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An aerolysin-like enterotoxin from Vibrio splendidus may beinvolved in intestinal tract damage and mortalities in turbot,

Scophthalmus maximus (L.), and cod, Gadus morhua L.,larvae

H L Macpherson 1 , Bergh 2,3 and T H Birkbeck 1

1 Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK 2 Institute of Marine Research, Bergen, Norway 3 Department of Biology, University of Bergen, Norway

Abstract

Vibrio splendidus is a pathogen that can cause majorlosses during the early stages of larval turbot rearing when live feed (rotifers or Artemia ) is used. Ashaemolytic bacteria have often been associated withlarval rearing losses, we studied the role of theV. splendidus haemolysin in infection of larvae.From a bank of over 10 000 transposon mutants of V. splendidus , two different types of haemolysin-negative mutants were obtained. Both had lostvirulence for larval sh, and immunohistochemistry showed that the transposon mutant studied colo-nized the turbot larval intestinal tract at a similarlevel to the wild-type organism but did not causedamage or signs of enteritis found with the wild-type organism. One transposon insertion site waslocated within a gene with high homology to aer-olysin, the cytolytic toxin produced by several Aeromonas spp. The haemolysin, which we havetermed vibrioaerolysin, had properties similar toaerolysin and osmotic protection studies showedthat it formed pores in the membranes of erythro-cytes of similar diameter to those of aerolysin. The

Tn10 insertion site of the second transposon mu-tant was in an adjacent ToxR-like gene, suggesting that this might control expression of the vibrioaer-olysin. The gastroenteritis caused by Aeromonas spp.in humans is considered to be due to production of

aerolysin causing cyclic AMP-dependent chloridesecretion in cells of the gastrointestinal tract.Damage to the intestinal tract of marine sh larvaecould occur in a similar way, and it is possible thatseveral Vibrio spp. found in the developing bacterialora of the larval sh gut can secrete aerolysin-liketoxins leading to death of larvae in the early rearing stages. Routine bacteriological screening on bloodagar plates of live feed is recommended with mea-sures to reduce the concentrations of haemolyticbacteria in rearing systems.

Keywords: aerolysin, Gadus morhua , gut microora,larval rearing, Scophthalmus maximus , Vibrio splendidus , vibrioaerolysin.

Introduction

The worldwide demand for sh cannot now be metfrom wild sheries alone, and aquaculture hasexpanded rapidly in the past three decades to meetthis demand (FAO 2006). Although the majority of aquaculture production is from freshwater, produc-tion of sh in marine environments now exceeds

17 million tonnes per annum (FAO 2006). There isincreasing interest in culturing other species such asturbot, Scophthalmus maximus (L.), halibut, Hip- poglossus hippoglossus (L.), and cod, Gadus morhua L., but this requires a signicant increase in thesupply of juvenile sh (Sva sand, Ottera & Taranger2004). Large losses still occur in larval rearing (Vadstein, Mo & Bergh 2004), and bacteria areconsidered to play a major role in such lossesbecause administration of antibiotics can enhance

Journal of Fish Diseases 2012, 35 , 153167 doi:10.1111/j.1365-2761.2011.01331.x

Correspondence T H Birkbeck, University Marine Biological Station, Millport, Isle of Cumbrae, Scotland KA28 0EG, UK (e-mail: [email protected]) DNA sequences have been deposited with EMBL under accession number AM157713.

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survival (Gatesoupe 1982, 1989; Verner-Jeffreys,Shields, Bricknell & Birkbeck 2004) and culture of turbot larvae in a bacteria-free rearing system canproduce very high survival (Munro, Barbour &Birkbeck 1995). Bacterial species linked withmortalities in larval turbot rearing include Vibrio splendidus (Gatesoupe, Lambert & Nicolas 1999;Thomson, Macpherson, Riaza & Birkbeck 2005),Vibrio pelagius (Villamil, Figueras, Toranzo, Planas& Novoa 2003) and Vibrio harveyi (Sun, Zhang,Tang, Wang, Zhong, Chen & Austin 2007). Of these, V. splendidus has been isolated from severaldifferent batches of turbot larvae over a number of years (Gatesoupe et al. 1999; Thomson et al.2005).

The pathogenic mechanisms of sh-pathogenicvibrios have so far been largely restricted to an

analysis of Vibrio anguillarum (Weber, Croxatto,Chen & Milton 2008) and V. harveyi (Sun et al.2007). Vibrio anguillarum has been studied exten-sively, and virulence determinants such as an iron-sequestering system (Crosa 1980), metalloprotease(Milton, Norqvist & Wolf-Watz 1992) and agella (Milton, O Toole, Horstedt & Wolf-Watz 1996;O Toole, Milton, Horstedt & Wolf-Watz 1997)have been identied, yet the causes of mortalities by this organism are still not completely understood.V. harveyi has been shown to produce a lethal,haemolytic phospholipase, and site-directed muta-genesis to inactivate this enzyme causes loss of lethality in turbot (Sun et al. 2007). Proteasesproduced by Vibrio alginolyticus (Nottage & Birk-beck 1987) and metalloprotease produced by V. splendidus (Le Roux, Binesse, Saulnier & Mazel2007; Binesse, Delsert, Saulnier, Champomier-Verges, Zagorec, Munier-Lehmann, Mazel & Le

Roux 2008) have been shown to be lethal to oysterlarvae.

