Veterinary Immunology and Immunopathology 131 (2009) 122–126

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Veterinary Immunology and Immunopathology

journal homepage: www.elsevier.com/locate/vetimm

Technical report

Characterization of two key molecules of teleost innate immunity

from rainbow trout (Oncorhynchus mykiss): MyD88 and SAA
Alexander Rebl a, Tom Goldammer a, Uwe Fischer b, Bernd Köllner 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 Südufer 10, 17493 Greifswald-Insel Riems, Germany

A R T I C L E I N F O A B S T R A C T

Article history: Toll-like receptors (TLR) are relevant for piscine innate immunity. TLR activation recruits
Received 14 October 2008 several downstream factors regulating the expression of immunorelevant genes. We have
Received in revised form 24 February 2009 characterized two key factors of innate immunity from rainbow trout: MyD88 as an
Accepted 6 March 2009
adaptor protein interacting directly with TLRs, and serum amyloid A as an effector
molecule induced by the activated Toll-like receptor signaling cascade.
Keywords: Both factors share a remarkable high degree of structural conservation with their
Pathogen
mammalian orthologs suggesting that innate immune defense mechanisms may also
Acute-phase protein
functionally be conserved between fish and mammals.
Toll-like receptor
TIR domain ß 2009 Elsevier B.V. All rights reserved.

Toll-like receptors (TLR) are relevant for piscine innate
immunity (Stein et al., 2007). These pattern recognition
molecules bind to exogenous pathogen- and endogenous
damage-associated molecular patterns; PAMP and DAMP
(Krishnan et al., 2007). TLR activation recruits several
downstream factors regulating the expression of immu-
norelevant genes. Our laboratory analyses the structure
and functional interaction of factors contributing to the
piscine TLR signaling cascade (Rebl et al., 2007, 2008). In
order to reconstitute the teleost TLR signal transduction
pathway 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 encoding
serum amyloid A (SAA) from trout to eventually provide an
effector gene of innate immunity which is known to be
regulated by the mammalian TLR signaling cascade.
* Corresponding author. Tel.: +49 38208 68 713; fax: +49 38208 68 702.
E-mail address: Seyfert@fbn-dummerstorf.de (H.-M. Seyfert).
Abbreviations: aa, amino acid; MyD88, Myeloid differentiation factor
88; PCR, polymerase chain reaction; RT-PCR, PCR following reverse tran-
scription of RNA; SAA, serum amyloid A; TIR, Toll-like/interleukin-1
receptor resistance; TLR, Toll-like receptor; UTR, untranslated region;
VHSV, viral hemorrhagic septicemia virus.
MyD88 is a key factor of the TLR signal transduction
pathway in mammals (Arancibia et al., 2007) and fish (van
der Sar et al., 2006). This adapter protein has a bipartite
structure. The C-terminal Toll-like/interleukin-1 receptor
resistance (TIR) domain binds directly to the TIR domain of
activated TLRs (Subramaniam et al., 2004). The N-terminal
death domain interacts with the kinase IRAK-1 (IL-1R-
associated kinase-1) recruiting further downstream fac-
tors. Infection ultimately increases the abundance of the
mRNA molecules encoding acute-phase proteins (APP).
The APPs represent a group of serum proteins produced
and released predominantly by hepatocytes, primarily
protecting the host from an overwhelming inflammatory
response (Eckersall et al., 2007). Serum amyloid A is a
major APP reported to exert a number of immune functions
in mammals (Uhlar and Whitehead, 1999). Recently, it has
been discussed that SAA may even constitute an endo-
genous TLR4 ligand. Only few studies document a similar
importance of SAA for piscine defense (Lin et al., 2007). A
short 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). This
sequence lacks the 30 -UTR and the 50 -UTR with the N-
terminal part of the coding sequence.

