Journal of Fish Diseases 1993, 16, 73-78

Production of the lethal acetylcholinesterase toxin bydifferent Aeromonas hydrophila strains

L. A. R O D R I G U E Z , A. I. G. F E R N A N D E Z & T. P. NIETO Departmento de BiologiaFundamental. Facultad de Ciencias. Campus de Orense. Orense, Spain

Abstract. Seven fish pathogenic isolates of Aeromonas hydrophila. one A. sobria and oneA. caviae were investigated for produetion of the fish lethal acetylcholinesterase toxin(AcChE-toxin). Western blotting was used for screening the ECP of these strains with arabbit antiserum prepared against the purified toxin of strain B32 and all the isolates (exceptA. sobria) gave positive results with different patterns of bands. The AcChE-toxin appearsto be secreted as a protein of high molecular weight which is stable at -20"C, and in 90%of the strains tested, it appears to be split into lower molecular weight fragments by theaction of other eotnponents present in the ECP. The smallest, stable and highly activefragment has a MW of lSkDa.

Introduction

Aeromonas hydrophila is a prolific producer of extracellular products and many of them havebeen described as important factors in pathogenesis; for example, haemolysins (Ljungh,Wretlind & Molley 1981; Rodriguez, Ellis & Nieto 1992a); proteases (Nieto & Ellis 1986;Rodriguez ct al. 1992a); cytotoxins (Asao, Kinoshita, Kozaki, Uemura & Sakaguchi 1984),and enterotoxins (Santos, Toranzo, Barja, Nieto & Villa 1988).

In a previous study, the most toxic component so far recognized was purified and charac-terized as an acetylcholinesterase (AcChE-toxin) from the extracellular products (ECP) ofA. hydrophila, strain B32, and studies of the in vivo effects in rainbow trout, Oncorhynchusmykiss (Walbaum), indicated this toxin produced typical neurotoxic symptoms (Nieto, Santos,Rodnguez & Ellis 1991; Rodriguez, Ellis & Nieto 1992b).

The aim of the present work was to establish the distribution of the AcChE-toxin amongother fish pathogenic strains of Aeromonas. To do this, the purified AcChE-toxin of strain B32was used to produce a rabbit anti-AcChE-toxin antiserum which was used to probe WesternBlots of crude ECP from several different strains of fish pathogenic motile Aeromonas.

Materials and methods

Pure AcChE-toxin

The AcChE-toxin was purified from the ECP of ^4. hydrophila, strain B32 (Nieto et al. 1991).This toxin was toxoided by adding formalin 40% (w/v) (formaldehyde) to a concentration of3% (v/v) and incubating for 48 h at 22 °C (f-toxin) and dialysed overnight against distilled water.

Rabbit anti-toxin antiserum

A New Zealand white rabbit weighing approximately 2 kg was used for immunization, andanother one to obtain normal serum as control.

Correspondence: T. P. Nieto, Departmento dc Biologia Fundamental, Facultad de Cieneias, Campus de Orense,As Lagoas, 32004 Orense, Spain.

73

74 L. A. Rodriguez ct al.

Pure toxoided AcChE toxin N)//g was emulsified with an equal volume of Freund's com-plete adjuvant (FCA-Difco) prior to subcutaneous injection at eight dorsal sites. Five weeksafter immunization, a similar booster injection was given using incomplete Freund's adjuvant.Three weeks later, serum was collected and stored at -20''C until use.

Bacterial strains

Seven highly and weakly virulent (in fish) strains of A. hydrophila (Table 1), including B32,were used in this study. One strain of A. sobria (P26t) and one of A. caviae (P322) wereincluded for comparison (Santos et al. 1988). Stock cultures were maintained on tryptone soyaagar (TSA) (Oxoid) slants at 4°C under mineral oil.

Extracellular products

The extracellular products (ECP) from different strains were obtained according to Liu (1957)as follows. Log phase cultures in tryptone soya broth (TSB) (Difco) were inoculated ontocellophane sheets overlaying TSA and incubated at 22°C for 24 h. The cultures were suspendedin phosphate buffered saline (PBS), pH 7-3, and centrifuged at lOOOOg for 30min, and thesupernatant (ECP) was sterilized by filtration (0-221.1m membrane, Millipore) and stored at-20"C. In addition, ECP of strains B32 and THO were harvested at different incubation times(16, 24 and 48 h), and used freshly prepared or after maintenance for 24 h at room temperature.

