min at 4°C), resuspended in the same volume of fresh 1 x A medium, and grown for an additional 15 min at 32°C. After that, [35S]methionine (1,111 Ci/mmol; Dupont, NEN Research Products, Boston, Mass.) was added at a concentration of 100 ,uCi/ml, and the incubation was continued for an additional 1 min (labeling). The labeling was terminated by the addition of an excess of unlabeled methionine (150 ,ug/ml) for 2 min (chase). The cells were then incubated on ice for 10 min and centrifuged at 15,000 x g for 5 min. The supernatant was separated, and the proteins in the supernatant were precipitated by the addition of 50 jig of bovine serum albumin (BSA) and an equal volume of 10% cold trichloroacetic acid. The pellet was fractionated as previously described (22). The proteins in the periplasmic, cytoplasmic, and membrane fractions were precipitated by the addition of 50 jig of BSA and an equal volume of 10% cold trichloroacetic acid. Immunoprecipitation experiments were performed as previously described (15, 22). Immunoprecipitated materials were analyzed by SDS gels (12%) and fluorography (15). Pronase treatment experiments. Cells were labeled with [35S]methionine for 1 minute, and then unlabeled methionine was added for 2 min. At the end of the chase period, the cells were centrifuged and suspended in the pronase digestion buffer (10 mM Tris HCl [pH 7.5], 10 mM MgCl, 250 jig of chloramphenicol per ml) to half of their original volume. Pronase was added to a final concentration of 100 jig/ml, and cell suspensions were incubated at room temperature for 20 min. At the end of the pronase digestion, cell suspensions were washed extensively (six times) with the pronase buffer (to remove the pronase enzyme), and toxin A-related materials were immunoprecipitated as described above. In the non-pronase-treated control, the samples were treated exactly the same way but without the addition of pronase. For the positive control experiment, 5 jil of toxin A immunoprecipitated from the supernatant of P. aeruginosa PA103 (toxin A hyperproducer) was mixed with the pellets of the excretion-deficient mutants, and the mixture was suspended in 500 ,ul of pronase buffer. At the end of pronase digestion (100 ,uglml for 20 min at room temperature), the sample was centrifuged and the pellet was discarded. Proteins in the

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supernatant were precipitated by the addition of BSA and 10% trichloroacetic acid followed by incubation on ice for 10 min. Control samples were treated in the same way except that the addition of pronase was omitted. For experiments with variable periods of chase, the samples were labeled with [35S]methionine for 1 min and then an excess of unlabeled methionine was added for variable periods (2, 10, 30, and 45 min). At the end of pulse-labeling, samples were treated with pronase, and toxin A-related materials were immunoprecipitated. Examining the disulfide bonds of toxin A. The disulfide bonds of toxin A were examined basically as described previously (26). About 5 ,ul of toxin A immunoprecipitated from either the supernatant of PA103 or the pellets of the excretion-deficient mutants was denatured by the addition of SDS to a final concentration of 1%. For the reduction of the disulfide bonds, dithiothreitol (DTT) was added to the toxin A-SDS samples to a final concentration of 20 mM. Both denatured and denatured and reduced toxin A samples were incubated at room temperature for 15 min. At the end of the incubation period, iodoacetamide was added (to a final concentration of 100 mM) to prevent the reformation of the disulfide bonds, and the samples were immediately loaded on 5% SDS-PAGE gels. Toxin A-related materials were visualized by autoradiography. Enzyme assays. The ADP-ribosyltransferase assay (to measure toxin A activity) was done as previously described (15). Assay of the exoenzyme S activity was done as previously described (32). The phospholipase C and alkaline phosphatase assays were done as previously described (3, 5). The elastase Congo red assay (to determine the elastolytic activity of the elastase) was done as described by Schad et al. (39). The dot-immunobinding assay for elastase was done as previously described (15) with specific elastase antiserum. ,B-Lactamase, glucose 6-phosphate dehydrogenase, and succinate dehydrogenase assays were performed as previously described (1, 28, 36).

