INFECTION AND IMMUNITY, May 1990, p. 1133-1140 0019-9567/90/051133-08$02.00/0 Copyright C) 1990, American Society for Microbiology

Vol. 58, No. 5

Secretion of Toxin A from Pseudomonas aeruginosa PAO1, PAK, and PA103 by Escherichia coli ABDUL N. HAMOOD, MARY JO WICK, AND BARBARA H. IGLEWSKI*

Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 Received 11 September 1989/Accepted 15 January 1990

The exotoxin A gene (toxA) from Pseudomonas aeruginosa PAO1 was expressed from the lac promoter in Escherichia coli, and the localization of the toxin A protein was determined. Throughout the growth cycle, the ADP-ribosyltransferase activity of toxin A was gradually reduced in the periplasm of E. coli, with no apparent degradation of the toxin A protein. This suggests the presence of an E. coli periplasmic factor that interferes with the ADP-ribosyltransferase activity in toxin A. Such an inactivating factor was found in the periplasmic extract from control E. coli cells. The processing of toxin A in E. coli was examined by pulse-chase immunoprecipitation experiments. Mature toxin was detected in both the periplasm and cytoplasm, whereas the membranes contained both mature and precursor forms. Toxin A precursor appears to be processed in both the cytoplasm and the periplasm of E. coli. Toxin A proteins from P. aeruginosa PAO1, PA103, and PAK were compared for their secretion in E. coli. Despite the differences in the amino acid sequences of their leader peptides, toxin A proteins from strains PAO1, PA103, and PAK were processed and secreted to the periplasm of E. coli.

Pseudomonas aeruginosa produces several extracellular virulence-related factors (18), among which is toxin A (24). The excreted mature form of toxin A ADP-ribosylates eucaryotic elongation factor 2, which results in the inhibition of protein synthesis (12). DNA sequence analysis revealed that the toxA gene encodes a 71-kilodalton precursor protein with a 25-aminoacid leader peptide (10). The leader peptide is removed during the processing and excretion of toxin A in P. aeruginosa, giving rise to the mature extracellular 66-kilodalton protein (10). In Escherichia coli, toxin A is not expressed from its own promoter; however, when it is expressed from a promoter recognized by E. coli, an enzymatically active and immunologically cross-reactive protein is formed (8, 19). Toxin A from P. aeruginosa PAK was shown to be processed and secreted to the periplasm of E. coli (8, 19). Toxin A appears to bypass the periplasmic space during its excretion in P. aeruginosa. Lory et al. (20) suggested that toxin A is excreted in P. aeruginosa through regions of inner-outer membrane fusions (Bayer junctions). When toxA was expressed from the lac promoter in P. aeruginosa, the majority of the synthesized toxin A was excreted to the extracellular environment. However, some of the mature toxin was seen in the periplasmic space (A. Hamood, M. Wick, and B. Iglewski, Abstr. Pseudomonas 89, 1989, 1-22, p. 19). This suggested that upon saturation of the regular pathway of toxin A excretion in P. aeruginosa, some of the toxin is processed and secreted to the periplasm. The mechanism of toxin A secretion to the periplasm of P. aeruginosa may be similar to that of toxin A secretion in E. coli. In this study, we examined the localization of toxin A throughout the growth cycle of E. coli. Toxin A processing in E. coli was examined by pulse-chase immunoprecipitation experiments. We also compared the secretion of toxin A from P. aeruginosa PA01, PA103, and PAK in E. coli. *

Corresponding author. 1133

MATERIALS AND METHODS Strains and media. E. coli TB1 [endAl gyrA69 thi hsdRJ7 supE44 relAl A(lac-proAB) rpsL] (3) was used for initial cloning. E. coli MC4100 (F- lacUJ69 araD139 thiA rspL relA) (13) was obtained from Donald Oliver (State University of New York at Stony Brook, Stony Brook). LB medium (25) was used to culture E. coli strains. For immunoprecipitation, minimum medium A (25) containing 1% glycerol was used. Plasmids used in this study are listed in Table 1. DNA manipulations. P. aeruginosa chromosomal DNA was prepared by the method of Goldberg and Ohman (9). DNA sequencing was done by the dideoxy method of Sanger et al. (32), using 7-deaza-dGTP in place of dGTP (2, 26). Plasmid DNA was isolated by the alkaline lysis procedure of Birnboim and Doly (4) and was purified by isopycnic CsCl centrifugation as previously described (23). Restriction enzymes and T4 DNA ligase were used according to the recommendations of the supplier (Bethesda Research Laboratories, Inc., Bethesda, Md.). Labeling, immunoprecipitation, and gel electrophoresis. Cells were grown in medium A at 37°C to the late logarithmic phase (optical density at 540 nm [OD540] = 1.5), centrifuged, and resuspended in fresh medium. After 10 min of incubation at 37°C, [35S]methionine (1,111 Ci/mmol, 41.11 TBq/mmol; Dupont, NEN Research Products, Boston, Mass.) was added at a concentration of 50 ,XCi/ml for 2 min, followed by an excess of nonradioactive methionine (150 ,ug/ml). When ethanol was used, it was added at the concentration indicated below 2 min before the addition of the radioactive methionine. Immunoprecipitation experiments were performed as described previously (15) except that after removal of the insoluble materials, the radioactive solution was diluted 1:5 in immunoprecipitation buffer (0.05 M Tris hydrochloride [pH 7.5], 0.15 M NaCl, 10 mM EDTA, 0.5% Triton-X-100, 0.5 mg of bovine serum albumin per ml). Immunoprecipitated proteins were analyzed by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis (17), followed by fluorography with Amplify (Amersham Corp., Arlington Heights, Ill.).

