- Email: [email protected]
Polymorphisms of chicken toll-like receptor 1 type 1 and type 2 in different breeds
Polymorphisms of chicken toll-like receptor 1 type 1 and type 2 in different breeds W. K. Ruan*† and S. J. Zheng*1 *State Key Laboratory of Agrobiotechnology, Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China; and †College of Animal Science and Technology, Beijing University of Agriculture, Beijing 102206, China 27 amino acid polymorphism sites in ChTLR: 14 sites in type 1 (8 sites in the extracellular domain, 5 sites in the cytoplasmic domain, and 1 site in the transmembrane domain) and 13 sites in type 2 (11 sites in the extracellular domain, 1 site in the cytoplasmic domain, and 1 site in the transmembrane domain). These results demonstrate that ChTLR1 type 1 and type 2 genes are polymorphic among chicken breeds, suggesting a varied resistance among breeds of chicken. This information provides new consideration for future poultry breeding and breed improvement.
Key words: chicken, leucine-rich repeat, polymorphism, toll–interleukin-1 receptor, toll-like receptor 2011 Poultry Science 90:1941–1947 doi:10.3382/ps.2011-01489
INTRODUCTION Infectious diseases are major threats to the modern poultry industry. In addition to viral diseases, bacterial diseases such as salmonellosis, colibacillosis, and fowl cholera, which are associated with causative organisms, lead to heavy economic losses (Leveque et al., 2003). The causative agents for these diseases, such as Salmonella, Escherichia coli, and Pasteurella multocida, are resident opportunistic microorganisms that are difficult to eradicate from flocks. Therefore, the genetic improvement chickens’ resistance to these opportunistic pathogens may be considered as an alternative control measure against these bacterial infections. Toll-like receptors (TLR) play a crucial role in host immune response via recognition of pathogen-associated molecular patterns (PAMP), such as lipopolysaccharides, lipopeptides, flagellins, double-stranded RNA, or CpG DNA motifs. So far, at least 10 members of the chicken TLR family have been identified, includ-
©2011 Poultry Science Association Inc. Received March 17, 2011. Accepted May 8, 2011. 1 Corresponding author: [email protected]
ing TLR1 type 1 and type 2, TLR2 type 1 and type 2, TLR3, TLR4, TLR5, TLR7, TLR15, and TLR21 (Fukui et al., 2001; Yilmaz et al., 2005; Temperley et al., 2008). Because the amino acid sequences of 2 identified chicken TLR (ChTLR) are almost equally homologous to sequences of human TLR1 and TLR6, it is difficult to determine which of these 2 receptors is ChTLR1 or ChTLR6. Thus, they were named ChTLR1 type 1 (long form) and ChTLR1 type 2 (short form; Yilmaz et al., 2005). Chicken TLR1 type 1 and type 2 are expressed in almost all tissues (Yilmaz et al., 2005). The ChTLR1 cluster is located on chromosome 4 and is believed to result from gene duplication (Beutler and Rehli, 2002). In humans, TLR1 interacts with TLR2 in a heterodimeric complex, which increases their capability to recognize ligands, such as lipids or proteins of extracellular pathogens as well as several synthetic peptides such as N-palmitoyl-S-dipalmitoylglyceryl and Cys-Ser-(Lys)4 (Takeuchi et al., 2002; Buwitt-Beckmann et al., 2006; Jin et al., 2007; Jin and Lee, 2008). In addition, TLR1 family cluster might be involved in the recognition of other unknown ligands (Jin and Lee, 2008). A few studies focused on the identification of ChTLR1 type 1 and type 2 (Yilmaz et al., 2005; Keestra et al., 2007; Higuchi et al., 2008), and it was found that ChTLR1 type 1 and type 2 cooperate with ChTLR2 to recognize pepti-
1941
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on June 14, 2015
ABSTRACT Toll-like receptors (TLR) mediate immune responses via recognition of pathogen-associated molecular patterns, thus playing important roles in the host defense. Polymorphisms of TLR may affect their recognition of pathogen-associated molecular patterns, leading to varied host resistance to pathogenic infections. However, little is known regarding the polymorphisms of chicken TLR (ChTLR) among breeds. In this study, we cloned ChTLR1 type 1 and type 2 genes from 9 chicken breeds (2 commercial breeds and 7 Chinese native breeds) and analyzed their sequences. We found
1942
Ruan and Zheng
MATERIALS AND METHODS Source of Chicken Breeds Chickens used in this study were obtained from commercial breeders and were from the same genetic background. Nine breeds were chosen (see Introduction). Four individual repeats were arranged in each breed.