RESULTS Characterization of the toxin A excretion-deficient mutants. Toxin A-deficient mutants were obtained by the chemical mutagenesis (ethylmethyl sulfonate and NTG) of P. aeruginosa PAO1. Single colonies from the mutagenized cultures were plated on TSB-DC plates, and the toxin A-deficient mutants were identified by the agar gel immunodiffusion assay as previously described (34) with toxin A-specific antiserum. Of the 6,000 colonies screened, 25 that represented potential toxin A-deficient mutants were isolated. Mutants isolated by the agar immunodiffusion assay could carry one of three types of mutations: in a toxin A excretionrelated gene, in a toxin A regulatory gene, or in the upstream region of toxA. A mutation in a toxin A excretion-related gene would result in intracellular accumulation of the toxin. To define toxin A excretion-deficient mutants, all toxin A-deficient mutants were grown in TSB-DC broth and the amounts of ADP-ribosyltransferase activity in their supernatants, lysates, and membranes were determined. Four potential toxin A excretion-deficient mutants were isolated (PAO-EXC1, PAO-EXC2, PAO-EXC3, and PAO-EXC4). Compared with the activity of their parent strain, PAO1, the amount of ADP-ribosyltransferase activity in the supernatant of each mutant was greatly reduced (Table 1). However, levels of ADP-ribosyltransferase activity in their lysates and membrane fractions were higher than those in the lysate and membranes of PAO1 (Table 1). The presence of cell-associ-

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TABLE 1. Toxin A production in the culture supernatants, cell lysates, and membranes of P. aeruginosa PAO1 and PA01 excretion-deficient mutantsa ADP-ribosyltransferase activityb (cpm) in: Lysate Membranes

P. aeruginosa strain

Supernatant

PAO1 PAO-EXC1 PAO-EXC2 PAO-EXC3 PAO-EXC4 PA103C

950.0 15.0 20.0 5.0 14.0 8,350.0

120.0 150.0 160.0 125.0 130.0 620.0

107.0 300.0 283.0 223.0 310.0 512.0

a For maximum toxin A production, cells were grown in TSB-DC (34) at 32°C with maximum aeration. Cells were grown for 18 h to an OD540 of 3.3 to 3.5. b The ADP-ribosyltransferase assay was done as previously described (15), and activity was determined as counts per minute per 10 microliters of fraction. Background counts were subtracted. Lysates and membrane fractions were prepared from cells suspended at 10% of their original volumes. Values of toxin A in these fractions were corrected to that of the original volume of the culture. Due to the variation in the protein content of different fractions, values of toxin A activity were not calculated as counts per minute per microgram of protein. c Toxin A-hyperproducing strain (positive control).

ated toxin A protein was determined by immunoblotting experiments using toxin A-specific antiserum. Intact toxin A protein was detected in the lysates of all mutants (Fig. 1). This confirms that these mutants have no defect in toxin A synthesis but are defective in the excretion of the synthesized toxin. To further characterize these mutants, we compared their growth patterns with that of the PAO1 strain. There was no marked difference between the growth patterns of any of the 1

2

3 4

5 6

7 8

9 10

11

68.043.0-

29.0-

18W414.3-

FIG. 1. Detection of toxin A protein in the supernatants of P. aeruginosa PAO1 and PAO1 excretion-deficient mutants. Cells were grown in TSB-DC medium, and the supernatants and lysates were obtained as described in Materials and Methods. Toxin A protein was detected by immunoblotting experiments with specific toxin A antiserum. Lanes: 1, toxin A (positive control); 2, PAO1 supernatant; 3, PAO-EXC1 supernatant; 4, PAO-EXC2 supernatant; 5, PAO-EXC3 supernatant; 6, PAO-EXC4 supernatant; 7, PA01 lysate; 8, PAO-EXC1 lysate; 9, PAO-EXC2 lysate; 10, PAO-EXC3 lysate; 11, PAO-EXC4 lysate. Each lane of the SDS-PAGE gel was loaded with 40 to 50 ,ug of protein. Molecular size standards (in kilodaltons) are shown on the left side of the blot.