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INFECT. IMMUN.

HAMOOD ET AL. TABLE 1. Plasmids used in this study

pUC19 pMJ21 pAM21 pAM21-1 pMJ41 pAM41 pAM41-1 pMS151

pAH140 pAH141

Reference

Construction

Plasmid

Ampr, general cloning vector A 2,700-bp PstI-EcoRI fragment of P. aeruginosa PAO1 DNA, containing an intact toxA gene, cloned into PstI-EcoRI sites of pUC18 A 2,424-bp EcoRV-EcoRI fragment of pMJ21 cloned into SamI-EcoRI sites of pUC19 A 2,000-bp NruI-EcoRI fragment of pAM21 cloned into Smal-EcoRI sites of pUC19 A 2,700-bp PstI-EcoRI fragment of P. aeruginosa PA103 DNA, containing an intact toxA gene, cloned into PstI-EcoRI sites of pUC18 A 2,424-bp EcoRV-EcoRI fragment of pMJ41 cloned into SmaI-EcoRI sites of pUC19 A 2,000-bp NruI-EcoRI fragment of pAM41 cloned into SmaI-EcoRI sites of pUC19 A 2,424-bp EcoRV-EcoRl fragment of P. aeruginosa PAK DNA, containing an intact toxA gene, cloned into SmaI-EcoRI sites of pUC18 A 2,445-bp PstI-EcoRI fragment of pMS151 cloned into PstI-EcoRI sites of pUC19 A 2,000-bp NruI-EcoRI fragment of pAH140 cloned into Smal-EcoRI sites of pUC19

Cellular fractionation. E. coli cells were fractionated by the cold osmotic shock procedure of Koshland and Botstein (16). E. coli MC4100 containing plasmid pAM21-1, pAH141, or pAM41-1 was grown in L broth containing ampicillin (40 ,ug/ml) at 37°C. One-milliliter portions were taken from the culture at different times, and the cells were processed as described previously (16). After separation of the periplasmic fractions, the spheroplasts were broken by sonication and centrifuged, and the supernatants were saved as the cytoplasmic fraction. The remaining pellets were suspended in distilled water and saved as the membrane fraction. To examine the in vitro effect of the E. coli periplasmic contents on the enzymatic activity of purified toxin, MC4100 was grown and periplasmic fractions were obtained at different points of growth. A 10-,ul portion of each periplasmic fraction was mixed with 300 ng of purified toxin A (Swiss Serum and Vaccine Institute, Bern, Switzerland) in a total volume of 20 p.1. The mixtures were incubated at 37°C for 1 h and assayed for ADP-ribosyltransferase activity. Immunoblotting. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the proteins were electrophoretically transferred to nitrocellulose as described by Towbin et al. (35). After transfer, the papers were blocked for 1 h with 1% Blotto (1% nonfat dry milk in Towbin saline [TS] [150 mM NaCl, 50 mM Tris chloride, pH 7.5]), washed several times with TS, and incubated with toxin A antiserum in 1% Blotto for 1 h at 37°C. The filters were washed with TS, incubated with '25I-protein A (Dupont, NEN) for 1 h at 37°C, washed, dried, and exposed to X-ray film (X-Omat AR; Eastman Kodak Co., Rochester, N.Y.). n-]