Routine culture of larval turbot on a large scalewithout recourse to the use of antibiotics could beachieved if the microbial ora was regulated(Munro et al. 1995; Verschuere, Rombaut, Sorge-loos & Verstraete 2000; Birkbeck 2004; Gram &Ring 2004) and growth of pathogens prevented. With this in mind, we have begun a study of themechanisms of pathogenesis of V. splendidus inlarval turbot and cod. Here, we have usedtransposon mutagenesis to prepare V. splendidus mutants decient in haemolysin production. Weshow that a haemolysin-decient mutant is notvirulent to larval turbot and does not cause theenteritis associated with infections caused by thewild-type organism. In addition, the DNA sequence

of the haemolysin gene has been determined, andthis shows the haemolysin to be unrelated to thoseknown to be produced by Vibrio species but to beclosely related to aerolysin (Parker, van der Goot &Buckley 1996), the rst time that expression of this type of toxin has been detected outside of the Aeromonas genus.

Materials and methods

Bacteria and transposon Tn10

The bacteria used in this study are shown inTable 1. V. splendidus DMC-1 was of biovar 1(Thomson et al. 2005) and was resistant to ampi-cillin (25 l g), tetracycline (25 l g) and streptomy-cin (10 l g) but sensitive to chloramphenicol(50 l g) and kanamycin (30 l g) when screenedwith Mastring S discs (Mast Diagnostics Ltd).

Table 1 Bacterial strains used in this study

Bacterial strains andcharacteristics References

Escherichia coli S17-1 k pir Tp r Sm r recA thi pro hsd R - M + RP4-2-Tc::Mu::Km Tn7 k pir Simon, Priefer & Puhler (1983)Escherichia coli SM10 k pir thiL thrL leuB6 supE44 tonA21 lacY1 recA ::RP4-2-Tc:: Mu Km r Simons, Houman & Kleckner (1987)Vibrio splendidus DMC-1 Pathogen of larval turbot isolated from a hatchery in Spain Thomson et al. (2005)Vibrio splendidus DMC-1-M2,

DMC-1-M3 and DMC-1-M4Haemolysin-negative Tn10- transposon mutants of DMC-1 This study

Vibrio splendidus DMC-2,DTC-5, DTR-2, DTY-1,LTS-3, LTS-4, LMS-1,LMS-2, LMS-3,LMS-4, LTH-1, LTH-3,LTH-4, HNF-8

Isolated from larval turbot inSpain

Thomson et al. (2005)

Vibrio anguillarum 91079 Isolated from moribund juvenile turbot in the U.K. Horne, Richards, Roberts& Smith (1977)

Roseobacter sp. HNF-1 Isolated from turbot larvae from a hatchery in Spain Thomson et al. (2005)

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with trypsin (Howard & Buckley 1985a) and wastested similarly.

Turbot tissue culture cell assays

The turbot tissue culture cell line TV1-S4 (Fern-a ndez-Puentes, Figueras & Novoa 1993) was kindly supplied by Dr C. Dopazo, University of Santiago,Spain, and cells were maintained in Eagles MinimalEssential Medium (Invitrogen) with 10% new borncalf serum and 2 mm glutamine at 15 C. Concen-trated lter-sterilized supernatant from a 24-hculture of V. splendidus DMC-1 (haemolysin titreof 1024 HU mL

) 1 ) was diluted from 1/2 to 1/2560 in the above medium and added to monolayercultures of TV1-S4 cells in a 24-well cell cultureplate (Nunc, Fisher Scientic). Assay plates were

examined using an inverted microscope, and cellswere assessed visually hourly for the rst 4 h andagain after 24 h for cytopathic effects.

SDS-PAGE

SDS-PAGE was carried out by the method of Laemmli (1970) using Novex 12% acrylamide Bis-Tris 8 8 cm mini-gels (Invitrogen).

Transposon mutagenesis

Vibrio splendidus DMC-1 was subjected to muta-genesis with the transposon Tn10 located in theplasmid vector pBSL181 in E. coli Sm10k pir .Optimization of conditions for transposon muta-genesis led to the following protocol. The donorstrain E. coli SM10k pir was grown overnight in50 mL LB broth in a bafed 250-mL Erlenmeyerask at 37 C and V. splendidus DMC-1 was grownovernight in 50 mL marine broth in a bafed 250-mL Erlenmeyer ask at 20 C. The optical density of each culture was measured to estimate cellconcentrations and appropriate dilutions spread onLB or marine agar plates to establish actual cellconcentrations. The donor strain, E. coli SM10k pir ,was spotted on to the surface of agar plates (LB agarcontaining 1.5% NaCl and 10 mm MgSO4(LB15 + MgSO4 ) in 20 l L volumes and the platesdried in a laminar ow hood. The recipient strain,V. splendidus DMC-1, was then spotted on top of the donor strain in the same volume and the platesdried again. Several ratios were set up based onestimated cell concentrations to obtain an actualratio as close as possible to 1:1. After overnight

incubation at 20 C and once the viable counts foreach strain had been established, only the plateswith a viable count ratio of 1:1 were processedfurther. The bacterial growth was scraped from theplate surfaces and resuspended in microfuge tubesin Nine Salts Solution (NSS, Sambrook, Fritsch &Maniatis 1989) +5 mm MgSO4 . The bacterialsuspension was spread on selective plates, marineagar containing 5% sheep blood, 50 l g mL

) 1

chloramphenicol and 100 l g mL) 1 ampicillin,

and incubated at 20 C. Plates were inspected daily for 5 days, and non-haemolytic colonies werepicked, Gram-stained, inoculated on to thiosul-phate citrate bile salts sucrose agar, subjected to theoxidase test and tested with antiserum to V. splendidus to conrm their identity.

Conrmation of transposon insertion inhaemolysin-negative mutants

Genomic DNA was obtained from bacteria using a Wizard Genomic DNA Purication Kit (Promega).The presence of transposon Tn10 in mutants wasconrmed by PCR on genomic DNA with E. coli Sm10 k pir (pBSL181:miniTn10Cm) and V. splendidus DMC-1 acting as positive and negativecontrols, respectively, with cat forward (5-GCGTGTTACGGTGAAAACCT-3 ) and catreverse (5- ATCACAAACGGCATGATGAA-3)primers (Alton & Vapnek 1979) specic for thechloramphenicol resistance cassette in Tn10.