Molecular Cloning of Chicken TLR1 Type 1 and Type 2 The total RNA was obtained from the spleen using TRIzol (Invitrogen Corp., Carlsbad, CA) per the manufacturer’s instructions and was used for cDNA synthesis. The 20 μL of reaction mixture contained 5 μg of total RNA, 0.5 μg of oligo dT primer (16–18 mer), 40 U of RNasin, 1,000 μM of deoxyribonucleotide triphosphate mix, 10 mM of dithiothreitol, and 5 U of M-MLV reverse transcriptase (Promega, Madison, WI) in 1× reverse transcriptase buffer. The reaction mixture was incubated at 37°C for 1 h. The cDNA synthesis was confirmed by amplifying the β-actin amplicon in PCR. One pair of primers used to amplify the full open reading frame was designed based on the consensus TLR1 mRNA sequences of Gallus gallus TLR1LA (NM_001007488.3) and Gallus gallus TLR1LB (NM_001081709.2) (type 1 primer: forward, 5′-GGATCCCTTACAAGTATCTACGTTT-3′; reverse, 5′-CACTTAATCAGACGTTGGGTTATAC-3′; type 2 primer: forward, 5′-TACTTCAGGTTGTATGGCACTCA-3′; reverse, 5′-CAAGGAGAAGAAGAAACTGCATTA-3′). The ChTLR1 type 1 and type 2 genes were amplified in PCR using the synthesized cDNA. The reaction conditions for amplification in PCR were the same for both types. The 25 μL of PCR reaction contained 50 pmol for each forward and reverse primer, 2 μL of Template cDNA, 200 μM each of deoxyribonucleotide triphosphate mix, and 2.5 U of Pfu DNA polymerase (Promega) in 1× Pfu DNA polymerase buffer. Amplification conditions were as follows: initial denaturation at 94°C for 2 min; 35 cycles at 94°C for 30 s, annealing at 56 to 60°C for 30 s, and extension at 72°C for 5 min; and a final extension at 72°C for 10 min. The PCR amplicons verified by 1% agarose gel electrophoresis were purified and ligated into pEASY-Blunt simple cloning vector (TransGen, Beijing, China) following the manufacturer’s instructions. Recombinant plasmids were characterized by PCR using gene-specific and vector primer pairs. The ChTLR1 type 1 and type 2 gene inserts in recombinant plasmids were sequenced from both ends by ABI 3730XL sequencer from SinoGenoMax Co. Ltd. (Beijing, China).
Sequence Analysis Nucleotide and amino acid sequences of ChTLR type 1 and type 2 were aligned with the analysis software MegAlign (DNAstar Inc., Madison, WI). The extracellular, transmembrane, and cytoplasmic domains of these protein sequences were detected with the analysis tools provided at http://smart.embl-heidelberg.de and http://split.pmfst.hr.