This study This study 37 This study This study 11, 19 This study This study

The protein content of each cellular fraction was determined by the method of Lowry et al. (21). The amount of toxin A protein in each fraction was determined by the dot immunobinding assay of Jahn et al. (14), using purified toxin A as a standard. Enzyme assays. P-Lactamase and glucose-6-phosphate dehydrogenase assays were performed as previously described (1, 22). ADP-ribosyltransferase assays were essentially done as previously described (36) with slight modifications. Briefly, toxin A was activated by the addition of 10 ,ul of 8 M urea-2% dithiothreitol (DTT) (Bethesda Research Laboratories) to an equal volume of each sample for 15 min at 25°C. The ADP-ribosyltransferase assay was initiated by the addition of 25 RI of wheat germ elongation factor 2, 25 ,ul of reaction buffer (125 mM Tris hydrochloride [pH 7.0], 100 mM EDTA), and 5 ,ul of [14C]NAD (533 mCi/mmol; Dupont, NEN). After 15 min of incubation at 25°C, proteins were precipitated by the addition of 200 ,ul of 10% trichloroacetic acid and the mixtures were filtered onto nitrocellulose filters. The filters were washed with ethanol and air dried, and the amount of radioactivity on each filter was determined with a liquid scintillation counter. RESULTS Plasmid constructions. Lory et al. (19) previously described cloning and expression of the toxA gene from P. aeruginosa PAK in E. coli. Similarly, we cloned the toxA gene from strain PAO1 in pUC19. Plasmid pAM21 was constructed by cloning the 2,424-base-pair (bp) EcoRVEcoRI fragment from pMJ21 into SmaI-EcoRI sites of

200 Dp

ATG

toxA gene

PstI

EcoRV

Nrul

EV TG

lac -

EooRV

Ilac --*

38 37

A

Nrul

WTG

EcoRI

D-1 EooRI

ni

I

Nrul

EooRI

pAM21

pAM41

pAH14O pAM21 -1 -1 ~~~~~~~~~~~~pAM41 pAH141

FIG. 1. Schematic diagram of P. aeruginosa toxA gene. Regions of toxA harbored by the plasmids discussed in the text are shown. Symbol O, toxin A translation stop codon. Arrows indicate the direction of transcription of the lac gene.

TOXIN A SECRETION BY E. COLI

VOL. 58, 1990

1

23

4

5

6 7

FIG. 2. Immunoblot analysis of the periplasmic fractions of E. coli MC4100 containing different recombinant plasmids. Cells were grown to an OD540 of 2.0 to 2.5 and fractionated as described in the text. Lanes: 1, purified toxin (standard); 2 and 5, MC4100 containing the toxA gene from strain PAO1 on pAM21 and pAM21-1, respectively; 3 and 6, MC4100 containing the toxA gene from strain PA103 on pAM41 and pAM41-1, respectively; 4 and 7, the toxA gene from strain PAK on pAH140 and pAH141, respectively. Details of plasmid construction are shown in Table 1.

pUC19 (Table 1, Fig. 1). Although toxin A protein encoded by pAM21 was processed and secreted to the E. coli periplasm (Fig. 2, lane 2), the amount of ADP-ribosyltransferase activity was low (Table 2). This was most likely due to the fact that pAM21 retained 400 bp from the upstream region of the toxA gene, resulting in the inefficient expression of toxA from the lac promoter. To express the toxA gene directly from the lac promoter, we constructed plasmid pAM21-1 (Table 1). Plasmid pAM21-1 was constructed by cloning the 2,000-bp NruI-EcoRI fragment, which contains the coding sequence for the 71-kilodalton precursor protein, into the SmaI-EcoRI sites of pUC19 (Fig. 1). This resulted in the addition of 17 amino acids (encoded by the multiple TABLE 2. ADP-ribosyltransferase activity in different fractions of MC4100(pAM21) and MC4100(pAM21-1) Fraction'

MC4100(pAM21) Periplasm Cytoplasm Membranes

MC4100(pAM21-1) Periplasm Cytoplasm Membranes

ADP-ribosyltransferase activity (cpm/,ug of protein)

6.0 36.0 14.5 17.0 420.0 214.0

a Cells were grown to an OD540 of 1.5 and fractionated as described in Materials and Methods.

