by Aeromonas salmonicida - Journal of Bacteriology - American ...

Vol. 171, No. 5

JOURNAL OF BACTERIOLOGY, May 1989, P. 2523-2527

0021-9193/89/052523-05$02.00/0 Copyright © 1989, American Society for Microbiology

Extracellular Secretion of Cloned Aerolysin and Phospholipase by Aeromonas salmonicida KEVIN R. WONG, MARGARET J. GREEN, AND J. THOMAS BUCKLEY*

Department of Biochemistry and Microbiology, University of Victoria, Box 1700, Victoria, British Columbia, Canada V8W2Y2 Received 28 November 1988/Accepted 1 February 1989

The promoterless structural genes for aerolysin and the extracellular phospholipase of Aeromonas hydrophila inserted into a multi-host-range expression vector and transferred into Aeromonas salmonicida and Escherichia coli. In both species, gene expression was under the control of the inducible tac promoter of the vector. Neither the phospholipase nor the aerolysin was released by intact E. coli. Instead, both proteins accumulated in the periplasm, leading to reduced growth and eventual cell death. When the aerolysin gene inserted into the vector contained its own promoter, the toxin was expressed constitutively by A. salmonicida but not by E. coli. Production of aerolysin and the phospholipase by A. salmonicida did not affect cell growth, and the proteins were correctly processed and exported by intact cells. Both proteins could also be detected in the periplasm, where their concentrations were considerably higher than they were outside the cells. Periplasmic aerolysin was rapidly released when cells were transferred to fresh medium, indicating that this compartment is part of the normal export pathway and that the protein is not shunted there as a consequence of overproduction. Plasmid-coded aerolysin did not appear to compete with the cell proteins for export components, as even when very large quantities of aerolysin were being exported by A. salmonicida, there was no effect on chromosomal protease release and only a modest reduction in the export of chromosomal phospholipase. were

in its secretion (14). Protein processing apparently is not involved in the export of aerolysin or cholera toxin, however, because the periplasmic and extracellular forms of both proteins seem to be identical (5, 10). Hirst and Holmgren (4, 5) have suggested that the large difference between the periplasmic and extracellular concentrations of choleragen might account for the unidirectional movement of the toxin out of V. cholerae; however, the details of how it crosses the outer membrane are unknown. It seems possible that other gene products are required to export both aerolysin and choleragen, perhaps to act as helpers or to form specific pores. The need for other gene products is supported by the observation that pleiotropic export mutants of A. hydrophila can be obtained which accumulate aerolysin in the periplasm (9). We have cloned and sequenced the structural genes for both aerolysin and the extracellular glycerophospholipid: cholesterol acyltransferase (GCAT) of A. hydrophila, and we have shown that E. coli does not contain the necessary apparatus to export either of these outside the cell (12, 13, 25). Depending on growth conditions, Aeromonas salomonicida releases a variety of proteins, including an acyltransferase which is homologous to A. hydrophila GCAT (16, 17, 19); however, it does not express a hole-forming toxin of any kind. We studied the specificity of export and examined how A. salmonicida deals with large quantities of either A. hydrophila GCAT or aerolysin, which are expressed on multiple-copy plasmids.

