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Cellular components of probiotics control Yersinia ruckeri

infection in rainbow trout, Oncorhynchus mykiss

(Walbaum)

A Abbass1,2, S M Sharifuzzaman2,3 and B Austin2*

1 Department of Fish Diseases and Management, Faculty of Veterinary Medicine, Banha University, Egypt

2 School of Life Sciences, Heriot-Watt University, Edinburgh, UK

3 Institute of Marine Sciences and Fisheries, University of Chittagong, Bangladesh

Abstract

Subcellular components of the probiotics Aeromonas sobria GC2 and Bacillus subtilis JB-1,when administered to rainbow trout, Oncorhyn- chus mykiss , conferred protection against a new biogroup of Yersinia ruckeri . Thus, intraperitonealor intramuscular injection of rainbow trout withcell wall proteins (CWPs), outer membraneproteins (OMPs), lipopolysaccharides (LPS),whole cell proteins (WCPs) and live cells fol-lowed by challenge on day 8 with Y. ruckeri ledto 80–100% survival compared with 10% survivalin the controls. Sodium dodecyl sulphate-poly-acrylamide gel electrophoresis (SDS-PAGE) pro-files of WCPs and OMPs from GC2 had 10 and5 variable protein bands in comparison to 11 and5 bands in the WCPs and CWPs from JB-1.Proteomic analyses were employed following SDS-PAGE to categorize one dominant proteinof 104.7 kDa from the CWPs of JB-1 andequated it with Bacillus spp. endoglucanase witha Mascot score >69. These results point to thepotential of using cellular components of probi-otics for protection of fish against bacterial

diseases.

Keywords: probiotics, rainbow trout, subcellularcomponents, Yersinia ruckeri .

Introduction

The increase in demand for aquaculture productshas been accompanied by a similar increase in thesearch for environmentally friendly alternatives toantibiotics of which probiotics feature prominently in the list of possibilities (Gatesoupe 1999; Kesar-codi-Watson, Kaspar, Lategan & Gibson 2008). Todate, a wide range of probiotics including Gram-positive and Gram-negative bacteria (Irianto &

Austin 2002) have been evaluated, and conferprotection against a range of pathogens, including

Aeromonas salmonicida (Nikoskelainen, Ouwehand,Bylund & Salminen 2001) and Yersinia ruckeri (Kim & Austin 2006), often resulting in enhance-ment of the innate immune responses. Moreover,oral administration of probiotics may lead toantibody production (Maassen, Boersma, van Hol-ten-Neele, Classen & Laman 2003). For example, ithas been recognized that feeding fish with probiot-ics produced cross-reactive antibodies against Vibrio harveyi (Arijo, Brunt, Chabrillon, Diaz-Rosales &

Austin 2008). Previously, we focused on the use of live cells of the probiotics Aeromonas sobria GC2and Bacillus subtilis JB-1 as feed supplements in

fish, and confirmed protection against multiplebacterial diseases including those caused by A. salmonicida, Lactococcus garvieae , Streptococcus iniae,Vibrio anguillarum, V. ordalii and Y. ruckeri (Brunt, Newaj-Fyzul & Austin 2007). Therefore,this study sought to determine which part of the cellis responsible for protection against Y. ruckeri infection in rainbow trout, Oncorhynchus mykiss (Walbaum).

Journal of Fish Diseases 2010, 33, 31–37 doi:10.1111/j.1365-2761.2009.01086.x

Correspondence Professor B Austin, School of Life Sciences,

John Muir Building, Heriot-Watt University, Riccarton, Edinburgh

EH14 4AS, UK

(e-mail: [email protected])

*Present address: Institute of Aquaculture, University of Stirling,

Stirling FK9 4LA, UK

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Materials and methods

Fish

Rainbow trout (average weight = 12 g) wereobtained from a commercial fish farm in Scotland.

The fish were maintained in aerated dechlorinatedfresh water at 12 C, and fed with commercialpelleted diet (Skretting) at 2% of body weightdaily. The health of the fish was checked initially upon receipt and then at 6- to 8-week intervals(Austin & Austin 1989).

