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Technical report
Characterization of two key molecules of teleost innate immunityfrom rainbow trout (
Oncorhynchus mykiss
): MyD88 and SAA
Alexander Rebl
a
, Tom Goldammer
a
, Uwe Fischer
b
, Bernd Ko¨llner
b
, Hans-Martin Seyfert
a,
*
a
Research Institute for the Biology of Farm Animals (FBN), Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany
b
Friedrich-Loeffler-Institute (FLI), Federal Research Institute for Animal Health, Institute of Infectiology, Am Su¨ dufer 10, 17493 Greifswald-Insel Riems, Germany
Toll-like receptors (TLR) are relevant for piscine innateimmunity (Stein et al., 2007). These pattern recognitionmolecules bind to exogenous pathogen- and endogenousdamage-associated molecular patterns; PAMP and DAMP(Krishnan et al., 2007). TLR activation recruits severaldownstream factors regulating the expression of immu-norelevant genes. Our laboratory analyses the structureand functional interaction of factors contributing to thepiscine TLR signaling cascade (Rebl et al., 2007, 2008). Inorder to reconstitute the teleost TLR signal transductionpathway in a cell model, we have isolated and character-ized the TLR adapter protein MyD88 from rainbow trout.Moreover, we have also characterized the gene encodingserumamyloidA(SAA)fromtrouttoeventuallyprovideaneffector gene of innate immunity which is known to beregulated by the mammalian TLR signaling cascade.MyD88 is a key factor of the TLR signal transductionpathway in mammals (Arancibia et al., 2007) and fish (van der Sar et al., 2006). This adapter protein has a bipartitestructure. The C-terminal Toll-like/interleukin-1 receptorresistance(TIR)domainbindsdirectlytotheTIRdomainof activated TLRs (Subramaniam et al., 2004). The N-terminaldeath domain interacts with the kinase IRAK-1 (IL-1R-associated kinase-1) recruiting further downstream fac-tors. Infection ultimately increases the abundance of themRNA molecules encoding acute-phase proteins (APP).The APPs represent a group of serum proteins producedand released predominantly by hepatocytes, primarilyprotecting the host from an overwhelming inflammatoryresponse (Eckersall et al., 2007). Serum amyloid A is amajorAPPreportedtoexertanumberofimmunefunctionsin mammals (Uhlar and Whitehead, 1999). Recently, it hasbeen discussed that SAA may even constitute an endo-genous TLR4 ligand. Only few studies document a similarimportance of SAA for piscine defense (Lin et al., 2007). Ashort segment of the mRNA-encoding rainbow trout SAA(rtSAA) was the first APP detected in a salmonid species(GenBank accession: X99387) ( Jensen et al., 1997). Thissequence lacks the 3
0
-UTR and the 5
0
-UTR with the N-terminal part of the coding sequence.
Veterinary Immunology and Immunopathology 131 (2009) 122–126
A R T I C L E I N F O
 Article history:
Received 14 October 2008
Received in revised form 24 February 2009
Accepted 6 March 2009
Keywords:
PathogenAcute-phase proteinToll-like receptorTIR domain
A B S T R A C T
Toll-like receptors (TLR) are relevant for piscine innate immunity. TLR activation recruitsseveral downstream factors regulating the expression of immunorelevant genes. We havecharacterized two key factors of innate immunity from rainbow trout: MyD88 as anadaptor protein interacting directly with TLRs, and serum amyloid A as an effectormolecule induced by the activated Toll-like receptor signaling cascade.Both factors share a remarkable high degree of structural conservation with theirmammalian orthologs suggesting that innate immune defense mechanisms may alsofunctionally be conserved between fish and mammals.
ß
2009 Elsevier B.V. All rights reserved.
* Correspondingauthor.Tel.:+493820868713;fax:+493820868702.
E-mail address:
Seyfert@fbn-dummerstorf.de(H.-M. Seyfert).Abbreviations: aa,aminoacid;MyD88,Myeloiddifferentiationfactor88; PCR, polymerase chain reaction; RT-PCR, PCR following reverse tran-scription of RNA; SAA, serum amyloid A; TIR, Toll-like/interleukin-1receptor resistance; TLR, Toll-like receptor; UTR, untranslated region;VHSV, viral hemorrhagic septicemia virus.