Many gram-negative bacteria release proteins into the growth medium. Often, this is a consequence of cell death or leakage of a periplasmic pool through a compromised outer membrane. The colicins (24) and heat-labile enterotoxin of Escherichia coli (6, 8) are examples of proteins which are released in this way. Some proteins nevertheless may be released by intact cells. These include the Escherichia coli alpha-hemolysin, Vibrio cholerae enterotoxin, Klebsiella pneumoniae pullulanase, Neisseria gonorrhoeae immunoglobulin A (IgA) protease, Pseudomonas aeruginosa exotoxin A and elastase, and Aeromonas hydrophila aerolysin. Remarkably, the limited evidence available, which has been summarized recently by Hirst and Welch (7), suggests that the release pathway for each of these proteins is unique. For some proteins, the periplasm is part of the normal route of export, whereas for others it is not. The alpha-hemolysin of E. coli is somehow exported directly to the medium from the cytoplasm (21). It has no amino-terminal signal sequence (1); in fact, only the C terminus is required for release, suggesting that the toxin does not cross the inner membrane in the same way as periplasmic and outer membrane proteins do. Exotoxin A appears to cross the inner and outer membranes of P. aeruginosa simultaneously (15), and K. pneumoniae pullulanase is a lipoprotein which may be released by membrane vesiculation (23). Other proteins seem to enter the periplasm on their way out of exporting cells, although they may cross the outer membrane in different ways. A precursor of IgA protease enters the outer membrane of N. gonorrhoeae from the periplasm. Release is accomplished by autoproteolytic cleavage of the mature enzyme from the portion of the molecule which is responsible for insertion and crossing of the outer membrane (22). Proteolysis is also required for the release of elastase by P. aeruginosa. Before proteolysis, the precursor is bound to another protein, which may play a role *

MATERIALS AND METHODS Bacterial strains and plasmids. Genotypes and sources of the bacterial strains and plasmids used in this study are listed in Table 1. Cell growth. Bacteria were grown in LB medium (GIBCO Laboratories, Grand Island, N.Y.) supplemented with 0.2% glucose (unless otherwise specified) and the appropriate

Corresponding author. 2523

2524

WONG ET AL.

J. BACTERIOL.

TABLE 1. Bacterial strains and plasmids used in this study Strain or

Genotype or

plasmid

description

Strains Aeromonas salmoni(ida Rif-1 Escherichia coli K-12 HB101

KUR1296

Plasmids pRK2013 pMMB66EH

pMMB66HE

pPH501 pHEC2.2 pJT-1

pKW2

pNB5 pJT2

pHEC2.2

Source or reference

M13mpl9

ATCC 14174 Rif-1

This laboratory

rec A13 hsdS20 ara-14 proA2 lac Yl ga/K2 leuB6 rpsL20 xyl-5 mntl-l supE44 thi thir leia lac Y galK galT ara tonA tsx dam duim supE44

E. E. Ishiguro

Kmr, conjugative helper plasmid RSF1010 A(Pstl-P'iiIll, 2.8 kb) l(/acJp tauP rrnB bla NruI-AhaIII, 3.0 kb)Apr Inverted polylinker of pMMB66EH Tcr Ql(pBR322, Pstl-Pstl geatA, 1.2 kb)Tc' M13 mpl9Ql(pHEC2.2, Pstl-PstI, 1.2 kb) pMMB66EH fl(pPH501, EcoRI-PstI, 3.4 kb)Apr pMMB66HE Ql(pPH501, NsiI-BclI, 1.7 kb)Ap' pMMB66EH fQ(pJT-1,

E. W. Nester

pPH501

pMMB66HE

[Bc,B]

2

FIG. 1. Plasmid construction. Only relevant restriction sites are shown. Restriction site abbreviations: E, EcoRI; P, Pstl: H, HindllI; N, Nsil; B, BanmHl; Bc, BclI. Numbers in parentheses

indicate the molecular sizes (in kilobases). 2 12 25 25

This study This study This study

antibiotics. Growth was at 37°C for E. coli HB101 and 27°C for AS440. The presence of glucose in the medium greatly reduced the production of the extracellular protease of A.