mutants and that of the PAO1 parent strain (data not shown). The four mutants were checked for a possible defect in the excretion of other extracellular products. The supernatants of the mutants were examined for the presence of elastase and alkaline protease proteins by immunoblotting experiments using elastase- and alkaline protease-specific antisera, respectively. The supernatants were also examined for the presence of phospholipase C, alkaline phosphatase, and exoenzyme S activities as previously described (3, 5, 32). Intact alkaline protease protein was detected in the supernatants of all mutants (data not shown). Similarly, exoenzyme S activity was detected in the supernatants of the mutants (Table 2). Compared with the PAO1 parent strain, two of the mutants (PAO-EXC2 and PAO1-EXC4) produced significantly higher levels of exoenzyme S activity (Table 2). However, very low levels of alkaline phosphatase and phospholipase C activities were detected in the supernatants of the mutants (Table 2). The supernatants of the mutants were also devoid of any elastolytic activity as determined by the previously described elastin Congo red assay (39) (data not shown). Moreover, no elastase protein was detected in the supernatants of the mutants (Fig. 2). Rather, the elastase was found to be cell associated (Fig. 2). These results indicate that while the mutants excrete alkaline protease and exoenzyme S normally, they are defective in the excretion of alkaline phosphatase, phospholipase C, elastase, and toxin A. Although alkaline phosphatase is predominantly a periplasmic protein, this study and previous studies have shown that a considerable level of alkaline phosphatase activity is usually detected in the supernatant of P. aeruginosa (32, 45). We also tried to determine if the cell-associated (unexcreted) products were secreted to the periplasm of the excretiondeficient mutants. The mutants were fractionated, and their periplasmic fractions were examined for both alkaline phosphatase and phospholipase C activities. In all fractionation experiments, about 90% of the 3-lactamase activity (periplasmic marker) was localized to the periplasmic space and 80 to 85% of the glucose phosphate dehydrogenase activity (cytoplasmic marker) was localized to the cytoplasm (data not shown). Both alkaline phosphatase and phospholipase C activities were detected in the periplasmic fractions of all mutants (Table 2). Because the intracellular elastase is elastolytically inactive (8), we decided to determine the amount of elastase present in different cellular fractions by using both immunoblotting and dot-immunobinding assays (15). A 33-kDa mature elastase protein was detected in the periplasmic spaces of all mutants (data not shown). However, a significant amount of the intracellular elastase was membrane associated (data not shown). The accumulation of alkaline phosphatase, phospholipase C, and elastase in the periplasms of the mutants suggests the presence of a defect in the final process of translocating these extracellular products across the outer membrane. Such a defect could arise from a missing outer membrane protein. Inner and outer membranes of the mutants and the PAO parent strain were separated on a 50 to 70% sucrose density step gradient (16), and their protein profiles were examined on 10 to 20% SDS-PAGE gels. As seen in Fig. 3, proteins of variable sizes were missing from the outer membranes of the mutants. For example, while the PAOEXCl outer membrane was missing a 90-kDa protein, the PAO-EXC4 outer membrane lacked a protein of about 70 kDa (Fig. 3). However, no common outer membrane protein appeared to be missing in all mutants (which would indicate the existence of a common mutation). There were no noticeable differences between protein profiles of the mutants'

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TABLE 2. Alkaline phosphatase, phospholipase C, and exoenzyme S activities in different fractions of P. aeruginosa PAO1 and PAO1 excretion-deficient mutants Alkaline phosphatase activityab' (U/mi) in:

P. aeruginosa strain

PAO1 PAO-EXC1 PAO-EXC2 PAO-EXC3 PAO-EXC4 388d

Phospholipase C activity' (U/ml) in:

Exoenzyme S activity' (cpm) in:

Supernatant

Periplasm

Supernatant

Periplasm

Cytoplasm

Supernatant

Periplasm

Cytoplasm

30.0 4.0 2.0 2.0 3.0 ND

35.0 22.0 18.0 15.0 25.0 ND

13.0 2.0 1.0 0.8 0.5 ND

5.0 15.0 10.0 12.0 19.0 ND

4.0 12.0 8.0 9.0 14.0 ND

1,560.0 8,486.0 50,778.0 1,684.0 26,062.0 39,274.0

19.0 181.0 3,650.0 82.0 370.0 1,533.0

16.0 119.0 243.0 27.0 110.0 175.0

a Average of three experiments. In all experiments, the cultures were adjusted to an OD540 of 2.5 before fractionation. Assays for phospholipase C and alkaline phosphatase activities were done as previously described (3, 5). ND, not determined. b Alkaline phosphatase is inactive in the cytoplasm (30). Cells were grown in the exoenzyme S-defined medium to an OD540 of 2.3 to 2.5 and fractionated, and the amount of exoenzyme S activity in 10 p.l of each fraction was determined as previously described (32). Both cytoplasmic and periplasmic fractions were prepared from cells suspended at 10% of their original volumes. Values of exoenzyme S activities in these fractions were corrected to that of the original volume of the culture. Because of variations in the protein contents of different fractions, values of exoenzyme S activity were not calculated as counts per minute per micrograms of protein. d P. aeruginosa 388, which is an exoenzyme S hyperproducer (32), was used as a positive control in the exoenzyme S assay.