Bacterial cultures

Yersinia ruckeri EX5, which was obtained froma diseased rainbow trout in England and representsa novel biogroup of the pathogen (Austin, Robert-

son & Austin 2003) and two probiotics, A. sobria GC2 and B. subtilis JB-I (Brunt & Austin 2005),were obtained from the bacterial culture collectionof the School of Life Sciences, Heriot-Watt Uni-versity. Authenticity was verified by comparison of phenotypic traits and by DNA sequence homology in accordance with criteria in the original publica-tions (Austin et al. 2003; Brunt & Austin 2005).Stock cultures were stored in tryptone soya broth(TSB; Oxoid) containing sterile (121 C for15 min) 20% (v/v) glycerol at )70 C. Cultureswere routinely grown on tryptone soya agar (TSA;Oxoid) plates or in TSB at room temperature.

Isolation of whole cell proteins (WCPs)

The method of Wilcox, Cook, Thickett, Eley &Spencer (1992) was used to isolate the WCPs fromGC2, with modifications. Thus, GC2 was grown inbrain heart infusion broth (BHIB; Oxoid) on a shaker at 4 g for 18 h at 28 C, and collected by centrifugation at 2550 g for 20 min at 4 C. Thecells were resuspended in 10 mL autoclaved(121 C for 15 min) distilled water (DW) to anoptical density of 0.5 at 590 nm (OD590), and then

pelleted, mixed with 150 lL of double strengthsample buffer [4 g sodium dodecyl sulphate (SDS);20 mL glycerol; 10 mL 2-mercaptoethanol;12.5 mL 0.5 TRIS-HCl, pH 6.8, per 100 mL of Millipure water (Millipore)] and kept over ice for30 min. The lysate was centrifuged at 5000 g for10 min at 4 C, and retained. JB-1 were grown onTSA and incubated at 28 C for 24 h. A loopful of bacteria was inoculated in 10 mL heart infusionbroth (HIB; Oxoid) and incubated overnight, then

1 mL was suspended in 100 mL of HIB andincubated on a shaker at 4 g for 16 h at 28 C.The cells were harvested by centrifugation at 9500 g for 5 min at 4 C and washed twice in Millipurewater, then resuspended in Millipure water con-taining 1 mm phenylmethylsulphonyl fluoride(PMSF) and frozen at )20 C. The suspensionwas disrupted by sonication and mixed with anequal volume of lysis buffer (4 g SDS; 20 mLglycerol; 10 mL 2-mercaptoethanol; 12.5 mL 0.5TRIS-HCl, pH 6.8, per 100 mL Millipure water)and kept over ice for 30 min. The supernatantcollected following centrifugation represents the

WCPs. Precipitation of proteins was done following the methanol–chloroform method (Wessel & Flu-gge 1984). Briefly, 0.4 mL of methanol was addedto 0.1 mL of lysate, vortexed well, then 0.1 mL

chloroform was added, and vortexed again beforeaddition of 0.3 mL DW. The mixture was vortexedand spun for 2 min at 8950 g at 4 C. The topaqueous layer was carefully removed and 0.3 mL of methanol added, vortexed and centrifuged at8950 g for 5 min at 4 C to pellet the proteins.The protein pellet was air dried and suspended inphosphate-buffered saline (PBS; Sigma) at pH 7.2and stored at )20 C for subsequent use.

Extraction of outer membrane proteins (OMPs)

Outer membrane proteins were extracted essentially using the modified method of Wilcox et al. (1992).Briefly, GC2 were cultured in BHIB on a shaker(4 g ) at 28 C for 18 h and cells were collected by centrifugation at 2550 g for 20 min at 4 C. Then,the cells were resuspended in 5 mL of 0.5 TRIS-HCl (pH 7.4) to OD590 1.5 and sonicated for fivecycles for 1 min over ice. Cell disruption waschecked by microscopic examination. Cell debriswas removed by centrifugation at 2550 g for10 min at 4 C. Then, 0.5 mL of 20% N-lauroyl-sarcosine sodium salt (Sigma) was added and keptat room temperature for 30 min. The OMPs were

pelleted by centrifugation at 17 650 g for 1 h at4 C, washed twice with PBS, suspended in freshPBS and frozen at )20 C.