Nucleotide Sequence Accession Numbers The coding sequences for the avian TLR1 type 1 and type 2 have been deposited in GenBank. The acces-
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on June 14, 2015
doglycan and lipoprotein with high efficiency (Higuchi et al., 2008). Polymorphisms of TLR may profoundly influence the response of a host to a wide range of pathogens and are associated with resistance and susceptibility to diseases (Hawn et al., 2007, 2009; Misch and Hawn, 2008). The TLR1 gene family cluster including TLR1, TLR6, and TLR10 is associated with the susceptibility of bacterial infections in human and mice. It was found that variation in the inflammatory responses to bacterial lipopeptides is regulated by a common TLR1 transmembrane domain polymorphism that could potentially affect the innate immune response and clinical susceptibility to a wide spectrum of pathogens (Hawn et al., 2007). Consistently, the hypermorphic genetic variation in TLR1 is associated with increased susceptibility to organ dysfunction, death, and gram-positive bacterial infections in sepsis (Wurfel et al., 2008). In addition, human TLR1 I602S variant is associated with a decreased incidence of leprosy (Johnson et al., 2007) and the response to N-palmitoyl-S-dipalmitoylglyceryl, a synthetic ligand for TLR1 (Hawn et al., 2007; Misch and Hawn, 2008). Furthermore, human TLR6 S249P polymorphism was associated with lower left ventricular wall thickness and inflammatory response in hypertensive women (Sales et al., 2010). These findings suggest that TLR polymorphisms are associated with host susceptibility to diseases. Although it is well known that the susceptibility of chickens with different genetic backgrounds to pathogenic infection varies, little is known regarding the genetic factors contributing to the susceptibility of chickens to diseases. Based on the above information and our clinical observations, we hypothesize that ChTLR1 type 1 and type 2 genes may have polymorphisms that affect chicken susceptibility. In this study, we used 2 commercial breeds (White Leghorn chicken and Hy-Line Brown chicken, which was used for eggs) and 7 Chinese native breeds (Beijing White 939 chicken, which was used for brown eggs; Nongda no. 3 chicken, which is a dwarf and was used for eggs; Luhua chicken, which was raised for meat and eggs; White-Feather Silky chicken, which had the characteristics of white feathers and black bones, used for dual purposes; and Beijing Fatty chicken, Royal chicken, and Laiwu Black chicken, which were characterized by their shape and good meat quality). We cloned and sequenced the full length of ChTLR1 type 1 and type 2 genes from different breeds of chickens with an objective to understand the genetic contributions to the polymorphisms of ChTLR1 type 1 and type 2.
1943
POLYMORPHISMS OF CHICKEN TOLL-LIKE RECEPTORS 1
Figure 1. Chicken toll-like receptor TLR (ChTLR) type 1 and type 2 structures. Dark rectangles represent the transmembrane domain and gray bands indicate leucine-rich repeats (LRR) involved in pathogen-associated molecular pattern recognition. Gray circles represent C terminal LRR (CTLRR). TIR = toll–IL-1 receptor. Analysis tool provided at http://smart.embl-heidelberg.de.