0165-2427/$ – see front matter ß 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetimm.2009.03.006
 A. Rebl et al. / Veterinary Immunology and Immunopathology 131 (2009) 122–126
For amplification and cloning of full-length SAA and
MyD88 cDNA sequences, we used clinically healthy
rainbow trout (Oncorhynchus mykiss, Forellenzuchtbetrieb
Uckermark/Brandenburg, Germany) weighing 80–100 g.
Fish were fed commercial dry pellets and maintained at
15 8C on a simulated natural photoperiod. For RNA
isolation, livers were flash frozen in and subsequently
powdered under liquid nitrogen in a mortar. RNA was
extracted from the powder using TRIzol1 reagent (Invi-
trogen, Karlsruhe, Germany), as prescribed. We identified
the rainbow trout EST 1RT70O09_A_H05 (CA358468)
harboring a partial MyD88-sequence in BLAST analysis
at the NCBI browser. For 50 -RACE experiments, we
transcribed in reverse 500 ng of total RNA from rainbow
trout liver following the instructions of the Gene RacerTM
Kit (Invitrogen, Karlsruhe, Germany) to generate a cDNA
template. PCR amplifications were performed using the
gene-specific 50 -RACE primer 50 -GAGGGCAAACTTTGTCTG-
GAAG-30 (antisense) and proof reading FastStart Taq
polymerase (Roche, Mannheim, Germany). PCR conditions
consisted of 1 min at 94 8C; 1 min at 60 8C, 2 min at 72 8C
for 35 cycles.
Similarly, we used the incomplete segment of the SAA
cDNA sequence from rainbow trout (533 bp, X99387) to
deduce the SAA-specific antisense primer 50 -CCATTACT-
GATGACTGTTGCTG-30 for 50 -RACE experiments. We estab-
lished a genomic walking library (BD Genome WalkerTM
Universal Kit; BD Biosciences, Erembodegem, Belgium)
from liver DNA of rainbow trout to retrieve and
characterize the SAA gene. Segments of the SAA gene
sequence were amplified in successive steps with the Fast
Start Taq Polymerase applying a collection of gene-specific
primers, which all were derived from our full-length SAA
cDNA sequence.
Protein domains were identified after conceptual
translation of the SAA-encoding cDNA sequence with the
ProtSweep v3.1 software, available at the DKFZ-Heidelberg
(Germany). Multiple sequence alignment was conducted
with the ClustalW2 program (Chenna et al., 2003).
Potential transcription factor binding sites were identified
within the proximal promoter of the rtSAA gene using the
MatInspector program (Cartharius et al., 2005).
In order to analyze the infection-related activation of SAA
gene expression, we infected healthy rainbow trout with
viral hemorrhagic septicemia virus (VHSV) strain Fi-13
propagated on EPC (epithelioma papillosum carpio) cells
and titrated on RTG-2 (rainbow trout gonad) cells. Fish was
anaesthetized in benzocain (10 mg/L water) and infected
intraperitoneally with 104 tissue culture infectious doses
(TCID50) in 100 ml of tissue culture medium. An anal
application of 103 TCID50 was given in support. Liver
biopsies were taken from three trout, each at days 3 and 16
post-infection. Three naı̈ve fish served as controls. For
quantitative real-time RT-PCR (qRT-PCR), we transcribed in
reverse 5 mg of oligo-dT-primed total liver RNA utilizing the
Super ScriptTMII kit (Invitrogen, Karlsruhe, Germany). We
used trout SAA-specific primers 50 -GACATGTGGCGTGCA-
TATGGC-30 (sense) and 50 -CCATTACTGATGACTGTTGCTG-30
(antisense) to amplify a 136 bp segment of the SAA cDNA for
PCR analysis using the LightCycler Instrument and FastStart
DNA MasterPLUS SYBR Green I Kit (Roche, Basel, Switzer-
123
land). Thermal cycling parameters for the 40 cycles included
melting (15 s at 95 8C), annealing (10 s at 60 8C), elongation
(20 s at 72 8C) and acquiring fluorescence (5 s at 84 8C). Copy
numbers were calibrated against amplifications of serial