Protein determination

The total protein of crude ECP was measured by Bradford's method (1976), using bovineserum albumin as standard.

SDS-PAGE

Sodium dodecyl sulphate 12% polyacrylamide gels were used for separating ECP components,according to Laemmli (1970). Five to 15/ g of protein of each crude ECP were used to prepare

Table 1. Source and virulenee of Aeromonas strains

Strain

A. hydrophila:t ^al

B32SOAi*80A,*

67P24*Bs,P212

A. sobria:P2M

A. caviae:P322

Origin

Oncorhynchus mykiss (Walbaum)0. mykiss0. mykiss0. mykissPlecoglossus ahivelis (Temminck & Schlegel)0. mykiss0. my kiss

0. mykiss

0. mykiss

Country

SpainSpainSpainSpainJapanSpainSpain

Spain

Spain

LDw

1

2-0 X 104-2 X 106-4 X 108-2 X IO'8-6 X 10*

1-4x10-'4-3 X 10

1-4x10^^

5-2 X 10

•^ Private collection.

A. hydrophila acetylcholinesterase toxin 15

the samples with fresh sample buffer. Bio Rad low molecular weight standards were used.The gels were prepared in triplicate sections; one part was stained with silver stain (Morrisey1987) and the other two parts were used to transfer the proteins (one probed by control rabbitserum and the other by AcChE-toxin antiserum).

Western blotting and iinmunostaining

The proteins were transferred to nitrocellulose membranes using a continuous buffer systemaccording to the Pharmacia electrophoretic transfer kit at a current of 1 mAcm^^ of transunitfor lh .

Immunostaining was performed according to the Bio Rad immuno-blot assay kit cat no.170—6509. The nitrocellulose membrane was incubated in the rabbit anti-toxin serum diluted1:100 in antibody buffer (2g gelatin and 200mI TBS) (TBS (21): 0-968g Tris, ll-696g NaCl,900 ml dH.O. Adjust pH to 7-5 with HCI. dH2O to 2000ml).

The second antibody solution was goat anti-rabbit Ig G conjugated to alkaline phosphatase(Bio-Rad) diluted 1:1000 in antibody buffer. The staining solution was a mixture of tworeagents:(solution A) 30mg of nitroblue tetrazolium (NBT) dissolved in 0 7ml N,N-dimethyl-formamide (DMF) mixed with 0-3 ml of distilled water; (solution B) 15 mg of 5-bromo-4-chloro-3-indolyl phosphate (BCIP) dissolved in 1ml of DMF. Both solutions were mixed with100ml of carbonate buffer pH 9-8 (8 4g sodium hydrogen carbonate, 0-203 g magnesiumchloride, in 900ml of distilled water). The colour development was stopped by transferring themembrane into distilled water, drying on filter paper and storing away from light.

In all cases a negative control with normal rabbit serum was used.

Results and discussion

The control rabbit serum did not stain any of the bands in the Western blots. Immunostainingof pure AcChE-toxin (Fig. 1) and the original B32 ECP harvested at 48 h of incubation andmaintained 24 h at room temperature (Fig. 2, lane C) with specific anti-AcChE-toxin serumgave a positive result with a single band of 15kDa.

When Western blots of ECP of different Aeromonas strains were probed with the anti-AcChE-toxin rabbit antiserum, positive reactions were obtained with ECP of seven strains of

Figure I. Western blot of purified AcChE-toxin (2//g) with anti AcChE-toxin serum (diluted 1/20).

76 L. A. Rodriguez cl al.

kDa

B CFigure 2. Western blot of crude ECP (10//g) using two A. hydrophila ECP strains and the original ECP B32strain used for toxin purification with anti-AcChE-toxin (diluted 1:20) antiserum: (A) Tj (, harvested after 24hincubation: (B) B51 har\'ested after 24h incubation: and (C) B32 harvested after 48h and maintained for 24liat room temperature.