RESULTS AND DISCUSSION Chicken TLR1 type 1 and type 2 are located on chromosome 4 71561343–71566597 and 513–2199 (Table 1). The full-length open reading frame of type 1 contains 2,457 nucleotides encoding 818 amino acids, and type 2 contains 1,590 nucleotides encoding 516 amino acids. The predicted results also show that 593 amino acids of type 1 and 317 amino acids of type 2 are located in the extracellular domain whereas 177 amino acids of type 1 and type 2 are located in the cytoplasmic domain. Using analysis software, we predicted that ChTLR type 1 and type 2 are type I transmembrane proteins, with a large extracellular domain comprising leucine-rich repeats (LRR) flanked by C-terminal cap motifs, a transmembrane domain, and a cytoplasmic toll–IL-1 receptor (TIR) domain (Figure 1). Similar to human or mouse LRR, the LRR of chickens are motifs of 20 to 30 amino acids in length that fold into a horseshoe shape and are involved in recognition of PAMP, mostly via interaction with other proteins (Jin and Lee, 2008; Werling et al., 2009). The TIR domain is similar to that of the IL-1 receptor, which is highly conserved and interacts with adapter proteins such as
myeloid differentiation factor (MyD88) and TIR domain containing adaptor protein (TIRAP, also known as Mal; Verstak et al., 2009). Ligand binding triggers homo- or heterodimerization of TLR and subsequent recruitment of intracellular adapter proteins (Higuchi et al., 2008; Jin and Lee, 2008). Multiple intermediates such as tumor necrosis factor receptor associated factor 6, IL-1 receptor-associated kinase 4 family proteins, Ik-kinase family proteins, mitogen-activated protein kinase, and transcription factors including nuclear factor κB family proteins are involved in TLR-mediated signal transduction, leading to the expression of inflammatory cytokines, chemokines, and adhesion molecules (Beutler, 2005; Vandewalle, 2008). The structural analysis of ChTLR1 type 1 reveals that it contains 5 LRR motifs, 1 C terminal LRR (LRRCT), 2 low compositional complexities, 1 transmembrane domain, and 1 TIR domain. The ChTLR1 type 2 structure includes 3 LRR, 1 LRRCT, 1 low compositional complexity, 1 transmembrane domain, and 1 TIR domain (Figure 1). It also shows transmembrane helix preference, β preference, and modified hydrophobic moment index as determined by online software SPLIT server (http://split. pmfst.hr; Figure 2). There is intensive hydrophobicity in ChTLR1 type 1 structures in the N terminal, which is predicted as a signal peptide domain, and other intensive hydrophobicity in the middle, which is predicted as a transmembrane domain. The sequences of ChTLR type 1 and type 2 from the 9 breeds showed varied sequence patterns (Tables 2 and 3). The amino acid sequence analysis of ChTLR1 type 1 and type 2 from 9 chicken breeds indicates that there are 27 sites of amino acid sequence with varied amino acids (Figure 3), including 14 sites in type 1 and 13 sites in type 2. Eight polymorphic sites are located
Table 1. Chicken toll-like receptor 1 (ChTLR1) type 1 and type 2 details1 Name
Chromosome
Location
ChTLR1 type 1 ChTLR1 type 2
4 4
71561343–71566597 513–2199
1AA
= amino acids.
Length (nucleotides)
Length (AA)
Extracellular AA
Cytoplasmic AA
2,457 1,590
818 516
593 317
177 177
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on June 14, 2015
sion numbers for type 1 and type 2 are, respectively, JF7823968 and JF7823977 (Royal chicken), JF823969 and JF823978 (Beijing White 939 chicken), JF823970 and JF823979 (White Leghorn chicken), JF823971 and JF823980 (Laiwu Black chicken), JF823972 and JF823981 (Hy-Line Brown chicken), JF823973 and JF823982 (Luhua chicken), JF823974 and JF823983 (Nongda no. 3 chicken), JF823975 and JF823984 (White-Feather Silky chicken), and JF823976 and JF823985 (Beijing Fatty chicken).
1944
Ruan and Zheng
Table 2. Chicken toll-like receptor 1 type 1 polymorphism details1 Position (AA)
Structural characteristic
Majority AA (codon)
Polymorphic AA (codon)
Chicken breed2
Synonymous substitution
36 38 130 144 149 207 245 249 359 431 447 465 471 477 499 502 519 527 556 560 611 642 645 650 711 730 735 746 747 749 760 761 790