Contents lists available atScienceDirect
Veterinary Immunology and Immunopathology
journal homepage: www.elsevier.com/locate/vetimm
0165-2427/$ – see front matter
ß
2009 Elsevier B.V. All rights reserved.doi:10.1016/j.vetimm.2009.03.006
 
For amplification and cloning of full-length SAA andMyD88 cDNA sequences, we used clinically healthyrainbow trout (
Oncorhynchus mykiss
, ForellenzuchtbetriebUckermark/Brandenburg, Germany) weighing 80–100 g.Fish were fed commercial dry pellets and maintained at15
8
C on a simulated natural photoperiod. For RNAisolation, livers were flash frozen in and subsequentlypowdered under liquid nitrogen in a mortar. RNA wasextracted from the powder using TRIzol
1
reagent (Invi-trogen, Karlsruhe, Germany), as prescribed. We identifiedthe rainbow trout EST 1RT70O09_A_H05 (CA358468)harboring a partial MyD88-sequence in BLAST analysisat the NCBI browser. For 5
0
-RACE experiments, wetranscribed in reverse 500 ng of total RNA from rainbowtrout liver following the instructions of the Gene Racer
TM
Kit (Invitrogen, Karlsruhe, Germany) to generate a cDNAtemplate. PCR amplifications were performed using thegene-specific 5
0
-RACE primer 5
0
-GAGGGCAAACTTTGTCTG-GAAG-3
0
(antisense) and proof reading FastStart Taqpolymerase (Roche, Mannheim, Germany). PCR conditionsconsisted of 1 min at 94
8
C; 1 min at 60
8
C, 2 min at 72
8
Cfor 35 cycles.Similarly, we used the incomplete segment of the SAAcDNA sequence from rainbow trout (533 bp, X99387) todeduce the SAA-specific antisense primer 5
0
-CCATTACT-GATGACTGTTGCTG-3
0
for 5
0
-RACE experiments. We estab-lished a genomic walking library (BD Genome Walker
TM
Universal Kit; BD Biosciences, Erembodegem, Belgium)from liver DNA of rainbow trout to retrieve andcharacterize the SAA gene. Segments of the SAA genesequence were amplified in successive steps with the FastStart TaqPolymeraseapplyinga collectionof gene-specificprimers, which all were derived from our full-length SAAcDNA sequence.Protein domains were identified after conceptualtranslation of the SAA-encoding cDNA sequence with theProtSweepv3.1software,availableattheDKFZ-Heidelberg(Germany). Multiple sequence alignment was conductedwith the ClustalW2 program (Chenna et al., 2003).Potential transcription factor binding sites were identifiedwithin the proximal promoter of the rtSAA gene using theMatInspector program (Cartharius et al., 2005).Inordertoanalyzetheinfection-relatedactivationofSAAgene expression, we infected healthy rainbow trout withviral hemorrhagic septicemia virus (VHSV) strain Fi-13propagated on EPC (epithelioma papillosum carpio) cellsand titrated on RTG-2(rainbow trout gonad) cells.Fish wasanaesthetized in benzocain (10 mg/L water) and infectedintraperitoneally with 10
4
tissue culture infectious doses(TCID
50
) in 100
m
l of tissue culture medium. An analapplication of 10
3
TCID
50
was given in support. Liverbiopsiesweretakenfromthree trout, eachat days 3 and 16post-infection. Three naı¨ve fish served as controls. Forquantitativereal-timeRT-PCR(qRT-PCR),wetranscribedinreverse5
m
gofoligo-dT-primedtotalliverRNAutilizingtheSuper Script
TM
II kit (Invitrogen, Karlsruhe, Germany). Weused trout SAA-specific primers 5
0
-GACATGTGGCGTGCA-TATGGC-3
0
(sense) and 5
0
-CCATTACTGATGACTGTTGCTG-3
0
(antisense)toamplifya136 bpsegmentoftheSAAcDNAforPCRanalysisusingtheLightCyclerInstrumentandFastStartDNA Master
PLUS
SYBR Green I Kit (Roche, Basel, Switzer-land).Thermalcyclingparametersforthe40cyclesincludedmelting(15 s at95
8
C),annealing(10 sat60
8
C), elongation(20 sat72
8
C)andacquiringfluorescence(5 sat84
8
C).Copynumbers were calibrated against amplifications of serialdilutions of subcloned cDNAs serving as external standards(10
6
to 10
3
copies).Our investigations revealed that the MyD88 mRNAfrom rainbow trout (rtMyD88, GenBank accession:AJ878918) encodes 282 aa residues (Fig. 1). It featuresan N-terminal death domain (Fig. 1A, aa position 22–99),followed by an interdomain (aa positions 100–144).Functional relevance of the latter domain was provenafter the discovery of a human MyD88 splice variantlackingtheinterdomain.This‘‘MyD88short’’variantblocksNF-