A. hvdrophila and one strain of A. caviae (Figs 2 & 3). The staining reaction was weak withthe ECP of strain 8OA3 (Fig. 3, lane C), a virulent strain of A. hydrophila, and no reactionoccurred with ECP of A. sobria (P261) (Fig- 3, lane E). Different patterns of staining appearedamong the strains with bands which ranged from 94 Xo 15-5 kDa (Figs 2 & 3).

When freshly prepared ECP of strains B32 and THO with 16 h growth were Western blotted

2

1 4B D

Figure 3. Western blot of erudc ECP (lO//g) of different A. hydrophila, A. sohria and A. caviae strainshar\estcd after 24h incubation and stained with anti-AcChE-toxin antiserum (diluted 1:20): (A) P212; (B) 80A,;(C) 8OA3; (D) V,z2, (E) P2M; and (F)

A, hydrophila acetylcholinesterase toxin 11

Figure 4. Western blotting of crude ECP (16//g) of strains B^T and T,s(,, obtained at different incubation times,stained with anti-AcChE-toxin scrum (diluted 1/20): (A) Tso ECP obtained at I6h; (B) B32 ECP obtained at4iSh; and (C) B32 ECP obtained at 16h.

and probed with specific anti-AeChE-toxin serum, a single band with high molecular weight(67 kDa) was stained (Fig. 4, lanes A & C).

The same pattern was observed when the ECP of B32, harvested after 16 h incubation andstored at —20°C for 2 weeks, was blotted (data not shown); hence, the 67kDa MW form isstable at -20°C.

The ECP of B32 harvested after 48 h growth showed a pattern with a clear band and adiffuse band (Fig. 4, lane B).

From these results, it must be concluded that the AcChE-toxin is excreted as a protein ofhigh molecular weight, stable in the ECP at —20°C and present in 90% of strains tested; it iscleaved at room temperature, possibly by the presence of other components in the whole ECP,into a small stable polypeptide of 15 kDa.

The differences in the banding patterns between strains is probably due to differencesin the proteases present in the ECP of these strains and the optimal growth time for theirproduction (Nieto & Ellis 1991).

Further work is required to clarify which component plays a role in fragmenting the highmolecular weigkt form and if this is necessary to activate the toxin.

Acknowledgment

A. I. G. Fernandez and L. A. Rodriguez acknowledge the Conselleria de Educacion eOrdenacion Universitaria, Xunta de Galicia, Spain, for a research fellowship. This work wassupported by a grant of DGICYT (PB89-0547).

References

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Bradford N. M. (1976) A rapid and sensitive method for the quantification of microgram quantities of proteinutilising the principle of protein-dye binding. Analytical Biochemistry 72, 248-254.

Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature (London) 227, 680-685.

78 L. A. Rodriguez cl al.

Liu P. V. (1957) Survey of hcrnolysin production among species of pscudomonads. Journal of Bacteriology 74,718-727.

Ljungh A., Wrellind B. & Mollcy R. (1981) Separation and characterization of entcroloxin and two haemolysinsfrom Acromanas hydrophiJa. Acta Rathol. Microhiol. Scand. Sect B-89, 3X7-397.

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Niclo T. P. & Ellis A. E. (1991) Heterogeneity of extracellular proteases produced by different isolates ofAeromonas hydrophiUi and Aeromonas sobria pathogenic for fish. Journal of Fish Diseases 14, 229—235.

Nieto T. P., Santos Y., Rodriguez L. A. & Ellis A. E. (1991) An extracellular acetyleholinesterase produced byAeromonas hydrophila is a major lethal toxin for fish. Microbial Pathogenesis I I , 101 — 110.

Rodriguez L. A., Ellis A. E. & Nieto T. P. (1992a) Purification and characterisation of an extracellularmctalloprotease, serineprotease and haemolysin of Aeromonas hydrophila strain B32: all are lethal for fish.Microbial Pathogenesis. in press.

Rodriguez L. A., Ellis A. E. & Nieto T. P. (1992b) Effects of the acetylcholinesterase toxin of Aeromonashydrophila on the central nervous system of fish. Microbial Pathogenesis 13, 17—24.

Santos Y., Toranzo A. E,. Barja J. L.. Nieto T. P. & Vila T. G. (1988) Virulence properties and enterotoxinproduction of Aerotnonas strains isolated from fish. Infection and Immunity 56, 3285—3293.