1135

cloning region of pUC19) to the amino-terminal region of the toxin A precursor protein. However, a toxin A encoded by pAM21-1 was processed and secreted to the E. coli periplasm (Fig. 2, lane 5), which indicates that the presence of these 17 extra amino acids had no effect on the processing and secretion of toxin A in E. coli. Furthermore, ethanol treatment of the MC4100(pAM21-1) cultures resulted in the inhibition of toxin A secretion to the periplasm (see below). Localization of toxin A protein in E. coli. The presence of toxin A protein in different fractions of MC4100(pAM21-1) grown to the late log phase (OD540, -2) was determined by both ADP-ribosyltransferase assay and Western blot (immunoblot) analysis using toxin A-specific antiserum. Considerable ADP-ribosyltransferase activity was detected in the cytoplasmic and membrane fractions of MC4100(pAM21-1) grown to the late logarithmic phase, but the periplasmic fraction contained a low level of enzymatic activity (Fig. 3A). In contrast, high levels of ADP-ribosyltransferase activity were detected in the periplasm, as well as in the cytoplasm and membrane fractions of MC4100(pAM21-1), when samples were taken at an early stage of growth (OD540, -0.35) (Fig. 3A). These results indicate that the reduction in the ADP-ribosyltransferase activity of toxin A in the periplasm of MC4100(pAM21-1) occurred only at late stages of growth. Most of the glucose-6-phosphate dehydrogenase activity (cytoplasmic marker) was localized to the cytoplasm, whereas most of the P-lactamase activity (periplasmic marker) was localized to the periplasm (Table 3), indicating that leakage of cellular materials through the cytoplasmic membrane was not a problem. To investigate this further, we examined the cellular distribution of toxin A activity throughout the growth cycle of MC4100(pAM21-1). Throughout the growth cycle, there was a gradual increase in the ADP-ribosylation activity of the cytoplasmic and membrane fractions but a gradual decrease in the ADP-ribosylation activity of the periplasmic fractions (Fig. 3A). In contrast to the decrease in toxin A activity, the amounts of toxin A protein present in the periplasm of MC4100(pAM211) at different points of growth increased (Fig. 4). Furthermore, intact toxin A protein was detected in the periplasm of MC4100 containing pAM21-1 even at a late stage of growth (OD540, -2.5) (Fig. 2, lane 5). These results showed that the growth-related reduction in the ADP-ribosylation activity of the MC4100(pAM21-1) periplasm was not due to the proteolytic degradation of toxin A protein or to an inhibition of toxin A secretion to the periplasm. Since toxin A was not degraded in the periplasm of MC4100, it is possible that at late stages of growth there may be some factors that inhibit its enzymatic activity. To test this possibility, samples from E. coli MC4100 were obtained at different points of growth and were fractionated. A 10-,ul portion of each periplasmic fraction was mixed with purified toxin A. After 1 h of incubation at 37°C, the mixtures were assayed for ADP-ribosyltransferase activity. The enzymatic activity of purified toxin was reduced when mixed with periplasmic fractions from late stages of growth of MC4100 (Fig. 5). The same mixtures, incubated under the same conditions (37°C for 1 h) were examined for toxin A protein by Western blot analysis. A stable, undegraded, toxin A protein was detected in all the mixtures (data not shown). In a control experiment, activated purified toxin A (treated with urea and DTT) was incubated with the periplasmic fractions of MC4100 and no degradation of toxin A was detected (data not shown). These results indicate that the reduction of ADP-ribosyltransferase activity of toxin A in MC4100 periplasm at late stages of growth was not due to the degradation

HAMOOD ET AL.

1136

INFECT. IMMUN.

A

C

B

80

80 :._

.U

2

U

60

.)

co

0

Laaco

0

U)

0

L.

C 0 U)

4-c

40

60

S

S

0

40

n

&

0 s

CL

0.

d.

20

20

0

49

4c

'4

'40 0

0 0

2

0

OD540

2

1

3

3

0

OD540

OD540

FIG. 3. ADP-ribosyltransferase activity of toxin A in different fractions throughout the growth cycle of E. coli MC4100. Cells were grown and fractionated at different points of growth as described in the text. Toxin A activity in each fraction, determined as counts per minute per microgram of protein, is expressed as a percentage of the total activity. (A) MC4100 containing toxA from strain PAO1 on pAM21-1; (B) MC4100 containing toxA from strain PA103 on pAM41-1; (C) MC4100 containing toxA from strain PAK on pAH141. Symbols: C, periplasm; *, cytoplasm; x, membranes.