To identify the site of transposon insertion in theV. splendidus mutants, puried genomic DNA wasdigested with Sph I and the DNA fragments ligatedinto Sph I-digested and dephosphorylated pUC18(Sambrook et al. 1989). The ligated plasmid wastransformed into E. coli TOP10 cells using themanufacturer s instructions (Invitrogen), and bac-teria were cultured on LB agar plates containing 50 l g mL

) 1 chloramphenicol and 100 l g mL) 1

ampicillin, to select only clones containing pUC18with Tn10 inserts.

Plasmid purication and DNA sequencing

Plasmids were puried using a Quiaprep Purica-tion Kit (Qiagen), and DNA sequencing wasperformed by DBS Genomics at the University of Durham, using the PRIDM DyeDeoxy TerminatorCycle Sequencing Kit (Applied Biosystems) and an Applied Biosystems 373A. Primers used wereinitially M13F (5-GTAAAACGACGGCCAGT-

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3) and M13R (5-AACAGCTATGACCATG-3 )(Messing 1983), but to extend the V. splendidus sequences surrounding the transposon insertionsprimers were designed based on the sequencesdetermined so far. DNA sequences were analysedand contigs aligned using the Genebuilder sequenceanalysis programme.

Sequence alignments

The vibrioaerolysin amino acid sequence wasaligned with other aerolysin amino acid sequencesusing the DIALIGN program from the MRCRFCGR Bioinformatics Applications. Evolutionary analyses were conducted in MEGA4 (Tamura,Dudley, Nei & Kumar 2007), and the evolutionary history was inferred by using the maximum likeli-

hood method based on the JTT matrix-based model(Jones, Taylor & Thornton 1992).

Incidence of the vibrioaerolysin gene in isolatesof Vibrio splendidus

In addition to V. splendidus DMC-1, 14 otherV. splendidus strains (Table 1), V. anguillarum strain 91079 (Table 1), and Roseobacter strainHNF-1 (Table 1) were selected and their chromo-somal DNA extracted using a commercial kit(Biorad). PCR and agarose gel electrophoresis werecarried out as described above using primers specicfor the vibrioaerolysin gene (DMC-1 Hly forward,5-CAACTCGAATCGGAAGCTCT-3 and DMC-1Hly reverse, 5-AGCCGAAGAGCAAAAGAGTG-3)to amplify an 800-nt product from V. splendidus DMC-1.

Effect of bacteria on larval turbot

Three trials were carried out at the Institute of Marine Research, Bergen, in which larval turbotwere exposed to V. splendidus or the transposonmutants derived from strain DMC-1. In the rsttwo trials on turbot yolk-sac larvae, eggs wereobtained from the hatchery of Stolt Sea Farms,Kvinesdal, Norway The challenge procedure was asdescribed by Sandlund & Bergh (2008) andSandlund, Rdseth, Knappskog, Fiksdal & Bergh(2010), with adaptations (temperature and bacterialconcentrations). Briey, eggs were acclimatized to16 C and distributed individually into the wells of 24-well polystyrene dishes (Nunc) containing 2 mLautoclaved sea water (30& salinity). Immediately

after distributing the eggs, 100 l L sterile sea wateror bacterial suspension (1 106 or 1 108 mL

) 1

for trial 1 and 1 105 or 1 107 mL) 1 for trial 2)

was added to each well for control and test groups,respectively. Seventy-two larvae were assigned toeach group (three multi-well dishes). Survival ordeath of each larva was assessed daily for 6 dayspost-hatch. As the survival of each larva wasindependent of the survival of larvae in other wells,it was assumed that the data were binomially distributed and differences in mortalities onindividual days were tested by the Chi-squarecontingency table test using the Minitab program.

For the third trial, turbot larvae (1 day post-hatch) were obtained from the above hatchery, andapproximately 1500 larvae were introduced intoeach of six 160-L tanks containing 42 L of full-

strength (32& salinity) sea water containing Nan- nochloropsis (Reed Mariculture) to create a greenwater system (Alderson & Howell 1973) aeratedwith two airstones (1 4 cm). The water temper-ature was maintained between 15.7 and 17.1 C,and 0.5 g wet paste of Nannochloropsis was added toeach tank on days 13 and 68. A rotifer (Brachi- onus plicatilis ) suspension (13 L containing 600 -rotifers mL

) 1 ) supplied by Julie Skadal, University of Bergen, was cultured in 80-L conical tankscontaining 42 L of 22& salinity sea water at 24 C.Rotifers were maintained by daily feeding with 1 g fresh yeast per million rotifers and 0.1 g DC DHA Selco (INVE) g ) 1 of yeast with numbers of rotiferscounted and recorded daily.

For bacterial challenges, V. splendidus DMC-1and the haemolysin-negative mutants DMC-1-M2and DMC-1-M3 were precultured for 24 h inmarine broth and 0.4 mL was used to inoculate20 mL marine broth in conical asks at 20 Cshaken at 100 oscillations per min for 24 h.Bacteria were harvested by centrifugation, washedtwice with sterile sea water and resuspended insterile sea water to a cell density of approximately 2 108 cfu mL

) 1 (A600 nm = 0.3). Cell concen-trations were veried by plate counts on marineagar plates at 18 C.