salmonicida and GCAT without affecting the production or

pMMB66EH

W. Kusser

EcoRI-HindIII, 1.2 kb)Apr

export of plasmid-coded aerolysin

pJT1

or

GCAT (data not

shown). When indicated, isopropyl-p-D-thiogalactoside (IPTG) was added to a final concentration of 1 mM. Transformation and mobilization procedures. Strain HB101 was transformed by plasmid DNA as described by Maniatis et al. (18). The plasmid pMMB66 derivatives were mobilized from HB101 to A. salmonicida with the helper plasmid pRK2013 by using the filter mating method described by Harayama et al. (3). Transformants of HB101 were selected on human blood agar (HBA) plates containing 100 ,ug of ampicillin per ml, and transconjugants of AS440 were selected on HBA plates containing 100 pLg of ampicillin per ml and 40 ,ug of rifampin per ml. Plasmid construction. Three plasmids were produced by insertion of A. hydrophila DNA into pMMB66EH or pMMB66HE by the strategies depicted in Fig. 1. Plasmid pJT2 contains a 1.2-kilobase-pair (kb) fragment which we have shown codes for GCAT but which lacks a promoter region (25). Plasmid pKW2 contains a 3.4-kb insert which not only has the structural gene for aerolysin but also has some upstream information, including that for the aerolysin promoter and approximately 1.4 kb of DNA downstream of the gene which is uncharacterized (13). Plasmid pNB5 contains only the structural gene for aerolysin.

Cell fractionation. Shock fluids were obtained by the sucrose-EDTA method of Willis et al. (27). Intact or osmotically shocked cells were disrupted by passing them through a French pressure cell (1,100 kg/cm2) or by repeated freezing and thawing. Release of periplasmic aerolysin. A. salmonicida was grown in LB medium and washed and suspended in the same media containing 20 ,ug of chloramphenicol per ml. Cells were separated from culture supernatants by centrifugation at the specified time intervals and fractionated as described above. Toxin and enzyme assays. f-Lactamase activity was assayed as described earlier (11). Protease was determined with hide powder azure as the substrate by the method of Young and Broadbent (28). Titers were measured as reported previously in 0.4-ml volumes of 0.4% human erythrocytes in phosphate-buffered saline (11). Unless otherwise noted, all fractions were pretreated with 0.5 pg of trypsin per ml for 10 min to convert any protoxin to active toxin. Results are expressed as log2 values of the highest dilutions resulting in 100% cell hemolysis. Ten micrograms of pure, freshly prepared aerolysin gave a titer of 10 when it was measured in this way. Electrophoresis and Western blotting (immuiioblotting). Sodium dodecyl sulfate (SDS)-gel electrophore,is was carried out in 12% acrylamide slab gels by the mett!od of Neville (20). Proteins were stained with Coomassie blue or were transferred to nitrocellulose paper, reacted with anti-aerolysin antibody, and stained with goat anti-mouse horseradish peroxidase-conjugated second antibody as described by Towbin et al. (26). TABLE 2. Effect of induction of aerolysin on release of chromosomal protease and GCAT by A. salmonicida Enzyme in supernatant

GCAT Protease

Plasmid in AS440

% Uninduced

pNB5 pMMB66EH

66.1 + 8.4 (5) 92.2 ± 18.7 (4)

pNB5

pMMB66EH

activity'

118.0 ± 7.5 (4) 99 ± 17.9 (4)

Values are means ± standard errors of the mean (values in parentheses are the number of determinations).

EXTRACELLULAR SECRETION BY AEROMONAS SALMONICIDA

VOL. 171, 1989

2525

8 5

Wu4

-I

c

w 0

-J

04

3

2I 00 0 0 6 4 5 3 1 2 0

TIME AFTER INDUCTION

1

2

3

4

5

1

0

6

2

I, . 3

4

I

6

5

TIME AFTR INDUCTION

TIME AFTER INDUCTION

FIG. 2. Production of aerolysin by E. coli containing pKW2. (A) Hemolytic activity in the cells (U) and culture supernatants (0) of induced cells. No aerolysin was detected in uninduced cells or culture supernatants (O). (B) Growth of induced (0) and uninduced (0) cells. (C) ,3-Lactamase in culture supernatants of induced (0) and uninduced (0) cells.