inner membranes and that of the PA01 strain (data not shown). Localization and processing of toxin A in excretion-deficient mutants. Toxin A localization was examined by pulse-chase immunoprecipitation experiments as previously described (15, 22). Since all mutants had the same phenotype (regarding the defective excretion of toxin A), we chose to examine the localization and processing of toxin A in one of the mutants (PAO-EXC1) first. Cells were labeled with [35S]methionine for 1 min, and then an excess of cold methionine was added (chase). After that, cells were fractionated, and toxin A-related materials were immunoprecipitated by using toxin A-specific antiserum. Mature processed toxin A was detected only in the membrane fraction of PAO-EXC1 (Fig. 4). The absence of toxin A from all other fractions suggests that toxin A synthesis and processing are localized to the membranes. These results do not contradict our original observation, in which toxin A was found to be accumulated

in the lysate and the membranes of the mutants (Table 1). In the original experiments, we examined toxin A accumulation in cultures that were grown to a stationary phase of growth. Thus, accumulated toxin (which is usually released to the supernatant in a normal cell) was detected in both the lysate and the membrane fractions. In contrast, pulse-labeling experiments examined the synthesis of toxin A within a few minutes. A previous study showed that treatment of P. aeruginosa PA103 with 9.5% ethanol (a membrane-perturbing agent) results in the accumulation of a 71-kDa toxin A precursor on the outer surface of the outer membrane (27). We failed to detect any toxin A-related materials in repeated experiments in which PAO-EXC1 cells were treated with 9.5% ethanol before labeling. This is because, unlike strain PA103 (which is a toxin A hyperproducer), PA01 and its mutants produce lower levels of toxin A (Table 1). In addition, treatment of cells with ethanol not only interferes l 2 3 4 5 6

1

2

3

4

5

7 8

6

9 10 11

68 0-

92.5-

_

66.2-

*

43o0_

w

__4

:z ...*4

29.0-_

45.0--8

W

31.0-

I

18.414.3-

FIG. 2. Detection of elastase in the supernatants and lysates of P. aeruginosa PAO1 and PAO1 excretion-deficient mutants. Cells were grown in peptone-Trypticase soy broth, and the supernatants and lysates were obtained as described in Materials and Methods. To each lane of the SDS-PAGE gel was added 40 to 50 Fig of protein, and the elastase was detected by immunoblotting with specific elastase antiserum. Lanes: 1, elastase (positive control); 2, PAO1 supernatant; 3, PAO-EXCl supernatant; 4, PAO-EXC2 supernatant; 5, PAO-EXC3 supematant; 6, PAO-EXC4 supernatant; 7, PAO1 lysate; 8, PAO-EXC1 lysate; 9, PAO-EXC2 lysate; 10, PAO-EXC3 lysate; 11, PAO-EXC4 lysate. Molecular size standards (in kilodaltons) are shown on the left side of the autoradiogram.

21-5-

14.4-

O

FIG. 3. Coomassie-stained gel of the outer membrane proteins of P. aeruginosa PAO1 and PA01 excretion-deficient mutants. Cells were grown in TSB-DC medium, and the outer membranes were isolated as described in Materials and Methods. Each lane of the 10 to 20% SDS-PAGE gradient gel was loaded with 50 ,ug of protein. Lanes: 1, molecular size standards; 2, PAO1; 3, PAO-EXC1; 4, PAO-EXC2; 5, PAO-EXC3; 6, PAO-EXC4. Molecular size standards (in kilodaltons) are shown on the left side of the gel.

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1

2

INFECT. IMMUN.

3

4

5

6

6uo

FIG. 4. Localization of toxin A in the excretion-deficient mutant PAO-EXC1. Cells were grown in 1xA minimum medium, pulselabeled with [35S]methionine, and fractionated as described in Materials and Methods. Toxin A-related materials were immunoprecipitated by using specific toxin A antiserum. Fraction I represents the 0.2 M MgCl2 periplasmic extract, while fraction II represents the 0.01 M MgCl2 periplasmic shock fluid (6, 22). Lanes: 1, toxin A immunoprecipitated from the supernatant of P. aeruginosa toxin A-hyperproducing strain PA103 (positive control); 2, PAO-EXC1 supernatant; 3, PAO-EXC1 periplasm (fraction I); 4, PAO-EXCl periplasm (fraction II); 5, PAO-EXC1 cytoplasm; 6, PAO-EXCl membranes.