Rotifers from the growth vessel were rinsed with22& salinity sea water, and the suspension wasadjusted for daily feeding to turbot larvae from day 2 post-hatch. When required, bacteria were addedto the rotifer suspension to a concentration of 1 107 cfu mL

) 1 (from plate counts, the actualconcentrations of bacteria added were DMC-1 = 5 107 and 7 107 , DMC-1-M2 = 7.5

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107 and 4.4 107 , respectively, on the two chal-lenge days) and were incubated with rotifers for 1 hat 20 C. Bacteria-inoculated rotifers were fed tolarvae at a concentration of 2 rotifers mL

) 1 on days4 and 5 post-hatch, and thereafter, the density of rotifers was maintained at 46 rotifers mL) 1 by daily addition. Although six tanks were set upinitially, one tank crashed on day 2 and only onecontrol group was present in this trial.

Effect of bacteria on larval cod

As turbot larvae were not readily available, thefourth trial was carried out in Glasgow with codlarvae as these are also susceptible to V. splendidus DMC-1 (Reid, Treasurer, Adam & Birkbeck 2009). Larvae (14 days post-hatch) were obtained

from Viking Sea Farms, Ardtoe, and were chal-lenged with bacteria as described by Reidet al.( 2009). Briey, larvae were dispensed individ-ually into the wells of 6-well multidishes in 10 mLsea water. Rotifer suspensions were ltered, rinsedwith 22& salinity sea water and adjusted to a concentration of 50 rotifers mL

) 1 with 1 107

cfu mL) 1 added bacteria, which were prepared as

described above (actual concentrations of DMC-1and DMC-1-M4 = 12 107 on the three chal-lenge days). Each well received 1 mL of rotifersuspension containing no added bacteria (control),DMC-1 or DMC-1-M4 on days 1, 2 and 3 of thetrial with all wells receiving rotifers with no addedbacteria on days 4 and 5. Ten 6-well plates were setup for control larvae and 5 for V. splendidus DMC-1 and DMC-1-M4. Larvae were inspecteddaily and mortalities recorded when the trial wasterminated on day 6 post-infection.

Immunohistochemistry

In the third trial, samples of larvae were taken daily and processed for immunohistochemistry as de-scribed by Engelsen, Sandlund, Fiksdal & Bergh(2008). Briey, larvae were xed in 3.7% phos-phate-buffered formaldehyde at pH 7.0 for at least24 h, dehydrated in ethanol, embedded in parafnand serially sectioned at 3 l m. Sections weredewaxed in xylene and rehydrated in an ethanolbath series before washing in running water. Asprimary antibody, a rabbit antiserum made againstV. splendidus DMC-1, diluted to 1:2500, was used.Positive staining was visualized with the ABCcomplex reaction kit by DAKO (New Fuchsin

Substrate System), and haematoxylin was used forcounterstaining. Sections were examined using a Leica DMBE microscope and photographs takenusing a Leica Wild MPS52 phototube attached tothe microscope.

Results

Haemolytic and cytotoxic activities in Vibrio splendidus DMC-1 culture supernatant uid

Haemolytic activity in V. splendidus DMC-1-l-tered bacterial culture supernatant from marinebroth was detected against turbot (> 1/64), salmon(1/128), sheep (1/128), horse (1/128) and rabbit(1/256) erythrocytes, and activity was destroyed by treatment at 100 C for 10 min. Culture superna-

tant was concentrated to a titre of 1024 HU mL) 1

and this was highly cytotoxic towards turbot TV1-S4 tissue culture cells. At a dilution of 1/160, itcaused dendritic elongations in the cells, cellrounding, clustering of the rounded cells and,nally, detachment of cells from the plastic surface.

Preparation of transposon mutants of Vibrio splendidus

After optimization of conditions for mutagenesis,over 10 000 transconjugants were screened for lossof haemolytic activity and 3 hly- mutants (termedDMC-1-M2, DMC-1-M3 and DMC-1-M4) wereobtained. All 3 hly- mutants, but not the parentstrain, were shown by PCRs to contain thechloramphenicol resistance cassette associated withTn10, showing the presence of the transposon ingenomic DNA.

Biochemical and physiological properties of thehaemolysin-negative mutants

The growth rate of the mutants in marine broth wasindistinguishable from that of the wild-type parent

strain over 48 h, and the protein contents of theculture supernatants of wild-type DMC-1 and themutants DMC-1-M2, DMC-1-M3 and DMC-1-M4 were similar at 58, 58, 66 and 64 l g mL

) 1 ,respectively, with haemolysin titres of 1/256 for thewild type and none detected for the mutants. Against monolayer cultures of TVS1-S4 turbottissue culture cells, the culture supernatants of thehly- mutants lacked cytotoxicity, even when testedat a dilution of 1/2. When concentrated bacterial

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culture supernatants were compared by SDS-PAGE, no differences in banding patterns couldbe seen (results not shown), and identical resultswere obtained for the hly- mutants and DMC-1 forthe enzymes detected using the API-ZYM system(alkaline phosphatase, C4 esterase, C8 esteraselipase, leucine arylamidase, trypsin, acid phosphatase,naphthol-AS-BI-phosphohydrolase, b-galactosidase,a -glycosidase and N -acetyl-b-d -glucosaminidase).The only difference detected between the wild-type and mutant strains was in production of cytotoxin active against tissue culture cells anderythrocytes.

Identication of the transposon insertion site inthe mutants DMC-1-M2, DMC-1-M3 and DMC-1-M4

The Tn10-containing fragments cloned fromDMC-1-M2 and DMC-1-M3 were of 4.5 kB andfor mutant DMC-1-M4, 3.5 kB. Sequencing of these inserts showed Tn10 insertion sites in mutantsM2 and M3 to be identical and, therefore, mostlikely originating from the same mutagenesis event,with the insertion occurring in a ToxR-like geneadjacent to a gene with high homology to a sodium/alanine symporter from Vibrio spp. In contrast, thetransposon insertion site in mutant DMC-1-M4 wasin a gene with no signicant identity to genes of Vibrio spp. but with high homology with theaerolysin gene from several Aeromonas spp. As thehaemolysin appeared to be a novel protein unrelatedto known Vibrio haemolysins, it was named vibrio-aerolysin.