Preparation of lipopolysaccharides (LPS)

The LPS was prepared by proteinase digestion of whole-cell lysate (Hitchcock & Brown 1983) withmodification. Thus, GC2 were grown in TSB on a shaker at 4 g for 24 h at 28 C. The bacterial cells

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were harvested by centrifugation at 1250 g for10 min at 4 C, and resuspended in PBS to OD5251.0. The suspension was dispensed into 1.5 mLEppendorf tubes and pelleted by centrifugationbefore the cells were mixed with 50 lL of samplebuffer (2 g SDS; 10 mL glycerol; 4 mL 2-mercap-toethanol; 1 m TRIS-HCl, pH 6.8). The mixturewas kept on ice for 30 min, then placed in a heating block with 10 lL of proteinase K (2.5 g L)1) andincubated for 2 h at 60 C. Digested lysate wascentrifuged to remove remaining cell debris and theLPS was precipitated (Darvean & Hancock 1983)by adding 2· volumes of cold ethanol containing 0.375 m MgCl2, with mixing at )20 C for15 min. The preparation was then vortexed thor-oughly and centrifuged at 7850 g for 15 min at2 C. The precipitated LPS was suspended in PBS

and stored at )

20 C.

Collection of extracellular products (ECPs)

The ECPs of GC2 were collected by the cellophaneoverlay method (Austin & Rodgers 1981) wherebacteria were grown overnight in TSB at 28 Cbefore 200 lL of culture was spread on a sterilecellophane sheet overlayered on a TSA plate withincubation for 72 h at 22 C. The cells werescraped into 1 mL of ice-cold PBS and centrifugedat 5000 g for 15 min at 4 C. Then, the superna-tant containing ECPs was filtered through 0.22 lmpore-size Millipore Millex filters and stored at)20 C. For JB-1, the ECPs were collected asdescribed by Østensvik, Form, Heidenreich, OSul-livan & Granum (2004) with slight modification.In brief, bacteria were grown on TSA and incubatedat 28 C for 24 h. A loopful of culture wasinoculated in 10 mL BHIB and incubated over-night on a shaker at 28 C. Then, 0.1 mL wassuspended in 10 mL of BHIB and incubated in a shaker for 16 h at 28 C. Supernatant containing ECPs was collected by centrifugation at 5000 g for15 min at 4 C, filtered through a 0.22 lm

Millipore filter and stored at )

20 C.

Fractionation of cell wall proteins (CWPs)

JB-1 was grown on TSA with incubation at 28 Cfor 24 h. A loopful of bacteria was inoculated in10 mL of BHIB and incubated overnight, and then5 mL volumes transferred to 500 mL of BHIB andincubated on a shaker at 4 g for 16 h at 28 C. Thecells were collected by centrifugation at 2820 g for

15 min at 4 C, washed twice with 0.05 TRIS-HCl(pH 7.8), resuspended in 40 mL of 0.05 TRIS-HCl(pH 7.4) containing 1 mm PMSF and sonicated forfour cycles for 5 min over ice. Cell disruption waschecked by microscopy. The cells were spun toremove cell debris, and then the cell walls separatedby centrifugation at 17 650 g for 1 h at 4 C. Thepellet was resuspended in 0.1 m NaCl, washed twicein PBS and frozen at )20 C.

Live and dead preparations of probiotics

Preparations of live and formalized cells of theprobiotics were made according to the methodsdescribed by Brunt & Austin (2005). Formalizedcells were washed three times before use.

Challenge tests to verify the efficacy of cellularcomponents, and live and dead preparations of probiotics

Before challenge with Y. ruckeri, the protein con-centrations of cellular components were determined(Bradford 1976), and live and formalized cellsadjusted to 108 cells mL)1 using a haemocytometerslide (Improved Neubauer type; Merck) at a magnification of ·400. Yersinia ruckeri was pre-pared after Brunt et al. (2007) and adjusted to a predetermined dose of 2.5 · 106 cells mL)1.Groups of 10 fish were injected either intraperito-neally (i.p.) or intramuscularly (i.m.) with 0.1 mLof 2 mg mL)1 WCPs, CWPs, OMPs, ECPs;1 mg mL)1 LPS; and 108 cells mL)1 live andformalized cells and then challenged with 0.1 mL of Y. ruckeri on day 8. All fish groups were maintainedin dechlorinated fresh water at 19 C with 50%water exchange daily. Mortalities were recordeddaily over 7 days. All dead and moribund fish, andthe survivors, were examined clinically and bacte-riologically to determine the presence of Y. ruckeri (Austin & Austin 2007). Challenge experimentswere performed in triplicate.

