FEMS Microbiology Letters 142 (1996) 185-189

MICROBIOLOGY LETTERS

The role of the capsular polysaccharide of Aeromonas salmonicida in the adherence and invasion of fish cell lines

Susana Merino a, Alicia Aguilar a, Xavier Rubires a, Dolores Simon-Pujol b, Francisco Congregado bt*, Juan M. Tom&s a

a Deportomento de Microbiologio, Universitot de Barcelona. Avdo. Diagonal 645, 08071 Barcelona, Spoin b Departomento de Microbiologio y Porasitologio Sanitorias, Facultot de Farmdcia. Universitat de Barcelona, Avdo. Joan XXIII sin,

08028 Barcelona, Spain

Received 10 April 1996; revised 21 June 1996; accepted 28 June 1996

Abstract

The ability of several Aeromonas salmonicida strains grown under different conditions (capsulated and non-capsulated) to adhere to and invade two fish cell lines was compared. The level of adherence was slightly higher when the strains were grown under conditions promoting capsule formation than when the same strains were grown under conditions which did not promote capsule formation. However, the most significant difference among the wild-type strains grown under conditions promoting capsule formation was the ability to invade fish cell lines, which was significantly higher than when the same strains were grown under conditions which did not promote capsule formation. From these results we conclude that the capsular polysaccharide, in these strains, is an important factor for intracellular invasion.

Keywords: Aeromonos solmonicida; Capsule; Polysaccharide; Adherence; Invasion

1. Introduction

Aeromonas salmonicida is an important pathogen of salmonid fishes, producing the systemic disease furunculosis. The principal virulence factor described

for this pathogen appears to be an S-layer (A-layer), which is composed of a two-dimensional array of a crystalline tetragonal protein (A-protein of 49,000

* Corresponding author. Tel. : +34 (3) 402 1486; Fax: +34 (3) 411 0592; E-mail: juant@porthos.bio.ub.es

Da molecular mass) [l], tethered to the cell by LPS

[2]. Labelling studies have shown that the A-layer appears to cover most of the surface of virulent A. salmonicida [3], although some LPS may also be ex- posed [4]. This structure has been shown to protect the bacterium from serum killing in a manner that requires both LPS and the A-layer [5], but the

A-layer is not completely necessary for the bac- terium to be resistant to serum killing [5]. The con-

ditions under which A. salmonicida forms a capsule in vitro and also in vivo have recently been described

I&71. In the present study, we have investigated the role

of the capsule in the adherence and invasion by A. salmonicida strains of two cell fish lines.

0378-1097/96/%12.00 Copyright 0 1996 Federation of European Microbiological Societies. Published by Elsevier Science B.V.

PIISO378-1097(96)00263-7

186 S. Merino et al. IFEMS Microbiology Letters 142 (1996) 185-189

2. Materials and methods

2.1. Bacterial strains and media

volume of sample buffer (containing 4% SDS) and

boiled for 5 min; 10 1.11 portions were applied to the

gel. LPS bands were detected by the silver staining

method of Tsai and Frasch [14].

The A. salmonicida strains used here were three

typical A-layer positive strains which produced A- protein (A449, A450 and A451), a gift from W.W.

Kay (University of Victoria, British Columbia, Can-

ada), and strain ATCC 14174 a typical A-layer neg- ative strain which did not produce A-protein [8].

Cultures were maintained and grown as previously

described for non-capsulation conditions [9]; the medium used to obtain capsule formation was yeast

extract-peptone-glucose-mineral salts (YPGS) med-

ium [6]. The strains were also grown on an autoly-

sate of fish viscera medium (FVM) [lo].

2.4. Electron microscopy

Electron microscopy of negatively stained whole

cells and of thin sections labelled with polycationic ferritin was performed as previously described [ 151.

2.5. Antisera

2.2. Lipopolysaccharide and capsule isolation

Lipopolysaccharide (LPS) from A. salmonicidu

strains was purified by the method of Westphal and Jann [l l] as modified by Osborn [ 121. Capsular

polysaccharides were isolated from different A. sal-

monicidu strains as previously described [6].