of the toxin A protein and support the conclusion that an inhibitor of toxin A activity accumulates in the periplasm of E. coli during the late stages of growth. In all experiments, periplasmic samples from different stages of growth were not normalized to a uniform protein concentration before the assay. Consequently, the greater reduction in toxin A activity at late stages of growth reflects an absolute increase in the concentration of the inhibitor. Processing of toxin A. The processing of toxin A in E. coli was examined by pulse-chase-immunoprecipitation experiments (15). E. coli MC4100(pAM21-1) was grown to an OD540 of 1.5, labeled with [35S]methionine for 2 min, and chased with an excess of unlabeled methionine for 4 min. Labeled cells were fractionated, and the fractions were immunoprecipitated with toxin A antiserum. Both the periplasmic and cytoplasmic fractions contained mature toxin A, whereas the membrane fraction contained both the precursor and mature forms of the toxin (Fig. 6). The rate of processing of the membrane-associated precursor form of the toxin was examined by labeling MC4100(pAM21-1) for 2

min, followed by chasing with an excess of unlabeled methionine for different times. At the end of each chase period, the cells were centrifuged, the pellets were suspended in 10% trichloroacetic acid, and the toxin A protein was immunoprecipitated by using toxin A antiserum. The precursor disappeared only after 30 min of chase (Fig. 7), indicating a slow rate of processing to the mature form. The presence of the mature toxin in the cytoplasm of MC4100(pAM21-1) was consistent in repeated experiments. It was further confirmed by the determination of cytoplasmic and periplasmic markers (data not shown). Mature toxin A was also detected in the cytoplasm of MC4100(pAM21) (data not shown). To determine whether the presence of mature 100

-

80

-

60

-

40

-

TABLE 3. Localization of toxin A protein in E. coli

MC4100(pAM21-1) Fraction

% Activity l3-Lactamasea

G-6-P-Db

ADP-ribosyltransferase

NDC 85.0 10.0 5.0

ND 2.6 74.4 18.6

ND 44.5 26.3 29.2

OD540= 0.35 Supernatant Periplasm Cytoplasm Membranes

20

0 0

OD540 = 1.5

Supernatant Periplasm Cytoplasm Membranes

ND 79.0 14.0 7.0

ND 1.2 72.0 26.9

ND 4.7 48.6 46.7

a Periplasmic marker. b G-6-P-D, Glucose-6-phosphate dehydrogenase; cytoplasmic marker. c ND, Not determined.

2

1

3

OD540 FIG. 4. Quantity of toxin A protein detected in the periplasm throughout the growth cycle of MC4100 containing toxA from strain PAO1 on pAM21-1 (0), strain PA103 on pAM41-1 (A), and strain PAK on PAH141 (-). Toxin A protein was quantitated by the dot immunobinding assay (15), using toxin A antiserum. Purified toxin A was used as a standard.

TOXIN A SECRETION BY E. COLI

VOL. 58, 1990

100000

1137

-

2

1

3

4

5

10000 -

75 40 (L r.. 2

Chase

2 10 30 60 FIG. 7. Rate of processing of toxin A precursor to mature toxin in E. coli MC4100. Cells were grown to an OD540 of 1.5, pulsed with [35S]methionine for 2 min, and chased with an excess of unlabeled methionine. At the indicated chase time, samples were removed and immunoprecipitated with toxin A antiserum. Lane 1 contained toxin A obtained from the supernatant of P. aeruginosa PA103 (positive control). P, Toxin A precursor; M, mature toxin. (min.)

1000

-

100

-

%J %J

0

2

1

3

4

OD540 FIG. 5. In vitro effect of E. coli periplasm on the ADP-ribosyltransferase activity of purified toxin A. Portions of the periplasmic fraction of MC4100 obtained at different points of growth were mixed with purified toxin A at 37°C for 1 h, and the mixtures were assayed for ADP-ribosyltransferase activity. ll, Counts per minute for 300 ng of purified toxin A.

toxin in the cytoplasm of MC4100 relates to the export. of the toxin across the cytoplasmic membrane, MC4100(pAM21-1) was pulse-labeled in the presence of 9.5% ethanol. This concentration of ethanol is known to cause dissipation of the proton motive forces of the membrane, thereby interfering with the secretion process (28). Ethanol treatment resulted in the absence of mature toxin from the periplasm and the accumulation of the precursor form in both the cytoplasm and the membranes (Fig. 8). Thus, toxin A secretion to the periplasm, as well as its cytoplasmic processing, was inhibited by ethanol treatment. Secretion of toxin A from P. aeruginosa PA103, PAK, and PAO1 in E. coli. Previous work suggested that when the toxA gene from P. aeruginosa PA103 is expressed in E. coli, the toxin A precursor is localized predominantly in the cytoplasm (10). In contrast, toxin A protein encoded by the toxA gene from P. aeruginosa PAK is efficiently secreted to the periplasmic space of E. coli (19). The signal peptides of the toxin A proteins from strains PA103 and PAK are identical in size but differ in three amino acids (19). These differences were defined at positions -22, -5, and -3 from the putative alanine-alanine signal peptidase recognition site. Since we 1