RESULTS Production of aerolysin and GCAT by E. coli. Aerolysin was measured in E. coli carrying pKW2 a few minutes after induction with IPTG (Fig. 2A). The protein could not be detected in the absence of IPTG, indicating that the structural gene was strictly under the control of the tac promoter in the plasmid. Since pKW2 also contained the A. hydrophila aerolysin promoter, this observation indicates that this promoter is not recognized in E. coli. At early times, all of the toxin was associated with cells, but later it appeared in the culture supernatant. This was paralleled by the release of ,B-lactamase (Fig. 2C) and a decline in the number of viable cells (Fig. 2B), leading to the conclusion that cell disruption occurred. Very similar results were obtained for the production of GCAT by E. coli carrying pJT2. Until there were signs of cell disruption, all of the enzyme produced on induction was recovered in the cells (data not shown). Aerolysin and GCAT are released by intact A. salmonicida. In contrast to the results with E. coli, both aerolysin and GCAT appeared in cell-free culture supernatants of A. salmonicida a few minutes after induction with IPTG (Fig. 3). Cell-associated GCAT and aerolysin were only detected late in growth, when extracellular concentrations were very high. Both were released by osmotic shock, indicating that they were located in the periplasm. In no case was the appearance of protein in the culture supernatant the result of cell disruption, as all of the strains grew at the same rate whether or not they were producing plasmid-coded aerolysin or GCAT (Fig. 4), and most of the 1-lactamase activity remained associated with the cells (Fig. 5). Aerolysin was produced with and without induction with IPTG by the strain carrying pKW2 (Fig. 3A), indicating that, unlike E. coli, A. salmonicida can recognize the A. hydrophila aerolysin promoter.

i

B

-00

I

co

0

C

SDS-polyacrylamide gel electrophoresis of A. salmonicida culture supernatants. The results in Fig. 6 show that proaerolysin and GCAT were the major proteins in culture supernatants after induction of A. salmonicida containing the corresponding plasmid. The molecular weights of both proteins matched the molecular weights of purified samples, indicating that the signal sequences were correctly removed by A. salmonicida. Proaerolysin was present in the culture supernatant of the strain containing pKW2 whether it was grown with or without IPTG, confirming the results presented in Fig. 3A. Aerolysin expression in A. salmonicida does not affect protease release but may reduce GCAT export. A. salmonicida normally releases several of its own proteins into the culture supernatant during growth in LB medium without glucose, including protease and chromosomal GCAT (negligible quantities of GCAT were produced by this strain in the presence of glucose). Export of these enzymes by strain AS440 containing pNB5 was measured with and without induction in order to determine the consequences of the concomitant export of large quantities of aerolysin. The results of one experiment are shown in Fig. 7. Induction did not affect the appearance of protease in the culture supernatant; however, the export of chromosomal GCAT was reduced when aerolysin was induced in the late log phase. Such moderate decreases in GCAT export were consistently observed in several experiments, although the extent of the reduction varied widely (Table 2). Proaerolysin can be detected in the periplasm of A. salmonicida. The results presented in Fig. 3A demonstrate that aerolysin can also be detected in the periplasm of A. salmonicida when extracellular concentrations are high. In order to study this further, the periplasmic fraction was isolated from cells grown in LB medium without glucose and compared with the culture supernatant by SDS-polyacrylamide gel electrophoresis and immunoblotting. Under these condi-

0

I

A-----

IF

w

200I-

I

U

100

C) C, 0

4 6 2 TIME AFTER INDUCTION

8

0


8

FIG. 3. Production of aerolysin and phospholipase by A. salmonicida. (A) Aerolysin production by pNB5- and pKW2-containing strains. Shown are culture supernatants from pNB5 (U, O) and pKW2 (0, 0) and cells from pNB5 (A, A) and pKW2 (*). (B) GCAT production by pJT2-containing strains. Cells (A, A) and supernatants (N, Cl) were examined. Solid symbols refer to induced cells; open symbols refer to uninduced cells.

m

8.5

9

8.0

0


8

FIG. 4. Effect of extracellular protein production on growth of A. salmonicida. This is the same experiment as that for which the

results are shown in Fig. 3. Solid open symbols refer to uninduced squares, pKW2; triangles, pJT2.

symbols refer to induced cells; cells. Symbols: circles, pNB5,

2526

J. BACTERIOL.

WONG ET AL.

w

w 4w