Polyclonal antiserum against purified capsular

polysaccharide of strain A450 grown at 20°C was raised in adult New Zealand white rabbits as pre-

viously described [16], and rendered specific for cap-

sular polysaccharide by extensive adsorption using

the same strain grown on trypticase soy agar (TSA; non-capsulation conditions). Monoclonal

antisera against the A-protein or LPS [6] were also used.

2.6. Fish cell lines

2.3. Electrophoretic techniques

SDS-PAGE was performed by the procedure of Laemmli [13]. Samples were mixed with an equal

EPC cells (epithelioma papulosum of carp, Cypri-

nus carpio) were grown at 25’C [17], and SBL cells (sea bass larvae) were grown at 20°C [18].

Table 1

The adherence to EPC and SBL cells of A. salmonicida strains grown under different conditions

Strain Growth media” Capsule formation Adherence tab

EPC cells SBL cells

A449

A450

A451

ATCC14174

A449

A450

A451

ATCC14174

A449

A450

A451

ATCC14174

TSA

TSA

TSA

TSA

YPGS

YPGS

YPGS

YPGS

FVM

FVM

FVM

FVM

_ 0.89 f 0.29

_ 0.91 + 0.25

_ 0.88 C 0.28

_ 0.72 + 0.23 + 1.34f0.35

+ 1.36 + 0.33

+ 1.42 k 0.41

+ 1.37 + 0.29

+ 1.43 f 0.37

+ 1.52 + 0.41

+ 1.5150.33

+ 1.48 f 0.36

0.93 f 0.30

0.98 f 0.31

0.95 f 0.27

0.79 + 0.24

1.72 + 0.38

1.7920.32

1.82k0.36

1.68 + 0.34

1.89 + 0.39

1.9OkO.37

1.92 2 0.40

1.87+0.31

&TSA, trypticase soy agar; YPGS, yeast extract-peptone-glucose-mineral salts medium; FVM, fish viscera medium.

aPercentage adherence is the percentage of input bacteria adhering after extensive washing without gentamycin treatment. Numbers represent

the mean + standard deviation. The negative control for adherence was E. coli HBlOl which showed 0.16 +_ 0.08% adherence.

S. Merino et al. IFEMS Microbiology Letters 142 (1996) 185-189 187

2.7. EPC and SBL adherence and invasion assays

These assays methods were adapted from Oel- schlaeger et al. [19]. Briefly, approximately 2 X lo6 bacterial cells were layered onto confluent monolayer of approximately 7 x lo4 EPC or SBL cells sus- pended in Hank’s balanced salt solution (HBSS) per well in 24-well plates, and incubated at 25°C or 20°C for 2 h under an atmosphere of 5% COa/95% air. For determination of adherence, the cells were washed extensively in HBSS with strong agitation for 2 min, prior to lysis of the monolayer with 0.01% Triton X-100 and enumeration of total bacteria by plate count in TSA medium. For determination of invasion, the monolayer was washed extensively with HBSS, and fresh, prewarmed medium containing gentamycin (100 l.@nl) was added to kill extracellu- lar bacteria grown under both conditions (promoting or not promoting capsule formation, respectively). After 1 h incubation, the monolayer was washed twice with HBSS, and cells lysed with 0.01% Triton X-100 for 30 min; the released intracellular bacteria were enumerated by plate counting. The invasive ability was expressed as the percentage of the inocu- lum surviving the gentamycin treatment; adherent bacteria were expressed as the total number of bac- teria enumerated without antibiotic treatment. In some experiments coverslips were used in the 24- well trays, and after incubation the coverslids were

washed, fixed in 70% methanol and stained with Giemsa. Coverslips were then mounted on glass mi- croscope slides and adherence was assessed by bright-field microscopy. For each assay, bacteria ad- hering to 30 randomly selected cells from each of two monolayers were counted. The assays were per- formed at least in triplicate.

3. Results and discussion

All the A. salmonicida strains tested produced a capsular polysaccharide when grown in YPGS med- ium, detected either by electron microscopy of nega- tive staining whole cells or of thin sections labelled with polycationic ferritin, as previously described [6,15]. The same strains also produced capsular poly- saccharide when grown on FVM but not when grown on TSA, as shown previously [6]. The purified capsular polysaccharide of the strains, as well as the whole cells grown on FVM, cross-reacted with spe- cific antiserum against the capsular polysaccharide, as did the capsular polysaccharide, or whole cells, of the same strains grown in YPGS. However, no reac- tion was detected with this antiserum using whole cells of the same strains grown in trypticese soy broth (TSB).