with protein secretion but also reduces the rate of protein synthesis in general (41). We did not use other perturbing agents (like phenylethyl alcohol or procaine) because these agents do not interfere with the process of toxin A excretion in P. aeruginosa (27). Next, we tried to determine if toxin A is localized to the inner or the outer membranes of excretion-deficient mutants. The mutants, together with their PAO1 parent strain, were grown in TSB-DC medium, and their membranes were separated on 50 to 70% sucrose density step gradients (16). Proteins from bands representing inner and outer membranes were precipitated and examined later for ADPribosyltransferase activity. About 85% of the succinate dehydrogenase activity (inner [cytoplasmic] membrane marker) was detected in bands enriched for inner membrane proteins (data not shown). In all mutants, bands enriched for the outer membrane proteins contained most of the ADPribosyltransferase activity (Table 3). These results suggest that toxin A may be localized to the outer membranes of the mutants. We also tried to determine if toxin A accumulates within the outer membrane or on its extracellular surface. TABLE 3. Toxin A activity in the inner and outer membranes of PAO1 and PAO1 excretion-deficient mutantsa Toxin A activity (CPM/,ug of protein) in P. aeruginosa strain

PAO1 PAO-EXC1 PAO-EXC2 PAO-EXC3 PAO-EXC4

sucrose gradient bands enriched for: Inner membrane Outer membrane proteins proteins

8.0 44.0 17.0 25.0 19.0

13.0 128.0 52.0 150.0 230.0

a Inner and outer membranes were separated on 50 to 70% sucrose density step gradients as previously described (16). Proteins from each band were precipitated, suspended in distilled water, and assayed for ADP-ribosyltransferase activity.

FIG. 5. Examination of the exposure of toxin A to the extracellular surface of the excretion-deficient mutant PAO-EXC1. Cells were grown in minimum medium 1xA, pulse-labeled with [35S]methionine, pelleted, and treated with pronase enzyme as described in Materials and Methods. Toxin A was immunoprecipitated by using specific toxin A antiserum. (A) Lanes: 1, toxin A immunoprecipitated from the supernatant of P. aeruginosa toxin A-hyperproducing strain PA103 (positive control) (with pronase treatment); 2, toxin A immunoprecipitated from the supernatant of P. aeruginosa PA103 (without pronase treatment); 3, the pellet of PAO-EXC1 (with pronase treatment). (B) Cells were labeled with [35S]methionine as in panel A, chased for different periods (as indicated in the figure), and treated with pronase. Lanes: 1, toxin A immunoprecipitated from the supernatant of P. aeruginosa PA103 (positive control) (with pronase treatment); 2, toxin A from the supernatant of P. aeruginosa PA103 (without pronase treatment); 3 to 5, the pellet of PAO-EXC1 (with pronase treatment).

PAO-EXC1 cells were pulse-labeled and treated with pronase (100 F.g/ml) for 20 min, and toxin A was immunoprecipitated. Toxin A is sensitive to pronase enzyme (unlike chymotrypsin and carboxypeptidase, to which toxin A is resistant) (25). Thus, if toxin A is exposed to the extracellular surface of PAO-EXC1, its complete digestion by pronase will prevent the detection of any toxin A materials by immunoprecipitation. Mature toxin A immunoprecipitated from the supernatant of P. aeruginosa PA103 (which is a toxin A hyperproducer) was used as control. The toxin (from PA103) was mixed with the PAO-EXC1 pellet, treated with pronase, and immunoprecipitated again. Pronase treatment of PAO-EXC1 pellet did not cause a detectable degradation of toxin A (Fig. 5A). This suggests that toxin A is not exposed to the extracellular surface of PAO-EXC1. It is possible, however, that more time is required for toxin A localization to the extracellular surface than is permitted by the pulse-chase experiments (only 2 min of chase). To examine this possibility, samples of PAO-EXC1 were pulselabeled for 1 min, chased for variable periods (2, 10, 30, and 45 min), and treated with pronase, and toxin A was immunoprecipitated. As seen in Fig. 5B, there was no detectable degradation of toxin A in the PAO-EXC1 pellet even after 45 min of chase. Determining the status of disulfide bonds in toxin A from excretion-deficient mutants. Most secreted proteins are known to undergo certain conformational changes during their secretion (38). Intracellularly, these proteins are assumed to be in a partially folded conformation that is required to facilitate their secretion (38). Toxin A obtained from the supernatant of P. aeruginosa contains four intact disulfide bonds (44). These disulfide bonds contribute to the