Leucine-rich repeats Leucine-rich repeats Leucine-rich repeats Leucine-rich repeats C terminal leucine-rich repeats C terminal leucine-rich repeats Transmembrane domain Toll–IL-1 receptor Toll–IL-1 receptor Toll–IL-1 receptor Toll–IL-1 receptor Toll–IL-1 receptor Toll–IL-1 receptor Toll–IL-1 receptor Toll–IL-1 receptor Toll–IL-1 receptor Toll–IL-1 receptor
G (GGA) L (TTA) T (ACT) L (TTA) L (TTA) L (CTC) T (ACA) T (ACG) R (CGA) C (TGC) L (CTG) Q (CAA) N (AAT) T (ACT) R (AGG) T (ACA) A (GCT) S (AGC) G (GGC) G (GGC) P (CCG) R (CGG) A (GCT) A (GCC) S (TCG) K (AAG) N (AAC) P (CCG) I (ATC) P (CCA) K (AAG) A (GCT) N (AAC)
G (GGG) L (CTA) A (GCT) L (TTG) L (CTA) L (CTT) A (GCA) M (ATG) P (CCA) C (TGT) L (CTA) E (GAA) S (AGT) A (GCT) R (AGA) T (ACG) A (GCC) S (AGT) G (GGA) S (AGC) L (CTG) Q (CAG) T (ACT) T (ACC) S (TCC) R (AGG) N (AAT) P (CCA) I (ATT) S (TCA) K (AAA) A (GCC) N (AAT)
LH HL, LB RY LB BF LB LB LB BF NN3, WS, HL, LB BF BF NN3, BF HL, LB WS, HL, LB LH NN3, WS NN3 NN3 WS WS BF WL LB WS WS, HL, LB NN3, WS, HL, LB BW, WL, LH, RY BF NN3, WS, HL, LB LH NN3 NN3, BF, HL
Yes Yes No Yes Yes Yes No No No Yes Yes No No No Yes Yes Yes Yes Yes No No No No No Yes No Yes Yes Yes No Yes Yes Yes
1AA
= amino acid. = Beijing Fatty; BW = Beijing White 939; HL = Hy-Line Brown; LB = Laiwu Black; LH = Luhua; NN3 = Nongda no. 3; RY = Royal; WL = White Leghorn; WS = White-Feather Silky. 2BF
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on June 14, 2015
Figure 2. The structure of chicken toll-like receptor 1 (ChTLR1) type 1 and type 2 predicted results. Line A: transmembrane helix preference. Line B: modified hydrophobic moment index. Line C: beta preference. Gray boxes indicate predicted transmembrane helix position. Analysis tool provided at http://split.pmfst.hr.
POLYMORPHISMS OF CHICKEN TOLL-LIKE RECEPTORS 1
brane of type 1 and the polymorphic site L336P exists in the transmembrane of type 2; this might regulate signaling in response to the ligands for ChTLR1 type 1 and type 2. The variant sites in ChTLR may be involved in the resistance or susceptibility of chickens to infectious diseases. Although we found amino acid variation in T245A, T249M, R359P, Q465E, N471S, T477A, R642Q, and A645T in type 1 and in T27A, K33N, V56I, S83P, Q190E, S196N, T202A, G228S, and R367W in type 2, how these variations affect chicken response to pathogenic infections require further investigation. Among the chicken breeds we used in this study, White Leghorn chickens are well known for their resistance to salmonellosis (Wigley, 2004); however, the cause for the resistance of this breed to salmonellosis is unknown. Because innate immunity is the first line of host defense, it is possible that the allelic variation in ChTLR1 type 1 and type 2 may be connected with host defense. Therefore, we compared the amino acid sequences of ChTLR1 type 1 and type 2 with those of other chicken lines (JF7823968, JF823969, JF823971JF823978, and JF823980-JF823985). We found that there is 1 distinctive polymorphic site (A645T) in ChTLR1 type 1 and 1 distinctive polymorphic site (L275P) in ChTLR1 type 2. The L275P in LRRCT may functionally affect the recognizing ligands in ChTLR1 type 2 because the LRRCT domain is responsible for binding ligands. These findings may provide some clues toward the understanding of the resistance of White Leghorn chickens to salmonellosis. In addition, the polymorphic sites exist in other breeds of chickens. For example, Laiwu Black chickens
Figure 3. Polymorphism in chicken toll-like receptor 1 (ChTLR1) type 1 and type 2 proteins detected in 9 kinds of chicken. Rectangles indicate TLR. Gray boxes indicate putative signal peptides, transmembrane domains, leucine-rich repeats (LRR) and toll–IL-1 receptor (TIR) domains (predicted by SMART service; http://smart.embl-heidelberg.de). Positions of polymorphisms are indicated by the number of amino acids from the start in each protein.