2

used the toxA gene from strain PAO1 in this study, we determined the deduced amino acid sequence of the toxin A leader peptide from strain PA01 by nucleotide sequence analysis and compared it with the published leader sequences of strains PA103 and PAK (10, 19). The leader peptide of toxin A from strain PAO1 is similar in size and highly homologous, but not identical, to the leader peptides of toxin A from strains PA103 and PAK (Fig. 9). The differences in the three leader peptides are at positions -22, -5, -4, and -3 from the putative signal peptidase recognition site. While amino acid -22 is threonine in the toxin A proteins from both PAK and PAO1, it is replaced by isoleucine in the toxin A from PA103. Amino acid -5 is leucine in the PAK toxin and serine in the PAO1 and PA103 toxins (Fig. 9). Furthermore, the PAO1 toxin contains phenylalanine instead of serine at position -4. Alanine is the -3 amino acid in both PAO1 and PA103 toxins but is replaced by arginine in the PAK toxin. We determined whether these differences in the toxin A leader peptides from the three different strains affect secretion of toxin A in E. coli. The toxA genes from strains PA103

A 1

2

--

B 3

4

5

6

7

-r_.

3

FIG. 6. Location of the precursor and mature forms of toxin A in E. coli MC4100. MC4100(pAM21-1) cells were labeled for 2 min and fractionated, and the fractions were immunoprecipitated with toxin A antiserum and electrophoresed on a 12% polyacrylamide gel. Lanes: 1, MC4100(pAM21-1) periplasm; 2, MC4100(pAM21-1) cytoplasm; 3, MC4100(pAM21-1) membranes. P, Toxin A precursor; M, mature toxin A.

FIG. 8. Effect of ethanol treatment of E. coli on processing of toxin A precursor. E. coli MC4100(pAM21-1) cells were grown and labeled, and the fractions were immunoprecipitated as for Fig. 6. (A) Toxin A obtained from the supernatant of P. aeruginosa PA103

(positive control) (lane 1), MC4100(pAM21-1) periplasm (lane 2),

MC4100(pAM21-1) cytoplasm (lane 3), and MC4100(pAM21-1) membranes (lane 4). (B) Same as for panel A, but the cells were treated with 9.5% ethanol before labeling. Lanes: 5, MC4100 (pAM21-1) periplasm; 6, MC4100(pAM21-1) cytoplasm; 7, MC4100 (pAM21-1) membranes. P, Toxin A precursor; M, mature toxin A.

1138

HAMOOD ET AL.

INFECT. IMMUN.

P. aeruginosa Amino acid sequence of the toxin A leader peptide

strain -25

+

PAO01

|

Met-His-Leu-Thr-Pro-His-Trp- lle- Pro-Leu-Val-Ala-Ser-Leu-Gly-Leu-Leu-Ala-Gly-Gly-Ser- Phe-Ala-Ser-Ala|Ala

PAK

|

Met-His-Leu-Thr- Pro- His-Trp- lle- Pro-Leu-Val- Ala-Se r- Leu-Gly- Le u-Leu -Ala-G Iy-Gly- Leu-Ser-Arg -Ser-Ala- la

PA 103

Met-His-Leule-I Pro-H is-Trp-Ilie- Pro- Le u-Val-Ala-Ser- Leu-G ly- Leu- Leu-Ala-Gly-Gly- Ser-Ser-Ala-Ser-AI aAla

Hydrophilic region

Hydrophobic region

Leader peptidase recognition site

FIG. 9. Comparison of the deduced amino acid sequences of the toxin A leader peptides from P. aeruginosa PAO1, PA103 (10), and PAK (19). The first amino acid of the mature toxin is indicated by + 1. Amino acids that differ between the leader peptides are underlined.

and PAK were cloned into pUC19, generating plasmids pAM41 and pAH140, respectively (Table 1, Fig. 1). Mature toxin A protein was detected in the periplasm of MC4100 (pAM41) and MC4100(pAH140) (Fig. 2, lanes 3 and 4). Recombinant plasmids pAM41-1 and pAH141 were constructed by cloning the 2,000-bp NruI-EcoRI fragments from pAM41 and pAH140, respectively, into the SmaI-EcoRI sites of pUC19 (Table 1, Fig. 1). Similar to results obtained with pAM21-1, the secretion of toxin A encoded by pAM411 and pAH141 was not affected by the presence of the 17 amino acids encoded by the pUC19 multiple cloning region (Fig. 2, lanes 6 and 7). MC4100(pAM41-1) and MC4100 (pAH141) were fractionated at different points during the growth cycle, and the fractions were examined for ADPribosyltransferase activity. The high level of ADP-ribosyltransferase activity in the periplasmic fractions of MC4100 (pAM41-1) and MC4100(pAH141) at early stages of growth was followed by reduced activity at late stages of growth (Fig. 3B and C). This reduction in the ADP-ribosyltransferase activity was not observed when the cytoplasmic or membrane fraction of MC4100(pAM41-1) or MC4100 (pAH141) was assayed (Fig. 3B and C). Toxin A protein was neither decreased (Fig. 4) nor degraded in the periplasm of MC4100(pAH141) or MC4100(pAM41-1) at late stages of growth (Fig. 2, lanes 6 and 7).