The A. salmonicida strains showed identical LPS profiles when grown on the different media. Further-

Table 2

The invasion of EPC and SBL fish cells by A. salmonicida strains grown under different conditions

Strain Growth media” Capsule formation Invasion tab

EPC cells SBL cells

A449

A450

A451

ATCC14174

A449

A450

A451

ATCC14174

A449

A450

A451

ATCC14174

TSA

TSA

TSA

TSA

YPGS

YPGS

YPGS

YPGS

FVM

FVM

FVM

FVM

- 0.12 + 0.006

- 0.13 + 0.005

- 0.11 f0.003

_ 0.14 f 0.007

+ 0.91 f 0.09

+ 0.97 f 0.08

+ 0.96 fO.10

+ 0.95 f 0.06

+ 1.03 f 0.08 + 0.99fO.11

+ 1.05 f. 0.09

+ 1.07 + 0.08

0.17+0.005

0.16+0.006

0.17~0.005

0.19f0.005

1.22+0.11

1.24f0.10

1.20 + 0.08

1.25 k 0.08

1.30f0.12

1.37f0.10

1.33 + 0.09

1.42f0.13

“See Table 1.

bPercent invasion is the percentage of input bacteria surviving, after extensive washing, gentamycin treatment. Numbers represent the

mean + standard deviation. The negative control for invasion was E. coli HBlOl which showed 0.04 f 0.004% invasion.

188 S. Merino et al. IFEMS Microbiology Letters 142 (1996) 185-189

more, no relevant differences in the outer membrane

protein profile (except for the presence or absence of

the A-protein in appropriate strains) were observed when these strains were grown in the different media

(data not shown).

As shown in Table 1, strains grown under condi-

tions promoting capsule formation (YPGS or FVM) showed greater adherence (approx. l.%fold, in re-

producible experiments) to EPC and SBL fish cell lines than the same strains grown in TSB. The ad-

herence for all the strains to SBL was greater than to

EPC fish cells.

However, the most significant effect observed was

on the invasion of the fish cell lines by A. salmonicida

(Table 2). All strains grown in YPGS or FVM (pro- moting capsule production) showed a higher degree

of invasion than the same cells grown in TSB. The increase in invasion was at least g-fold for the A’

strains, and higher for strain ATCC 17174 (A-). As with adherence, the bacteria showed a higher degree

of invasion of SBL cells than of the EPC cells.

Prior treatment of these strains with decomple-

mented (treated at 56°C for 35 min, [16]) antiserum

against the capsular polysaccharide, abolished the

increase in invasion of the capsulated strains. No effect on invasion of the capsulated strains occurred

if the bacteria were treated with specific antiserum against the A-protein or LPS.

It is concluded that capsular polysaccharide is a

factor in these strains that promotes adherence and invasion of cells. As A. salmonicida strains grown in

vivo produce a capsular polysaccharide [7], we sug- gest that capsular polysaccharide may be an impor-

tant virulence factor for this bacterium, by promot- ing tissue invasion. Preliminary results (unpublished data, personal communication of R. Gaustad) sug-

gested that purified capsular polysaccharide isolated from cells grown on YPGS is an effective vaccine against furunculosis, which is superior to purified LPS or A-layer.

Acknowledgments

This study was supported by a Grant PB94-0906 from DGICYT (Ministerio de Education y Ciencia) to J.M.T. X.R. and A.A. are FPI fellowships from Ministerio de Education y Ciencia. We thank R.

Gaustad from the National Veterinary Laboratory

of Oslo (Norway) for unpublished results. We thank Maite Polo for her technical assistance.

References

[II

I21

[31

I41

[51

I61

Ishiguro, E.E., Kay, W.W., Ainsworth, T., Chamberlain, J.B.,

Buckley, T.A. and Trust, T.J. (1981) Loss of virulence during

culture of Aeromonas salmonicida at high temperature. J. Bac-

teriol. 148, 333400.