SDS-PAGE analysis

Whole cell proteins and OMPs of GC2 wereprepared according to Wilcox et al. (1992). OMPswere loaded on 4% stacking gel and run with 12%(w/v) acrylamide separating gel, whereas WCPs wererun with a separating gel consisting of 5 mL 1.5 mTRIS-HCl (pH 8.8), 8.8 mL dH2O, 200 lL 10%SDS, 5 mL 30% acrylamide, 100 lL ammonium

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persulphate and 20 lL N,N,N,N-tetramethyl-ethylenediamine TEMED (Laemmli 1970) using high molecular weight standard markers (Sigma).

For JB-1, the CWPs were extracted by resu-spending pelleted cell walls, prepared as above, in0.1 m NaCl, washing twice with 0.05 m TRIS-HCl,pH 7.8, then mixing with 2· sample buffer andheating in a heating block at 100 C for 10 min.The WCPs were isolated by mixing the bacterialsuspension, as previously prepared, with equalvolumes of sample buffer and heating at 100 Cfor 10 min. Gel electrophoresis (Laemmli 1970)was done with 12% (w/v) acrylamide gel using highmolecular weight standard markers (Sigma). Thegels were stained by Coomassie brilliant blue(R-250, Sigma) and destained with 40% (v/v)methanol and 10% (v/v) acetic acid.

Identification of proteins using MALDI-TOF-MS

Protein bands were identified by MALDI-TOFmass spectrometry. Thus, the protein bands of interest from SDS-PAGE and controls (protein freeregion) were picked by spot picker and immersed in100 lL of 25 mm ammonium bicarbonate(NH4HCO3) in 50% (v/v) acetronitrile (ACN)and vortexed for 10 min. The supernatant wasdiscarded and the wash/dehydration step repeatedthree times until the gels became pale blue. Then,the gels were immersed in 100 lL of 10 mm DTTin 25 mm NH4HCO3 and placed in a heating block at 56 C for 1 h, left to cool at room temperatureand the supernatant removed, before the addition of 100 lL of 55 mm iodoacetamide in 25 mmNH4HCO3 with incubation in the dark for45 min with occasional vortexing. The supernatantwas discarded before the gel pieces were washedwith 100 lL of 25 mm NH4HCO3 (pH 8) andvortexed for 10 min. Dehydration was with 100 lLof 25 mm NH4HCO3 in 50% (v/v) acetronitrile.This wash/dehydration step was repeated, the

supernatant removed, and the gel pieces dried in a vacuum centrifuge. The gels were rehydrated by addition of sequencing grade trypsin (10 lL at20 ng lL)1 in 25 mm NH4HCO3) and placing in a heating block at 37 C overnight. The trypticpeptides were removed from gel pieces by twoextractions with 25 lL of 50% (v/v) ACN in0.1% (v/v) trifluoroacetic acid (TFA). The pooledsupernatants were collected and the peptidesconcentrated by drying in a vacuum centrifuge

before resuspension in 5 lL of 50% (v/v) ACN in0.5% (v/v) TFA. The peptide mixture in 0.3 lLamounts was spotted directly onto Ettan MALDI-TOF-MS target slides and mixed with the samevolume of a-hydroxycinnamic acid [20 mg mL)1

in 50% (v/v) ACN in 0.5% (v/v) TFA]. Thepeptides were examined by MALDI-TOF-MS using an Ettan MALDI-Tof-pro (Amersham Biosciences)with the instrument set to 20 kv positive ionreflector mode with the pulsed extraction (focusmass 2500) and ion rejection set at 500. An eightshot sec)1 was used with three septica (250 shots)being acquired. Internal calibration was carried outwith trypsin autolysis product (m/z 842.51 and2211.10) and bovine serum albumin (BSA) as a control. Protein identification was performed by searching the non-redundant protein sequence data

base using Ettan MALDI software, with thefollowing research parameters used for data basesearches with peptide mass data: monoisotonic massaccuracy, 80 ppm; missed cleavage of trypsin 1;cysteine modified with iodacetamide (complete)and oxidation of methionine (partial).