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3

2

1

*

4

As-

FIG. 6. Examination for intact disulfide bonds in toxin A from the excretion deficient-mutant PAO-EXC1. Toxin A immunoprecipitated from PAO-EXC1 was either denatured (SDS) (lane 3) or denatured and reduced (SDS and DTT) (lane 4) as described in Materials and Methods. Toxin A immunoprecipitated from the supernatant of P. aeruginosa PA103 (positive control) was either denatured (SDS) (lane 1) or denatured and reduced (SDS and DTT) (lane 2). Treated samples were loaded on 5% SDS-PAGE gels.

conformational character of the excreted toxin. A difference in the status of the disulfide bonds (intact versus reduced) in excreted toxin and unexcreted toxin indicates a possible conformational change of toxin A during its excretion. We compared the relative electrophoretic mobility of toxin A in the presence of either a denaturing agent (SDS) or denaturing and reducing agents (SDS and DTT). A toxin A molecule with intact disulfide bonds will be more compact and will run faster on an acrylamide gel than a toxin A molecule with reduced disulfide bonds. Toxin A from P. aeruginosa PA103 was used as a control. As seen in the control experiment (Fig. 6), denatured toxin A migrated faster (on acrylamide gel) than denatured toxin in which all the disulfide bonds were reduced. In an experiment similar to the control experiment, the migration rate of a denatured toxin A obtained from PAO-EXC1 was faster than that of a denatured and reduced toxin A obtained from the same mutant (Fig. 6). The same results were obtained with toxin A from other excretion-deficient mutants (data not shown). These findings suggest that most of the four disulfide bonds of toxin A (which accumulates in the excretion-deficient mutants) are intact.

DISCUSSION Mutants described in this study have a pleiotropic defect in the excretion of several extracellular products, including toxin A, elastase, alkaline phosphatase, and phospholipase C (Tables 1 and 2; Fig. 1 and 2). This defect suggests the presence of a common step in the excretion pathways of these extracellular products. P. aeruginosa mutants with similar pleiotropic defects have been described previously (23, 45), and several genes (xcp genes) required for the excretion of P. aeruginosa extracellular proteins have been isolated and characterized (2, 9, 42). Bally et al. (2) have characterized the xcpA gene, which codes for a 31.8-kDa inner membrane protein. The xcpA gene is identical to the P.