dilutions of subcloned cDNAs serving as external standards
(106 to 103 copies).
Our investigations revealed that the MyD88 mRNA
from rainbow trout (rtMyD88, GenBank accession:
AJ878918) encodes 282 aa residues (Fig. 1). It features
an 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 proven
after the discovery of a human MyD88 splice variant
lacking the interdomain. This ‘‘MyD88short’’ variant blocks
NF-kB activation (Liew et al., 2005). The highly conserved
TIR domain comprises the C-terminal half of the protein
(Fig. 1A, aa position 145–282). RtMyD88 shares the highest
degree of similarity with MyD88 from salmon (90.8%) and
Japanese flounder (70.8%). Domain-specific amino acid
sequence comparison of MyD88 from human, chicken, and
salmonids (Fig. 1B) shows higher similarities for the TIR
domain (>75%) than for interdomain (>53%) and death
domain (>40%) suggesting that the TIR domain is
phylogenetically most highly conserved. Comparison with
the TIR domain of human MyD88 protein (NM_002468)
reveals clusters of conserved amino acids (Fig. 1A, boxes a–
c). These amino acids had previously been identified in the
human MyD88 protein as functionally important for TIR-
TIR interactions (Li et al., 2005). Our present alignment
emphasizes that additional amino acids are conserved in
piscine and human MyD88, suggesting their common
functional relevance.
The alignment of TIR domains was extended to those of
TLR factors, since TIR domains from both MyD88 and
receptor are known to interact with each other. To this end,
we included our previously characterized TIR domains
from salmonid fish (Rebl et al., 2007) and from the human
TLR4 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 and
man, but also across different factors. This extensive
breadth 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 amino
acids suggesting their superb functional relevance.
The TIR domains of TLR factors are longer by 8 aa
residues than those from MyD88 factors. A single aa
residue is found inserted behind position 162 of the
rtMyD88 sequence, and a string of 7 aa behind position 252
of the respective sequence. Interestingly, the amino acid
sequence of this insertion is conserved between salmon
and trout, but entirely different from that of the human TLR
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 be
common for salmonid TIR domains.
We isolated the entire rainbow trout SAA (rtSAA) cDNA
sequence in 50 - and 30 -RACE experiments. Our sequence
(AM422446) comprises 629 bp, including a run of 14
adenine nucleotides stemming from the poly(A) tail of the
 124
A. Rebl et al. / Veterinary Immunology and Immunopathology 131 (2009) 122–126
Fig. 1. (A) Amino acid sequence of the MyD88 factor from rainbow trout including the death domain (underlined), the linking interdomain (bold face letters), and a multiple alignment of TIR domain sequences from
salmonid and human MyD88 and TLR proteins, respectively. Sequences of rtMyD88 (CAI48087), salmonid TLRs (rainbow trout, Oncorhynchus mykiss; [Om] TLR22, CAF31506 and TLR22L, CAI48084; Atlantic salmon,
Salmo salar [Ss] TLR22a, CAJ80696; and TLR22b, CAR62394) as well as human [Hs] MyD88 (NP_002459), and TLR4 (AAC34135) were used for this comparison. Clusters of functional amino acids (a, b, c) within the TIR
domain relevant for the interaction of MyD88 and TLR factors are boxed. Identical and chemically equivalent aa residues are labeled by black or grey underlay, respectively. Conserved residues specific for salmonid
TIR 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) are
indicated.
 A. Rebl et al. / Veterinary Immunology and Immunopathology 131 (2009) 122–126 125