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on June 14, 2015
in the extracellular domain of type 1, including T130A, T245A, T249M, R359P, Q465E, N471S, T477A, and G560S. Five polymorphic sites are located in cytoplasmic domain of type 1, including R642Q, A645T, A650T, K730R, and P749S. One polymorphic site is located in transmembrane domain of type 1 (P611L). Eleven polymorphic sites are located in the extracellular domain of type 2, including T27A, K33N, V56I, S83P, F129L, Q190E, S196N, T220A, G228S, L275P, and G285S. One polymorphic site (R367W) is located in the cytoplasmic domain of type 2. One polymorphic site is located in the transmembrane domain of type 2 (L336P). It was reported that 5 amino acid variants (G225E, E301D, R343K, Y383H, and Q611R) are located within LRR of the extracellular domain of ChTLR4 and are associated with the susceptibility of chickens to salmonellosis (Leveque et al., 2003). Because LRR or LRRCT are the functional domains of recognizing ligands (Jin and Lee, 2008), the variation of amino acids in T130A and G560S in LRR or LRRCT domain of type 1 and F129L, C228S, L275P, and G285S in LRR or LRRCT domain of type 2 might affect the recognition of PAMP by ChTLR1 type 1 and type 2. It was found that a TIR domain variant (D96N) results in loss of MyD88 binding and reduced TLR2/TLR4 signaling (Nagpal et al., 2009). Because TIR is the functional domain of subsequent recruitment of intracellular adapter proteins (Werling et al., 2009), the variation of amino acids in A650T, K730R, and P749S in TIR domain of type 1 might be connected with the signal transduction mediated by ChTLR1 type 1. Similar to a previous study with human TLR1 (Hawn et al., 2007), the polymorphic site P611L also exists in the transmem-
1945
1946
Ruan and Zheng
Table 3. Chicken toll-like receptor 1 type 2 polymorphism details1 Structural characteristic
Majority AA (codon)
Polymorphic AA (codon)
Chicken breed2
Synonymous substitution
24 27 33 34 56 83 122 129 156 172 173 190 196 202 216 224 228 244 262 266 275 276 285 301 315 317 336 355 367 369 436 447 471 492
Leucine-rich repeats Leucine-rich repeats Leucine-rich repeats Leucine-rich repeats Leucine-rich repeats Leucine-rich repeats C terminal leucine-rich repeats C terminal leucine-rich repeats C terminal leucine-rich repeats C terminal leucine-rich repeats C terminal leucine-rich repeats C terminal leucine-rich repeats Transmembrane domain Toll–IL-1 receptor Toll–IL-1 receptor Toll–IL-1 receptor Toll–IL-1 receptor
V (GTC) T (ACT) K (AAA) T (ACA) V (GTT) S (TCT) E (GAA) F (TTC) C (TGC) L (CTG) T (ACA) Q (CAA) S (AGT) T (ACT) R (CGG) R (AGG) C (GGC) A (GCC) P (CCG) S (TCG) L (CTG) E (GAG) G (GGC) T (ACA) T (ACG) L (CTG) L (CTG) T (ACG) R (CGG) T (ACC) S (TCG) Y (TAC) P (CCA) T (ACG)
V (GTT) A (GCT) N (AAC) T (ACT) I (ATT) P (CCT) E (GAG) L (CTC) C (TGT) L (CTA) T (ACG) E (GAA) N (AAT) A (GCT) R (AGG) R (AGA) S (AGC) A (GCT) P (CCA) S (TCA) P (CCG) E (GAA) S (AGC) T (ACT) T (ACA) L (TTG) P (CCG) T (ACA) W (TGG) T (ACT) S (TCC) Y (TAT) P (CCG) T (ACC)
NN3, LB HL BF, HL BF, HL BW, RY HL BF, HL BF NN3, BF, HL, LB WL HL WS HL HL BF HL NN3, LB WL, HL, RY BF BW, BF, LH WL BF WS WS, HL BW, LH BF, WS HL, RY WL, RY LB NN3, LB BF, WS LH BF WL, BF, NN3, LB
Yes No No Yes No No Yes No Yes Yes Yes No No No Yes Yes No Yes Yes Yes No Yes No Yes Yes Yes No Yes No Yes Yes Yes Yes Yes
1AA
= amino acid. = Beijing Fatty; BW = Beijing White 939; HL = Hy-Line Brown; LB = Laiwu Black; LH = Luhua; NN3 = Nongda no. 3; RY = Royal; WL = White Leghorn; WS = White-Feather Silky. 2BF