aeruginosa pilin biogenesis

gene,

pilD, which codes for

a

515

membrane-associated endopeptidase (PiID) (33). A specific pilD mutation in P. aeruginosa resulted in the intracellular accumulation of some proteins, including toxin A (42). Nunn and Lory (33) suggested that PilD processes both the pilin precursor and the components of the protein excretion apparatus of P. aeruginosa. In addition, Filloux et al. (9) have isolated a 9.0-kb DNA fragment of P. aeruginosa chromosome that carries several excretion-related genes. This DNA fragment complemented the P. aeruginosa excretion mutations xcp-5 and xcp-51 to xcp-SS (9). Nucleotide sequence analysis and expression studies have shown that two of these genes (xcp Y and xcpZ) code for 18- and 26-kDa inner membrane proteins, respectively. It is thought that these proteins function in the transfer of excretion energy from the inner membrane to the outer membrane (9). Complementation analysis of our excretion-deficient mutants showed that neither plasmid pRK27 (which carries the xcpA-pilD gene) nor plasmid pAX24 (which carries other xcp genes on a 9.0-kb DNA fragment) complemented the excretion defect in PAO-EXCl (12). This indicates that PAOEXCl carries a unique excretion-defective mutation. Mutant PAO-EXC2 appears to have a mutation in one of the xcp genes described by Filloux et al. (9). The excretion defect in PAO-EXC2 was complemented by plasmid pAX24, which carries these genes (12). These results suggest the presence of at least one more unidentified P. aeruginosa excretionrelated gene. This gene may code for either an inner membrane protein that is indirectly involved in the excretion process or an outer membrane protein that functions in the direct translocation of proteins. In the absence of such an outer membrane protein, processed toxin A accumulates within the outer membrane (Fig. 5 and Table 3), while other extracellular products accumulate in the periplasm (Table 2). Protein profiles of the outer membranes of the mutants on Coomassie stained SDS-PAGE gels showed that proteins of variable sizes appear to be missing (Fig. 3). In agreement with the complementation analysis, the mutants did not lack a common major outer membrane protein (Fig. 3). Staining the SDS-PAGE gels by a more-sensitive staining method (silver staining) did not reveal the absence of more proteins or a common outer membrane protein (data not shown). Moreover, the protein profiles of the inner membranes of the mutants were not different from that of the PAO1 parent strain (data not shown). Further experiments, including mapping the mutation in PAO-EXC1 and identifying the gene that complements its defects, are necessary. Although the mutants were defective in the excretion of several extracellular proteins, both alkaline protease and exoenzyme S were excreted (data not shown; Table 2). Guzzo et al. (11) have shown that alkaline protease is excreted in P. aeruginosa by a specific pathway that is independent of the common pathway through which other proteins are excreted. The excretion of alkaline protease in P. aeruginosa is mediated by the function of several genes that are located adjacent to the alkaline protease gene (11). Similar to the alkaline protease, exoenzyme S appears to be excreted by another independent pathway. Unlike toxin A activity, exoenzyme S activity was detected in the periplasm of PAO1 and the mutants (Table 2). The only noticeable difference between the mutants and PAO1 is the significantly higher level of exoenzyme S activity produced by PAOEXC2 and PAO-EXC4 (Table 2). Since these mutants were generated by nonspecific mutagenesis (NTG or ethylmethyl sulfonate mutagenesis), it is possible that a separate mutation in an exoenzyme S-related gene was produced. Pulse-chase experiments with PAO-EXC1 showed the

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absence of toxin A from the periplasmic space of the mutant (Fig. 4). The same results were obtained in pulse-labeling experiments with the P. aeruginosa toxin A-hyperproducing strain PA103 (27). The inability to detect toxin A in the periplasm of P. aeruginosa is not due to an inherent problem with the fractionation or pulse-labeling experiments in P. aeruginosa. Similar pulse-labeling experiments (22) have shown that the elastase is present in the periplasmic space of P. aeruginosa. Moreover, a processed form of the elastase (proelastase) was shown to accumulate in the periplasmic spaces of elastase excretion-deficient mutants (22). Processed toxin A was detected in the periplasmic space of P. aeruginosa only when the toxA gene was expressed from the lac promoter (13). Similar results were obtained when Strom et el. (42) examined the localization of toxin A in a pilDdeficient mutant of P. aeruginosa containing the toxA gene that was expressed from the tac promoter. However, in both studies (13, 42), toxA was carried on a multicopy plasmid and was expressed from a strong promoter (lac or tac promoter). These results contrast with those of the present study, in which cells contained a single copy of toxA that was expressed from its own promoter. To explain these differences, we have suggested that overexpression of toxA in P. aeruginosa leads to the processing of toxin A to the periplasmic space (13). This processing may occur through a secondary pathway that is not functional under normal physiological conditions in the cell. Although processed toxin A is associated with the outer membranes of excretion-deficient mutants (Table 3), it is not localized to the extracellular surface of the outer membrane (Fig. 5). Toxin A from PAO-EXCI was not digested by the pronase enzyme even after 45 min of chase (Fig. 5). In the absence of a transporting protein, toxin A accumulates within and not on the extracellular surfaces of the outer membranes of PAO-EXC1. A less likely possibility is that the pronase resistance of toxin A is due to its abnormal localization within the membrane (as an indirect effect of the mutation in PAO-EXC1). Abnormally localized toxin A may be protected from the pronase digestion by outer membrane proteins of PAO-EXC1. The absence of toxin A from the periplasm of PAO-EXC1 and its accumulation within the outer membranes point to the unique mechanism through which toxin A is excreted in P. aeruginosa. According to the previously proposed model (27), toxin A is synthesized and processed on the inner membrane and translocated directly to the extracellular environment through regions of innerouter membrane fusion (Bayer junctions). If toxin A does not simply flow through the Bayer junctions, it must be translocated through the junction or the outer membrane by a specific toxin A-transporting protein. In the absence of such a transporting protein (as in PAO-EXC1), toxin A could accumulate in the Bayer junction or the outer membrane but not on the extracellular surface of the outer membrane (Fig. 5). Another possible model is that toxin A is processed in the inner membrane and transferred quickly through the periplasmic space to the outer membrane. This quick transfer prevents the detection of the toxin in the periplasmic space even by pulse-chase experiments. The ability of Escherichia coli to specifically process toxin A to the periplasmic space (7) and the presence of a processed toxin A in the periplasm of P. aeruginosa with an overexpressed toxA gene (13, 42) support this possibility. Thus, overproduced toxin A will accumulate in the periplasmic space if P. aeruginosa is unable to transfer it efficiently and quickly from the inner to the outer membrane. The absence of toxin A from the periplasm of P. aeruginosa and its association with the outer