Fig. 2. (A) Nucleotide sequence of the proximal promoter from rainbow trout SAA gene (240 bp of the 50 flanking region) featuring a TATA box (31 to
18 bp; black underlay), three consensus sequence motifs for attachments of NF-IL6 (88 to 74 bp and 199 to 185 bp, broken line boxes) and NF-kB
(84 to 72 bp, boxed with full line). (B) Exon intron organisation of the rainbow trout SAA gene. Grey underlay specifies the ORF, start and stop codon are
marked with triangles. Numbers indicate the DNA position relative to the transcriptional start. Below the scheme, all known piscine SAA protein sequences
and human SAA isoforms are aligned. Identical and chemically equivalent aa residues are labeled by black or grey underlay, respectively. The aa insertions of
Hs-SAA4 are boxed, and their position is marked with an arrow. The comparison is based on the sequences carp (Cc, Cyprius carpio; BAA36700), rainbow
trout (Om, Oncorhynchus mykiss; CAM12347), spotted green pufferfish (Tn, Tetraodon nigroviridis; CAF99678), zebrafish (Dr, Danio rerio; NP_001005599) as
well as human (Hs) SAA1 (AAA64799), SAA2 (NP_110381), and SAA4 (NP_006503).

mRNA. The open reading frame of 366 bp encodes 121 aa.
After definition of the transcriptional start site in those 50 -
RACE experiments, we used our genomic walking library to
extract 680 nucleotides of the proximal rtSAA gene
promoter. Computational analysis of the promoter
sequence revealed an element similar to a TATA box
(Fig. 2A; between position 31 and 18) and two
consensus sites for NF-IL6 binding (at 199 to 185 and
at 88 to 74). The proximal of these sites overlaps with a
binding motif for NF-kB attachment (at 84 to 72).
Studies of mammalian SAA promoters proved the impor-
tance of such promoter regions. The combined effect of NF-
kB and NF-IL6 is essential for induced transcriptional
activation of SAA (Li and Liao, 1992; Ray et al., 1995).
The rtSAA gene is segmented into three exons (Fig. 2B;
AM422447). Exon 1 encodes the majority of the 50 -UTR.
Exon 2 encodes the N-terminal 29 aa residues of the SAA
protein while exon 3 encodes the residual 92 aa.
The amino acid sequence of rtSAA proteins is remark-
ably conserved (Fig. 2B). Similarities between the protein
sequences of the piscine and the human SAA molecules
encoded by the two inducible SAA genes (hsSAA1, 2)
exceed 66%. The protein lengths are also similar, compris-
ing 121 aa in trout, zebrafish, and the pufferfish Tetraodon,
and 123 aa in carp as well as in hsSAA1, and hsSAA2 factors.
Moreover, the alignment shows clusters of highly con-
served amino acids, flanked by clusters of chemically
equivalent amino acids. This suggests that the SAA proteins
feature phylogenetically conserved functional domains.
Interestingly, a SAA multigene family apparently
evolved only in mammals. Teleosts and other non-
mammalian organisms including invertebrates such as
amphioxus and sea cucumber possess only one SAA-
encoding gene. All the SAA proteins encoded by those
genes are highly similar to those human SAA factors, which
are encoded by the inducible genes SAA1 and SAA2. Only
the constitutively expressed human SAA4 gene encodes a
protein featuring insertion of an additional tripeptide and a
pentapeptide sequence. These are located behind aa
position 88 and 91 of the rainbow trout SAA protein.
These structural features might indicate that the primor-
dial SAA gene was inducible and that the constitutively
expressed SAA-encoding genes evolved at later times,
within the mammalian lineage only.
We examined if the expression of the SAA-encoding
gene from rainbow trout is induced by viral infection.
Therefore, healthy rainbow trout were infected with VHSV.
SAA expression in livers was measured 3 and 16 days after
infection and compared to that found in uninfected,
healthy fish. SAA mRNA concentrations were low in
samples from control animals (0.02  0.004  106 copies
per cDNA equivalent, as derived from 500 ng total RNA) but
elevated by more than 85-fold, both at day 3
(1.64  0.94  106), and day 16 (1.68  1.03  106) after
infection. This observation is in line with previous studies in
salmonids reporting that SAA expression may be induced
after bacterial challenge (Jensen et al., 1997).
Taken together, our data show a remarkably high degree
of structural conservation of two important molecules of
innate immunity, the MyD88 adapter and the SAA effector
molecule, during vertebrate evolution. This suggests that
also the defense mechanisms based on innate immunity
126 A. Rebl et al. / Veterinary Immunology and Immunopathology 131 (2009) 122–126
might be conserved over a period of at least 450 Mio years,
dating back to the times when mammals and teleosts
branched away from their common ancestor.
Acknowledgements
The authors acknowledge Prof. E. Siegl for valuable
discussions and Marlies Fuchs for help with sequencings.
This work was supported by the European Community
through the EADGENE Network of Excellence and the
Deutsche Forschungsgemeinschaft through the FOR585
(Se 326/14-2).
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