Belland, R.J. and Trust, T.J. (1985) Synthesis, export and

assembly of Aeromonas salmonicida A-layer analyzed by trans-

poson mutagenesis. J. Bacterial. 163, 877-881.

Dooley, J.S.G., Engelhardt, H., Baumeister, W., Kay, W.W.

and Trust, T.J. (1989) Three-dimensional structure of an open

form of the surface layer from the fish pathogen Aeromonas

salmonicida. J. Bacterial. 171, 190-197.

Phipps, B.M. and Kay, W.W. (1988) Immunoglobulin binding

by the regular surface array of Aeromonas salmonicida. J. Biol.

Chem. 263, 9298-9303.

Munn, C.B., Ishiguro, E.E., Kay, W.W. and Trust, T.J. (1982)

Role of surface components in serum resistance of virulent

Aeromonas salmonicida. Infect. Immun. 36, 1069-1075.

Garrote, A., Bonet, R., Merino, S., Simon-Pujol, M.D. and

Congregado, F. (1992) Occurrence of a capsule in Aeromonas

ralmonicida. FEMS Microbial. Lett. 95, 127-132.

[7] Gardutio, R.A. and Kay, W.W. (1995) Capsulated cells of

Aeromonas salmonicida grown in vitro have different func-

tional properties than capsulated cells grown in vivo. Can. J.

Microbial. 41, 941-945.

[8] Gustafson, C.E., Thomas, C.J. and Trust, T.J. (1992) Detec-

tion of Aeromonas salmonicida from fish by using polymerase

chain reaction amplification of the virulence surface array

protein gene. Appl. Environ. Microbial. 58, 38163825.

[9] Merino, S., Alberti, S. and Tomas, J.M. (1994) Aeromonas

salmonicida resistance to complement mediated killing. Infect.

Immun. 62, 5483-5490.

[lo] Clausen, E., Gildberg, A. and Raa, J. (1985) Preparation and

u 11

L1-21

u31

u41

1151

testing of an autolysate of fish viscera as growth ubstrate for

bacteria. Appl. Environ. Microbial. 50, 15561557.

Westphal, 0. and Jann, K. (1965) Bacterial ipopolysacchar-

ides: extraction with phenol-water and further applications of

the procedure. Carbohydr. Chem. 5, 83-91.

Osborn, M.J. (1966) Preparation of lipopolysaccharide from

mutant strains of Salmonella. Methods Enzymol. 8, 161-164.

Laemmli, U.K. (1970) Cleavage of structural proteins during

the assembly of the head of bacteriophage T4. Nature (Lond.)

227, 680-685.

Tsai, CM. and Frasch, C.E. (1982) A sensitive silver stain for

detecting lipopolysaccharide in polyacrylamide gels. Anal.

Biochem. 119, 115-l 19.

Martinez, M.J., Simon-Pujol, D., Congregado, F., Merino, S.,

Rubires, X. and Tomas, J.M. (1995) The presence of capsular

polysaccharide in mesophilic Aeromonas hydrophila serotypes

0:ll and 0:34. FEMS Microbial. Lett. 128, 69-74.

S. Merino et al. IFEMS Microbiology Letters 142 (1996) 185-189 189

[16] Merino, S., Camprubi, S. and Tomas, J.M. (1991) The role of [18] Pinto, R.M., Jofre, J., Abad, F.X., Gonzalez-Dankaart and

lipopolysaccharide in complement killing of Aeromonas hydro- Bosch, A. (1993). Concentration of fish enveloped viruses phila strains of serotype 0:34. J. Gen. Microbial. 137, 1583- from large volumes of water. J. Virol. Methods 43, 3140. 1590. [19] Oelschlaeger, T.A., Guerry, P. and Kopecko, D.J. (1993).

[17] Wolf, K. and Mann, J.A. (1980) Poikilotherm vertebrate cell Unusual microtubuledependent endocytosis mechanisms trig- lines and viruses: a current list for fishes. In Vitro 16, 168

179.

gered by Campylobacter jejuni and Citrobacter freundii. Proc. Natl. Acad. Sci. USA 90. 6884-6888.