INFECT. IMMUN.

membrane (Fig. 4 and 5; Table 3) conform with both models. However, the results from this and the previous study (27) cannot be considered conclusive evidence because of the inherent limitation of the density gradient procedure in separating the membrane efficiently (37) and the possible contamination of the outer membrane with the Bayer junction region (in which toxin A may exist). Further studies, including the localization of toxin A in the membranes of PAO1 and PAO-EXC1 by using immunoelectron microscopy (43), will be required to confirm these results. Preliminary studies with toxin A from P. aeruginosa PA103 and the excretion-deficient mutants suggest that toxin A undergoes certain conformational changes during its excretion in P. aeruginosa. This possible conformational change in the toxin A molecule does not seem to affect the formation of most or all of the disulfide bonds (Fig. 6). However, a toxin A molecule with two or three intact disulfide bonds can still assume a certain partially folded excretion-competent conformation. ACKNOWLEDGMENTS This work was supported by Public Health Service grant A125669 from the National Institutes of Health. We thank Andree Lazdunski, Laboratoire de Chimie Bacterienne, Marseille, France, for her help in the complementation analysis. REFERENCES 1. Angus, B. L., A. M. Carrey, D. A. Caron, A. B. Kropinski, and R. B. Hancock. 1982. Outer membrane permeability in Pseudomonas aeruginosa: comparison of a wild type with an antibiotic supersusceptible mutant. Antimicrob. Agents Chemother. 21: 299-309. 2. Bally, M., G. Ball, A. Badere, and A. Lazdunski. 1991. Protein secretion in Pseudomonas aeruginosa: molecular cloning and characterization of the xcp-I gene. J. Bacteriol. 171:4342-4348. 3. Berka, R., G. L. Gray, and M. L. Vasil. 1981. Studies of phospholipase C (heat labile hemolysin) in Pseudomonas aeruginosa. Infect. Immun. 34:1071-1074. 4. Bever, R., and B. Iglewski. 1988. Molecular characterization and nucleotide sequence of the Pseudomonas aeruginosa elastase structural gene. J. Bacteriol. 170:4309-4314. 5. Brickman, E., and J. Beckwith. 1975. Analysis of the regulation of Escherichia coli alkaline phosphatase synthesis using deletion and (D80 transducing phages. J. Mol. Biol. 96:307-316. 6. Cheng, K. J., J. M. Ingram, and J. W. Costerton. 1971. Interaction of alkaline phosphatase and the cell wall of Pseudomonas aeruginosa. J. Bacteriol. 107:325-336. 7. Douglas, C., C. Guidi-Rontani, and R. J. Collier. 1987. Exotoxin A of Pseudomonas aeruginosa: active cloned toxin is secreted into the periplasmic space of Escherichia coli. J. Bacteriol. 169:4962-4966. 8. Fecyz, I. T., and J. N. Campbell. 1985. Mechanisms of activation and secretion of a cell associated precursor of an extracellular protease of Pseudomonas aeruginosa. Eur. J. Biochem. 146:35-42. 9. Filloux, A., M. Bally, G. Ball, M. Akrim, J. Tommassen, and A. Lazdunski. 1990. Protein secretion in gram-negative bacteria: transport across the outer membrane involves common mechanisms in different bacteria. EMBO J. 9:4323-4329. 10. Gray, G., D. Smith, J. Baldridge, R. Harkins, M. Vasil, E. Chen, and M. Hyneker. 1984. Cloning, nucleotide sequencing and expression in Escherichia coli of the exotoxin A structural gene of Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 81:2645-2649. 11. Guzzo, J., M. Murgier, A. Filloux, and A. Lazdunski. 1990. Cloning of the Pseudomonas aeruginosa alkaline protease gene and secretion of the proteases into the medium in E. coli. J.

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