DNA sequence surrounding the transposoninsertion site in Vibrio splendidus DMC-1mutants DMC-1-M2 and DMC-1-M4

The DNA sequence surrounding the transposoninsertion sites was determined, and this showed thatthe insertion sites in DMC-1-M2 and DMC-1-M4were in adjacent genes. In total, 8.7-kb DNA wassequenced and six signicant open reading frameswere identied (see EMBL sequence AM157713),four of which showed closest matches with genescommon to a range of vibrios (Fig. 1), the remain-ing two being the putative ToxR-like regulator andvibrioaerolysin. Figure 1 shows the gene arrange-ment in V. splendidus in comparison with the genespublished for V. splendidus LGP32 (Le Roux,Zouine, Chakroun, Binesse, Saulnier, Bouchier,

Zidane, Ma, Rusniok, Lajus, Buchrieser, Medigue,Polz & Mazel 2009) and V. harveyi, indicating a possible DNA insertion involving the vibrioaerol-ysin and ToxR-like genes into a common frame-work of Vibrio genes. The transposon insertion sitesidentied in the DMC-1-M2 and DMC-1-M4mutants are both within the region of insertedDNA, in the ToxR-like gene and vibrioaerolysingenes, respectively, resulting in haemolysin-negativephenotypes. To identify the possible sites at whichDNA insertion had occurred in the V. splendidus genome, the sequences prior to and after the ToxR and the haemolysin genes were analysed using theEMBOSS program palindrome to identify in-verted repeats in the nucleotide sequence. Fourpairs of inverted repeats were identied. Of these,inverted repeat two spanned the putative insertion

of vibrioaerolysin and the ToxR-like gene (Fig. 1).

Incidence of the vibrioaerolysin gene in isolatesof Vibrio splendidus

Of the 15 V. splendidus isolates from a turbothatchery isolated by Thomson et al. (2005), 9 of the 13 biotype 1 isolates yielded PCR products forthe vibrioaerolysin gene, but neither of the twoV. splendidus biotype 2 isolates gave a PCR product.

Comparison of the vibrioaerolysin and aerolysin

amino acid sequencesThe vibrioaerolysin amino acid sequence wasaligned with 12 aerolysin amino acid sequencesusing the DIALIGN program. For the aerolysingroup of sequences, all except Aeromonas salmoni- cida 17-2 and Aeromonas punctata possess a putativesignal peptide cleavage site between alanine residues23 and 24; the equivalent residues in vibrioaerolysinare asparagine and alanine that may represent thevibrioaerolysin signal peptide site (results notshown; see EMBL sequence AM157713).

Phylogenetic comparison of vibrioaerolysin and aerolysin

A maximum likelihood consensus tree comparing the vibrioaerolysin amino acid sequence with six selected aerolysin sequences from Aeromonas spp.and four sequences from genome sequences of Vibrio spp. currently being completed is shown inFig. 2. The sequences fall into four groups withvibrioaerolysin from V. splendidus DMC-1 being

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similar to sequences identied in the genomes of V. splendidus 12B01-1 and V. harveyi , whereasmost of the Aeromonas aerolysins form a separategroup (Fig. 2).

Effect of Vibrio splendidus DMC-1-M2 and DMC-1-M3 on turbot larvae

In the rst two trials, yolk-sac larvae were exposed

to wild-type V. splendidus DMC-1 and to themutants DMC-1-M2 and DMC-1-M3. Even whenexposed to 108 cfu bacteria mL

) 1 , mortalities werenot signicantly different from those of the controlgroups (Fig. 3). In subsequent experiments, rst-feeding turbot larvae were exposed to V. splendidus DMC-1 or strain DMC-1-M2, which were incor-porated in the rotifer live food. The parent strainDMC-1 gave signicantly higher mortalities thanthe control and DMC-1-M2 groups, with DMC-1-

M2 behaving similarly to the control group(Fig. 4). Immunohistochemistry showed thatDMC-1-M2 colonized the gut epithelium andwas present in the lumen, but did not damage orinvade the gut cells (Fig. 5, a12) to cause thecellular destruction that was seen in larvae infectedwith V. splendidus DMC-1 (Fig. 5, b2, arrow 5).

Effect of Vibrio splendidus DMC-1-M4 on cod larvae

Turbot larvae were not available in the later stagesof this work, and as V. splendidus DMC-1 has beenshown to be lethal to cod larvae (Reid et al. 2009)as well as turbot larvae, rst-feeding cod larvae werechallenged with V. splendidus DMC-1 or thehaemolysin-decient DMC-1-M4. V. splendidus DMC-1 gave signicantly greater mortalities thanthe control group from day 3 onwards (P < 0.05).

V. harveyi ATCCBAA-1116(CP000789)

VH2470 VH02472VH02471

VH02474VH02473VH02469

V. splendidus LGP32(FM954972)

V. splendidus DMC-1(AM157713)

VS1289VS1291 VS1290VS1294

VS2 Putative Na/ala symporter

VS5 Putative Threfflux protein

VS6 Putative carboxypeptidase(incomplete)