Results

Fish inoculated with viable probiotics and theirsubcellular components were highly resistant toclinical infection by Yersinia ruckeri . There were nomortalities in the groups which received WCPs,CWPs, OMPs and LPS of the probiotics. Incomparison, the mortalities for live cell-treated fishranged from 10 to 20%. These compared to 90%mortalities of the controls (Fig. 1). By contrast, fishthat received formalized cells and ECPs fared poorly (data not shown). All the survivors of the groupswhich received cellular protein and live cells didnot show any evidence of disease at the end of the experiments. Overall, these results suggest theeffectiveness of the subcellular components of probiotics in controlling bacterial fish diseases.

Furthermore, SDS-PAGE analysis of WCPs

from A. sobria GC2 showed 12 faint and 10 deeply stained protein bands ranging from 22.4 to177.8 kDa (Fig. 2a,b). Conversely, OMPs pos-sessed five darkly stained bands of between 24.6and 56 kDa, including one prominent protein of 38.9 kDa (Fig. 2c,d). WCPs of JB-1 had numerousbands, of which 11 protein bands stained well(range: 10.2 to 169.8 kDa) with the electrophoresis(Fig. 3a,b). This compares with five bands (range:43 to 104.7 kDa) for the CWPs (Fig. 3c,d).

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Although no common protein band was evident,there were two close major proteins of 43 and49.4 kDa in the WCPs and CWPs from JB-1(Fig. 3c,d).

The dominant protein band of 104.7 kDa fromCWPs of JB-1 was digested in-gel with trypsin andanalysed by MALDI-TOF. The output of proteinbands and a subsequent search in the data bank confirmed the identification as Bacillus spp.endoglucanase with a Mascot score >69 represent-ing a significant identification (P < 0.05) (Table 1).

Discussion

The results of this study confirm the view thatentire or components of probiotics are beneficial tothe host for protection against disease. The data supports previous studies which demonstrated thebeneficial effect of A. sobria GC2 and B. subtilis

JB-1 in controlling diseases of rainbow trout causedby Streptococcus iniae and Lactococcus garvieae (Brunt & Austin 2005), and A. salmonicida, Vibrio anguillarum and V. ordalii (Brunt et al. 2007). By contrast, formalized cells or ECPs offered lessprotection in rainbow trout. Similarly, dietary

administration of formalized, sonicated, heat-inac-

0

20

40

60

80

100

WCPs OMPs LPS Live cells Control

Aeromonas sobriaGC2

M

o r t a l i t y ( % )

WCPs CWPs Live cells Control

0

20

40

60

80

100

Bacillus subtilisJB-1

M o r t a l i t y ( % )

(a)

(b)

Figure 1 Mortalities (%) of rainbow trout (average

weight = 12 g) following infection via i.p. or i.m. injection with

2.5 · 106 cells mL)1 of Yersinia ruckeri after inoculation withcellular components and live cells of the probiotics (a) Aeromonas sobria GC2 or (b) Bacillus subtilis JB-1 for 7 days, compared withcontrols.

(a) kDa kDa

205 166

66

45

29

116

66

45

29

(b) (c) (d)

Figure 2 Coomassie blue stained SDS-PAGE analysis of pro-

teins extracted from the probiotic Aeromonas sobria GC2. Lanes:(a) whole cell proteins (WCPs), (c) outer membrane proteins

(OMPs) and (b, d) protein molecular weight markers. Arrow

points to dominant protein.

(a) kDa kDa

116116

66

45

29

66

4529

(b) (c) (d)

Figure 3 Coomassie blue stained SDS-PAGE analysis of pro-

teins extracted from the probiotic Bacillus subtilis JB-1. Lanes: (a)whole cell proteins (WCPs), (c) cell wall proteins (CWPs) and

(b, d) protein molecular weight markers. Arrow points to

dominant protein.