k
B activation (Liew et al., 2005). The highly conservedTIR domain comprises the C-terminal half of the protein(Fig.1A,aaposition145–282).RtMyD88sharesthehighestdegree of similarity with MyD88 from salmon (90.8%) and Japanese flounder (70.8%). Domain-specific amino acidsequencecomparisonofMyD88fromhuman,chicken,andsalmonids (Fig. 1B) shows higher similarities for the TIR domain (
>
75%) than for interdomain (
>
53%) and deathdomain (
>
40%) suggesting that the TIR domain isphylogenetically most highly conserved. Comparison withthe TIR domain of human MyD88 protein (NM_002468)revealsclustersofconservedaminoacids(Fig.1A,boxesa–c). These amino acids had previously been identified in thehuman MyD88 protein as functionally important for TIR-TIR interactions (Li et al., 2005). Our present alignmentemphasizes that additional amino acids are conserved inpiscine and human MyD88, suggesting their commonfunctional relevance.The alignment of TIR domains was extended to those of TLR factors, since TIR domains from both MyD88 andreceptorareknowntointeractwitheachother.Tothisend,we included our previously characterized TIR domainsfrom salmonid fish (Rebl et al., 2007) and from the humanTLR4 protein as an example of a mammalian TLR receptor.Thus, we have compared the primary structure of TIR domains not only between the MyD88 factors of fish andman, but also across different factors. This extensivebreadth of the comparison illustrates that those boxes(Fig. 1B, boxes a–c) are conserved in the TIR domains of different factors, suggestively across all vertebrates. More-over, we highlight further completely conserved aminoacids suggesting their superb functional relevance.The TIR domains of TLR factors are longer by 8 aaresidues than those from MyD88 factors. A single aaresidue is found inserted behind position 162 of thertMyD88sequence,andastringof7aabehindposition252of the respective sequence. Interestingly, the amino acidsequence of this insertion is conserved between salmonandtrout,butentirelydifferentfromthatofthehumanTLR factor. Conceivably, structural rather than chemical con-straints shape this feature. We note that the arginine-proline dinucleotide motif at aa position 263–264 may becommon for salmonid TIR domains.We isolated the entire rainbow trout SAA (rtSAA) cDNAsequence in 5
0
- and 3
0
-RACE experiments. Our sequence(AM422446) comprises 629 bp, including a run of 14adenine nucleotides stemming from the poly(A) tail of the
 A. Rebl et al./Veterinary Immunology and Immunopathology 131 (2009) 122–126 
123
 
Fig.1.
(A)AminoacidsequenceoftheMyD88factorfromrainbowtroutincludingthedeathdomain(underlined),thelinkinginterdomain(boldfaceletters),andamultiplealignmentofTIRdomainsequencesfromsalmonidandhumanMyD88andTLRproteins,respectively.SequencesofrtMyD88(CAI48087),salmonidTLRs(rainbowtrout,
Oncorhynchusmykiss
;[Om]TLR22,CAF31506andTLR22L,CAI48084;Atlanticsalmon,
Salmosalar 
[Ss]TLR22a,CAJ80696;andTLR22b,CAR62394)aswellashuman[Hs]MyD88(NP_002459),andTLR4(AAC34135)wereusedforthiscomparison.Clustersoffunctionalaminoacids(a,b,c)withintheTIR domainrelevantfortheinteractionofMyD88andTLRfactorsareboxed.Identicalandchemicallyequivalentaaresiduesarelabeledbyblackorgreyunderlay,respectively.ConservedresiduesspecificforsalmonidTIR domains are underlined. (B) The percentage of aa sequence identity of the respective MyD88 domains between trout and salmon (EF672332), chicken (NM_001030962) as well as man (NP_002459) areindicated.
A  .R  e  b   l    e  t   a  l    .  /    V  e  t   e r  i    n a r   y I    m m u n o  l    o   g   y  a  n d  I    m m u n o   p a  t   h   o  l    o   g   y 1  3 1   (   2  0  0  9   )   1 2 2 –1 2  6 
1  2  4  
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