VS1 Hypothetical protein

VS4 vibrioaerolysinVS3 Tox R-like regulator

0 1 2 3 4 5 6 7 8 9kb

IR2 IR2

Figure 1 Schematic diagram showing homologous genes in Vibrio splendidus strain DMC-1, V. splendidus strain LGP32 and Vibrio harveyi strain ATCC BAA-1116. Homologous genes in the different bacteria are shown with the same shading pattern. The genescovered are V. harveyi : VH02469, putative sodium/alanine symporter; VH02470 hypothetical protein; VH02471, hypothetical protein;VH02472, aerolysin-like protein; VH02473, hypothetical protein; and VH02474, putative carboxypeptidase. V. splendidus LGP32VS1289, carboxypeptidase; VS1290, hypothetical protein; VS1291, sodium/alanine symporter; and VS1294, hypothetical protein.VS1292 and VS1293 (non-coding RNA, glycine riboswitch) are not shown. V. splendidus DMC-1: VS1, hypothetical protein; VS2,putative sodium/alanine symporter; VS3, putative transcription activator ToxR; VS4, vibrioaerolysin; VS5, putative threonine efux protein; VS6, putative carboxypeptidase. The gene insertions that appear to have occurred in V. harveyi and V. splendidus DMC-1 in theregion common to V. splendidus LGP32 and vibrios such as Vibrio parahaemolyticus (AP005079) and Vibrio vulnicus (AE004220) areshown in lled arrows. The locations of inverted repeats IR2 are shown. EMBL accession numbers for nucleotide sequences are shown inparentheses.

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The mutant strain DMC-1-M4 gave signicantly fewer mortalities than the parent strain DMC-1(P < 0.01) and fewer mortalities than the controlgroup although the latter was not statistically signicant (Fig. 6). When the experiment wasterminated, the bacterial content of homogenatesof the larvae was analysed. The control groupcontained 1.9 104 cfu per larva of a diversebacterial ora, none of which resembled V. splendidus DMC-1. Larvae exposed to DMC-1 contained3.3 105 cfu per larva over 90% of which resem-bled DMC-1 in colony morphology. Five repre-sentative colonies were conrmed as V. splendidus by reaction with antiserum to V. splendidus DMC-1. The group challenged with DMC-1-M4 con-tained 9 104 cfu per larva, of which ca 30%resembled the colony morphology of V. splendidus and all isolates tested reacted with antiserum toV. splendidus .

Comparison of the action of vibrioaerolysin and Aeromonas hydrophila aerolysin on sheeperythrocytes

Vibrioaerolysin gave a doseresponse curve for lysisof sheep erythrocytes that was indistinguishablefrom that caused by aerolysin of A. hydrophila (Buckley, Halasa, Lund & Macintyre 1981) (resultsnot shown). As solutes such as PEG 1500 inhibitedlysis, it was concluded that haemolysis occurred by colloid osmotic lysis as is known to occur with

aerolysin (Buckley 1999). The diameters of thechannels induced in sheep erythrocytes, as measuredin osmotic protection experiments, were 1.86 nmfor vibrioaerolysin (95% CL 1.821.90) and1.93 nm for aerolysin (95% CL 1.862.00),respectively.

Discussion

Several studies have reported that V. splendidus isinvolved in infections in turbot (Myhr, Larsen,Lillehaug, Gudding, Heum & Ha stein 1991; Pazos,Santos, Magarinos, Bandin, Nunez & Toranzo1993; Angulo, Lopez, Vicente & Saborido 1994;Gatesoupe et al. 1999; Thomson 2001; Thomsonet al. 2005) and oysters (Gay, Renault, Pons & LeRoux 2004; Le Roux et al. 2007). In isolates frommoribund turbot, Gatesoupe et al. (1999) andThomson et al. (2005) were only able to distinguishbetween pathogenic and non-pathogenic isolates of V. splendidus by their effects on larval turbot andnot from physiological and biochemical properties.

As cytolytic toxins have been found to beimportant virulence determinants in many bacterialpathogens (Alouf & Freer 1999), the properties of the haemolysin of V. splendidus biotype 1 wereinvestigated. Mutagenesis with transposon Tn10resulted in identication of three V. splendidus mutants completely lacking in production of haemolysin and virulence towards marine sh larvaewith all other properties tested being the same. This

V splendidusDMC-1

Vsplendidus12B01-1

Vharveyi

Vsplendidus12BO1-2

VcorallyticusATCCBAA450

AveroniA8QIT4

AhydrophilaNLEP Q93CE6

AhydrophilaATCC7966

AtrotaAB3

AtrotaATCC49659

Aenteropelogenes O85370

0.1

Figure 2 Molecular phylogenetic analysis by the maximum likelihood method. The evolutionary history was inferred by using themaximum likelihood method based on the JTT matrix-based model (Jones et al. 1992). The tree with the highest log likelihood() 5340.5625) is shown. Initial tree(s) for the heuristic search were obtained automatically as follows. When the number of common sitesis < 100 or less than one-fourth of the total number of sites, the maximum parsimony method was used; otherwise, the BIONJ methodwith MCL distance matrix was used. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.The analysis involved 11 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 483 positions in the nal data set. Evolutionary analyses were conducted in MEGA4 (Tamura et al. 2007).

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suggests that the haemolysin plays a role in thevirulence of this organism.

Sequence determination of a region of thechromosome of V. splendidus DMC-1 showed a gene arrangement almost identical to those in otherVibrio species; the only major difference betweenV. splendidus DMC-1 and Vibrio cholerae (Gen-

bank AE004220), Vibrio vulnicus (AE016806)and Vibrio parahaemolyticus (AP005079) sequenceswas the section of the genome that contained thevibrioaerolysin and ToxR-like genes, and thisprovided strong evidence that there had been a recombination event in the V. splendidus chromo-some with the insertion of foreign DNA. Thecomplete genome sequence of V. splendidus strainLGP32, a pathogen of oysters, has been reportedrecently by Le Roux et al. (2009), and this lacks the

apparent insertion containing the ToxR-like andvibrioaerolysin genes. The sequences of V. splendidus LGP32 and DMC-1 correspond except that a 231-bp sequence of the V. splendidus LGP32genome between the sodium/alanine symporterand thermostable carboxypeptidase genes is absentin V. splendidus DMC-1 and is replaced by a 3767-bp section with the ToxR-like and vibrioaerolysin