Table 1 The sequence of peptide mass

fingerprints obtained from MALDI-TOF

mass-spectrometry identified as endoglu-

canase = Bacillus spp. A Mascot scoregreater than 69 indicates the protein

identification is significant (P < 0.05)

Observed

mass (kDa)

Calculated

mass (kDa) Position Peptide sequences

3063.5010 3062.5884 231–260 SPSEAGALQLVELNGQLTLAGEDGTPVQLR-

1608.7680 1607.6628 690–703 -VTEDDFESFGDGYK-

1429.5540 1428.6422 779–790 -QGWDWHTESGVK-

3694.7750 3693.7587 791–824 -TALTIEEANGSNALSWEYAYPEVKPSDGWATAPR

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tivated and cell free preparations of probiotics led toa low level of protection in rainbow trout andgilthead sea bream (Brunt & Austin 2005; Diaz-Rosales, Salinas, Rodriguez, Cuesta, Chabrillon,Balebona, Moriñigo, Esteban & Meseguer 2006).Certainly, many investigators have used whole cellsor components of bacterial cells, such as WCPs(Eldar, Sharpio, Bejerano & Bercovier 1995), LPS(Al-Harbi & Austin 1992; Selvaraj, Sampath &Sekar 2004) and OMPs (Fang, Ling & Sin 2000;Dumetz, Lapatra, Duchaud, Claverols & Henaff 2007), to enhance the immune response in variousfish species. Other workers have suggested the use of probiotics including L. lactis and Lactobacillus sp.(Kajikawa, Satoh, Leer, Yamamoto & Igimi 2007),and A. sobria (Arijo et al. 2008) as oral vaccines. Inthis study, the benefit of using probiotics GC2 and

JB-1 was confirmed and it is the first study in whichthe cellular components of probiotics have beenidentified and shown to be effective for the controlof yersiniosis in rainbow trout. Of especial relevancewas the effectiveness against a new biogroup of thepathogen that has been resistant to conventionalvaccines (Austin et al. 2003).

The two major proteins found in the WCPs andCWPs of JB-1 match previous work concerning shared proteins in whole cell lysates and the cell wallof Staphylococcus epidermidis (Patrick, Plaunt, Sweet& Patrick 1990). The identification of Bacillus endoglucanase in JB-1 was parallel to previouswork, which identified endoglucanase activities inB. subtilis (John, Rice & Preston 2006), Cellulo- monas, Bacillus and Micrococcus (Immanuel,Dhanusha, Prema & Palavesam 2006). Theseextracellular enzymes are secreted by Gram-positivespore-forming bacteria, and are required to liberateutilizable sugars from complex hemicellulose frac-tions (Pell, Taylor, Gloster, Turkenburg, Fontes,Ferreira, Nagy, Clark, Davies & Gilbert 2004).Certainly, cell wall components, which are surfaceexposed and available to interact with the host, may well be responsible for the probiotic activity of the

bacterial cell. Likewise, the major proteins in GC2may explain the beneficial effect of the probiotic.There is conclusive evidence that the non-specificdefence of vertebrates has evolved towards recogni-tion of conserved microbial structures, i.e. yeast/fungal cell wall b-glucan, bacterial LPS and pepti-doglycan, bacterial DNA and viral double-strandedRNA. These cell wall fragments also have the ability to enhance the host resistance against microbialdiseases (Robertsen 1999). Thus, injection with

OMPs of A. hydrophila was shown to controlinfection caused by pathogenic bacteria (Rahman& Kawai 2000). Therefore, it is of course possiblethat proteins, i.e. the endoglucanase-like com-pound, may well function by stimulating immunity in the fish. This possibility is worthy of furtherinvestigation.

In conclusion, OMPs, LPS and WCPs fromGC2, and CWPs and WCPs from JB1 werebeneficial for controlling yersiniosis in rainbow trout and may well explain the parts of the cellsinvolved in protection.

Acknowledgement

AA acknowledges funding from the EgyptianMinistry of Higher Education.

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Received: 26 January 2009 Revision received: 14 April 2009

Accepted: 30 April 2009

37 2009



abbas s 2010

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