DISCUSSION Our results show that while toxin A was secreted to the periplasm of E. coli, its ADP-ribosyltransferase activity was significantly reduced at late stages of growth (Fig. 3). One possible explanation for this reduction in toxin A activity is degradation of toxin A in the periplasm of E. coli. Misfolding of toxin A protein during its secretion could make it more liable to degradation by periplasmic and outer membrane proteases. Abnormal proteins in E. coli are usually degraded by several proteases (29). Strauch and Beckwith (34) recently described E. coli mutants in which unstable foreign proteins were not degraded in the periplasm. However, the reduction in the ADP-ribosyltransferase activity was not

accompanied by either degradation or quantitative decrease of toxin A protein (Fig. 2 and 4). Moreover, periplasmic fractions from MC4100 grown to a late logarithmic phase interfered with the enzymatic activity of the purified toxin in vitro (Fig. 5). It is less likely that the reduction in ADPribosyltransferase activity in the E. coli periplasm is due to an improperly folded, but undegraded, toxin A molecule. To determine the ADP-ribosyltransferase activity, all samples (including cytoplasmic and membrane fractions) were treated with urea-DTT to activate toxin A. This treatment with urea-DTT linearizes the toxin A molecule and exposes the enzymatically active site (36). The presence of certain ions in the E. coli periplasm could interfere with the ADPribosyltransferase reaction. However, upon dialysis of the MC4100(pAM21-1) periplasm (using membranes with a molecular weight cutoff of 3,000), no enhancement in the toxin A activity was observed (data not shown). In addition, the inhibitory effect of the MC4100 periplasm on the enzymatic activity of toxin A in vitro was not removed when the E. coli periplasm was dialyzed (data not shown). It is unlikely that a change in the pH of the E. coli periplasm was a determining factor in the observed reduction in toxin A activity, as Chung and Collier (5) demonstrated that toxin A remains enzymatically active over a wide range of pH. These results suggest that a certain factor that accumulates in the periplasm of E. coli at late stages of growth interferes with the ADP-ribosyltransferase activity of toxin A. This presumed factor could simply cause the degradation of the [14C]NAD substrate, resulting in a severe inhibitory effect on ADPribosyltransferase activity. The presence of toxin A precursor in the membrane of MC4100(pAM21-1) suggests that synthesized toxin A is not efficiently processed in E. coli (Fig. 6). The presence of toxA on a high-copy-number plasmid (approximately 24 copies per cell for ColEl plasmids [6]) combined with its expression from the lac promoter would result in a high concentration of newly synthesized toxin. This high concentration of toxin may lead to the saturation of the regular export sites on the cytoplasmic membrane, causing the accumulation of the

VOL. 58, 1990

precursor toxin on the membranes (Fig. 6). One unexpected finding in this study was the detection of the mature toxin in the cytoplasm of MC4100 (Fig. 6). The amount of trichloroacetic acid-precipitated 35S counts in the cytoplasm of MC4100 was about 80% of that in the periplasmic fraction (whereas only a small fraction of the periplasmic marker was detected in the cytoplasm). This confirms that the detection of the mature toxin in the cytoplasm of MC4100 was not a result of contamination from the periplasm. Moreover, when purified toxin A was added to MC4100 spheroplasts prior to sonication (a control experiment), only 10 to 20% of the toxin was recovered in the cytoplasm (data not shown). The inhibition of toxin A processing in MC4100(pAM21-1) by the addition of 9.5% ethanol to the culture before labeling was accompanied by the accumulation of toxin A precursor in the cytoplasm and membranes (Fig. 8). This suggests that the processing of toxin A in the cytoplasm of E. coli is either dependent on or related to the transfer of toxin A to the periplasm. This is different from the processing of other secreted proteins in E. coli. It is known that the leader peptides of secreted proteins are removed by leader peptidases after translocation of the proteins across the cytoplasmic membrane (31). Leader peptidases of E. coli span the cytoplasmic membrane, with their enzymatic portion located on the outer surface of the cytoplasmic membrane (7). The processing of the phosphate-binding protein (PhoS) in E. coli has some similarity to that of toxin A. Hyperproduction of PhoS in E. coli leads to the accumulation of PhoS precursor (prePhoS) in the cytoplasm (27). However, unlike toxin A, prePhoS is gradually and nonspecifically degraded in the E. coli cytoplasm even in the presence of a proton motive