Trial 2

0

25

50

75

100

Day 2 Day 3 Day 4 Day 5 Day 6

Post-hatch

% M

o r t a

l i t i e s

ControlDMC-1 10e5DMC-1 10e7M2 10e5M2 10e7M3 10e5M3 10e7

Trial 1

0

25

50

75

100

Day 2 Day 3 Day 4 Day 5 Day 6

Post-hatch

% M

o r t a

l i t i e s

Control

DMC-1 10e6

DMC-1 10e8

M2 10e6

M2 10e8

M3 10e6

M3 10e8

Figure 3 Turbot yolk-sac larval trials with wild-type Vibrio splendidus DMC-1 and haemolysin-negative mutants V. splendidus DMC-1-M2 and V. splendidus DMC-1-M3. Two separatetrials were conducted, and the concentration of bacteria to whichlarvae were exposed is shown in the legends.

0

25

50

75

100

4 5 6 7 8 9Days Post-hatch

%

M o r t a

l i t i e s

Tank 2 Control

Tank 3 DMC-1

Tank 4 DMC-1

Tank 5 M2

Tank 6 M2

Figure 4 Turbot larvae rst-feeding trials with wild-type Vibrio splendidus DMC-1 and haemolysin-negative mutant V. splendidus DMC-1-M2. Turbot larvae were challenged on days 4 and 5,with challenge bacteria added to the live food rotifers to give a nal bacterial concentration of 3 104 cfu mL

) 1 in eachchallenge tank.

0

10

20

30

40

50

60

70

80

1 2 3 4 5 6Days post infection

% T

o t a l m o r t a

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Figure 5 Cod larvae rst-feeding trials with wild-type Vibrio splendidus DMC-1 and haemolysin-negative mutant V. splendidus DMC-1-M4. Cod larvae were challenged on days 1 and 2,with challenge bacteria added to the live food rotifers to give a nal bacterial concentration of 2 106 cfu mL

) 1 in eachchallenge tank (- -, control; -j -, DMC-1; -m -, DMC-1-M4).

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genes, terminating 122 bp past the vibrioaerolysingene. The V. harveyi genome sequence is currently under completion, and this also contains a similarinsertion with an aerolysin-like gene (Genbank CP000789).

Several short inverted repeat sequences wereidentied before and after the vibrioaerolysin gene,and inverted repeat pair two spanned the differencein sequence between strains LGP32 and DMC-1,consistent with this section of DNA having under-gone a genetic mobilization event during theorganisms evolution, perhaps through a transposoninsertion or phage integration. Mobilization eventsleading to acquisition of haemolysin or toxin genesare common in Vibrio species such as V. cholerae (Faruque, Albert & Mekalanos 1998), V. parahaemolyticus (Terai, Baba, Shirai, Yoshida, Takeda &Nishibuchi 1991; Nishibuchi & Kaper 1995),Vibrio pommerensis (Jorres, Appel & Lewin 2003)

as well as in V. anguillarum for acquisition of thepJM1 genes involved in the anguibactin iron-uptake system (Tolmasky & Crosa 1995; DiLorenzo, Stork, Tolmasky, Actis, Farrell, Welch,Crosa, Wertheimer, Chen, Salinas, Waldbeser &Crosa 2003).

Motile aeromonads are widely distributed in theaquatic environment and are the causative agents of haemorrhagic septicaemia of sh, reptiles andamphibians (Austin & Austin 2007). Several extra-cellular toxins are recognized from Aeromonas spp. including haemolysins, enterotoxin, cytotoxin,acetylcholinesterase, phospholipid cholesterolacyltransferase and proteases. Phylogenetic analysisof vibrioaerolysin with other aerolysin sequencesindicates that the aerolysin toxins cluster into twodifferent groups with Aeromonas trota, A. hydrophila and Aeromonas enteropelogenes forming one group of the conventional aerolysins and those from vibrios

(a1) 100 magnification. (a2) 1000 magnification.

(b1) 100 magnification.



12

3

4

5

6

Figure 6 Immunohistochemistry of sections from rst-feeding larval turbot (trial 3, IMR Bergen). (a1 and a2) Vibrio splendidus vibrioaerolysin-negative mutant DMC-1-M2; (b1, b2 and b3) vibrioaerolysin-producing V. splendidus DMC-1-challenged rst-feeding larvae sampled on day 8. Arrows 1 and 3 show stained cells in the gut of larvae challenged with V. splendidus DMC-1-M2 and DMC-1,respectively; arrows 2 and 4 show individual bacterial cells, arrows 5 and 6 indicate damage to the brush border of larvae challenged with

V. splendidus DMC-1. In addition to individual bacterial cells, dense red-stained material can be seen, representing either clusters of bacteria or extracellular material from bacteria. The blue-stained globule-like structures in the lumen are the remains of rotifers.

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forming a second broad group with Aeromonas veroni . The V. splendidus DMC-1 aerolysin showshigh homology with database sequences fromsimilar genes of V. splendidus 12B01-1 and V. harveyi . A further sequence from V. splendidus (12B01-2) appears quite distinct from all other aerolysingenes.

Aerolysin is a well-characterized pore-forming toxin identied over 30 years ago (Bernheimer & Avigad 1974), and the crystal structure has beensolved (Parker et al. 1996). Conversion of inactive A. hydrophila proaerolysin into haemolytically ac-tive aerolysin occurs by cleavage at a site betweenarginine and leucine residues near the carboxy terminus (Howard & Buckley 1985a), and invibrioaerolysin, an arginineisoleucine bond in a similar position may serve as the equivalent site.