uncoupler (27). The presence of 9.5% ethanol in the growth medium interferes with the proton motive forces in the membranes. These proton motive forces may be required to maintain overall topology and organization of the membrane. Thus, it is possible that ethanol treatment of E. coli interfered with a protease in the cytoplasmic membrane, causing the accumulation of toxin A precursor in the cytoplasm. A less plausible explanation is that, like P. aeruginosa, E. coli may process toxin A at or close to regions of inner-outer membrane fusion (Bayer junctions). Mature toxin A has been detected in the cytoplasm of P. aeruginosa (11). Available evidence suggests that the toxin A leader peptidase in P. aeruginosa is located in the cytoplasmic membrane or in the Bayer junction region (20; A. N. Hamood and B. H. Iglewski, manuscript in preparation). In the absence of a necessary excretory machinery required to transfer it to the extracellular environment, processed toxin A may be released in the cytoplasm of E. coli. The general structure of the toxin A signal peptide shares many features with the signal peptides of other procaryotic secreted proteins. Toxin A from all three P. aeruginosa strains (PAO1, PAK, and PA103) was processed and secreted to the periplasm of MC4100 (Fig. 2 and 4). This was due to the fact that the major common characters of the leader peptides of toxin A from these strains were not affected by the differences in their amino acid compositions. The leader peptides of toxin A from all three strains retain the two basic residues (histidine) in their amino-terminal region (Fig. 9). The replacement of isoleucine (-22) with threonine in the PAO1 and PAK toxins did not change the net positive charge of the amino-terminal region of the leader peptide (Fig. 9). Changing the net positive charge of the amino-terminal part of the leader peptides of the E. coli secreted proteins lipoprotein and LamB to a net negative

TOXIN A SECRETION BY E. COLI

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charge affected both the secretion and rate of synthesis of these proteins (30, 33). The leader peptides of toxin A from all three strains have no change in the hydrophobic core region, the putative helical disrupting amino acid, glycine (-6), or the alanine residue (-1) at the signal peptidase recognition site (Fig. 9). However, the conserved small amino acid alanine (-3) is replaced with the larger basic amino acid arginine in PAK toxin. Previously reported differences in the secretion of toxin A from strains PA103 and PAK in E. coli (10, 19) could have been due to either the different E. coli strains that were used in each experiment or the different vectors that were used to express toxA from each strain. The similarities between the requisite primary leader peptide structures of E. coli secreted proteins and the leader peptide of toxin A reflect possible functional similarities in the respective leader peptidases. Thus, even though E. coli and P. aeruginosa secrete toxin A by two different mechanisms, they may have a common step in toxin A processing (i.e., the removal of the leader peptide from the toxin A precursor). In this regard, Lory et al. (20) showed that a toxin A precursor obtained from P. aeruginosa was not processed in vitro by a purified E. coli leader peptidase I. ACKNOWLEDGMENTS This work was supported by Public Health Service grant AI25669 from the National Institutes of Health. We thank Stephen Lory, University of Washington, Seattle, for providing plasmid pMS151. LITERATURE CITED 1. Angus, B. L., A. M. Carey, D. A. Caron, A. B. Kropinski, and R. B. Hancock. 1982. Outer membrane permeability in Pseudomonas aeruginosa: comparison of a wild type with an antibioticsupersusceptible mutant. Antimicrob. Agents Chemother. 21: 299-309. 2. Barr, P. J., R. M. Thayer, P. Laybourn, R. C. Najarian, F. Seela, and T. R. Tolan. 1986. 7-Deaza-2'-deoxyguanosine-5'triphosphate: enhanced resolution in M13 dideoxy sequencing. BioTechniques 4:428-432. 3. Bethesda Research Laboratories. 1984. E. coli host for pUC plasmids. Focus 6(4):7. 4. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523. 5. Chung, D. W., and R. J. Collier. 1977. Enzymatically active peptide from the adenosine diphosphate-ribosylating toxin of Pseudomonas aeruginosa. Infect. Immun. 16:832-841. 6. Clewell, D. B. 1972. Nature of Col El plasmid replication in Escherichia coli in the presence of chloramphenicol. J. Bacteriol. 110:667-676. 7. Dalby, R., and W. Wickner. 1986. The role of the polar, carboxy terminal domain of Escherichia coli leader peptidase in its translocation across the plasma membrane. J. Biol. Chem.

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