Aerolysin is synthesized as a preprotoxin containing an N-terminal signal sequence and a C-terminalactivation peptide (Howard & Buckley 1985a),where it is subsequently cleaved to form proaerol-ysin (Howard & Buckley 1985b) Proaerolysinbinds to glycosylphosphatidylinositol-anchoredproteins of the cell membrane such as Thy-1(Nelson, Raja & Buckley 1997), contactin (Diep,Nelson, Raja, Pleshak & Buckley 1998) or eryth-rocyte aerolysin receptor (Cowell, Aschauer,Gruber, Nelson & Buckley 1997), and a carboxy-terminal peptide of about 40 amino acids is cleavedby proteases to form active aerolysin (Howard &Buckley 1985b) that inserts into the membrane toform a heptameric transmembrane channel or poreleading to membrane depolarization and death of the cell. Estimates of the diameter of the trans-membrane channel depend upon the measuring procedure employed. The pore size in erythrocytesin this study produced by vibrioaerolysin andaerolysin of A. hydrophila (Buckley et al. 1981)was 1.86 nm for vibrioaerolysin and 1.93 nm for A. hydrophila aerolysin, respectively, which arecomparable with other estimates.

Aeromonas hydrophila can cause gastroenteritis,deep wound infections and septicaemia (Buckley 1999), and Aeromonas sobria is a related speciescausing acute gastroenteritis in adults and children(Tanoue, Takahashi, Okamoto, Fujii, Taketani,Harada, Nakano & Nakaya 2005). Aerolysin isrecognized as the major virulence factor for theseorganisms, and the aerolysin toxin of A. sobria hasbeen shown to cause uid accumulation in themouse ileal loop test and to activate cyclic AMP(cAMP)-dependent chloride secretion (Tanoue

et al. 2005) via prostaglandin E2 stimulation (Fujii,Tsurumi, Sato, Takahashi & Okamoto 2008)causing diarrhoea in a manner similar to that of V. cholerae and E. coli . Thus, the mechanism by which aerolysin causes damage to cells of thegastrointestinal tract mucosa in humans andsubsequent diarrhoea is well understood, andvibrioaerolysin may well have a similar, damaging effect when V. splendidus colonizes the intestinaltract of marine sh larvae in sufcient numbers.Unlike V. anguillarum , V. splendidus does notappear to be invasive. Turbot larvae seem unaf-fected when exposed to V. splendidus in water, andmortalities only occur when bacteria are associatedwith food such as rotifers and gain entry to thedigestive tract. Also, damage appears to be localizedto the gut epithelium as occurs for enteropathogens

such as V. cholerae (Faruque et al. 1998).The nine V. splendidus biotype 1 strains shownby PCR to contain the vibrioaerolysin geneincluded all four isolates shown to be pathogenicto turbot larvae by Thomson et al. (2005) and fourisolates found to be non-pathogenic. This suggeststhat in V. splendidus , as in most bacterial pathogens,virulence is multi-factorial (Smith 2000) and thatother factors are also required for virulence inV. splendidus .

The vibrioaerolysin gene was located immedi-ately downstream of an ORF encoding a ToxR-likegene, and transposon insertion into this ORF(mutants DMC-1-M2 and DMC-1-M3) renderedV. splendidus unable to produce haemolysin, tocause damage to the intestinal tract or to kill larvalturbot, which is possible evidence that this is a transcriptional factor involved in governing thelevel of haemolysin produced. Vibrio cholerae ToxR and similar molecules are transmembrane transcrip-tional activators that have a characteristic periplas-mic domain acting as a sensor, e.g., for changes inpH or ionic conditions, a transmembrane domainand a winged helix-turn-helix motif associated withDNA binding and regulation of gene transcription(Crawford, Krukonis & DiRita 2003).

Comparison of the V. splendidus ToxR homo-logue sequence with that of ToxR of E. coli showsthat it contains both putative helix-turn-helix motif and transmembrane domains but lacks a periplasmic domain. This may not be essential intransmembrane signalling as the periplasmicdomain is not required for transcriptional regu-lation of TcpP-dependent tox T, ompU and ompT genes in V. cholerae (Crawford et al. 2003). Thus,

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the V splendidus DMC-1 ToxR could well act asa sensor of environmental stimuli and a regulatorof vibrioaerolysin and possibly other virulencegenes.

Kamisaka, Jordal, Edvardsen, Kryv, Otterlei &Rnnestad (2010) have recently described a syn-drome, termed distended gut syndrome, that canaffect various stages in larval cod and is character-ized by low larval activity, reduced appetite and a gut that is distended and lled with uid and withan opaque lumen. The authors concluded that thepathophysiological basis of distended gut syndromeinvolves disturbed enteral water and electrolytebalance caused by toxins. Such an outcome isentirely compatible with damage caused by anaerolysin-like enterotoxin as described here. Thisdoes not indicate a role for V. splendidus in

distended gut syndrome as many other vibrioscould have acquired the genetic elements foraerolysin production. Based on the evidence hereof how some haemolytic bacteria could be harmfulto larval marine sh, it is advisable that hatcheriesshould attempt to reduce the concentration of haemolytic bacteria associated with live feed and therearing system in general by routine screening onblood agar plates and enhanced hatchery hygieneprocedures.

Acknowledgements

We thank the European Union for supporting thiswork through the PROBE project (ImprovedProcedures for Flatsh Larval Rearing through theuse of Probiotic Bacteria: Q5RS-2000-31457) andthrough the Improving Human Potential Programme for funding an exchange visit by HLM to theInstitute of Marine Research, Bergen. We alsothank the UK Seash Industry Authority and theBritish Marine Finsh Association for their support,Hari Rudra for help with the challenge experimentand Ingrid Uglenes Fiksdal for help with immuno-histochemistry.

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