have distinctive polymorphic sites in T245A, T249M, and A650T in ChTLR1 type 1 and R367W in ChTLR1 type 2. Beijing Fatty chickens have distinctive polymorphic sites in R359P, Q465E, and R642Q in ChTLR1 type 1 and F129L in ChTLR1 type 2. White-Feather Silky chickens have distinctive polymorphic sites in G560S and P611L in ChTLR1 type 1 and Q190E and G285S in ChTLR1 type 2. Royal chickens have distinctive polymorphic sites in T130A in ChTLR1 type 1. HyLine Brown chickens have distinctive polymorphic sites in T27A, S83P, S196N, and T202A in ChTLR1 type 2. Chinese native Nongda no. 3 chickens and Beijing Fatty chickens carry identical allelic variation sites in N471S in ChTLR1 type 1. Nongda no. 3 chickens and Laiwu Black chickens carry identical allelic variation sites in C228S in ChTLR1 type 2. Furthermore, it was found that each allelic variation site in both TLR1 type 1 and 2 switched between only 2 amino acids; for example, at 749 site in ChTLR1 type 1, the Pro changed to Ser and vice versa. These data demonstrated that the heterogeneity of chicken TLR proteins of various breeds was preserved in chicken breeds associated with poultry improvement (Leveque et al., 2003; Wigley, 2004).
In conclusion, we investigated polymorphic sites of ChTLR1 type 1 and type 2 among various breeds of chickens and demonstrated their variation of amino acids within the ChTLR1 type 1 and type 2 proteins. Based on TLR1 polymorphism and its correlation with the susceptibility of humans and mice to bacterial infections, we propose that ChTLR1 type 1 and type 2 polymorphisms may be associated with the resistance or susceptibility of chickens to infectious diseases. This information may help to further understanding of varied resistance among the breeds of chickens to diseases.
ACKNOWLEDGMENTS We thank the Earmarked Fund for Modern Agro-Industry Technology Research System (#NYCYTX-41, Beijing, China) National Natural Science Foundation of China (no. 30725026 and 31072117, Beijing, China) for financial support.
REFERENCES Beutler, B. 2005. The toll-like receptors: Analysis by forward genetic methods. Immunogenetics 57:385–392.
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on June 14, 2015
Position (AA)
POLYMORPHISMS OF CHICKEN TOLL-LIKE RECEPTORS 1
Misch, E. A., and T. R. Hawn. 2008. Toll-like receptor polymorphisms and susceptibility to human disease. Clin. Sci. (Lond.) 114:347–360. Nagpal, K., T. S. Plantinga, J. Wong, B. G. Monks, N. J. Gay, M. G. Netea, K. A. Fitzgerald, and D. T. Golenbock. 2009. A TIR domain variant of MyD88 adapter-like (Mal)/TIRAP results in loss of MyD88 binding and reduced TLR2/TLR4 signaling. J. Biol. Chem. 284:25742–25748. Sales, M. L., R. Schreiber, M. C. Ferreira-Sae, M. N. Fernandes, C. S. Piveta, J. A. Cipolli, C. C. Cardoso, J. R. Matos-Souza, B. Geloneze, K. G. Franchini, and W. Nadruz Jr. 2010. Toll-like receptor 6 Ser249Pro polymorphism is associated with lower left ventricular wall thickness and inflammatory response in hypertensive women. Am. J. Hypertens. 23:649–654. Takeuchi, O., S. Sato, T. Horiuchi, K. Hoshino, K. Takeda, Z. Dong, R. L. Modlin, and S. Akira. 2002. Cutting edge: Role of toll-like receptor 1 in mediating immune response to microbial lipoproteins. J. Immunol. 169:10–14. Temperley, N. D., S. Berlin, I. R. Paton, D. K. Griffin, and D. W. Burt. 2008. Evolution of the chicken toll-like receptor gene family: A story of gene gain and gene loss. BMC Genomics 9:62. Vandewalle, A. 2008. Toll-like receptors and renal bacterial infections. Chang Gung Med. J. 31:525–537. Verstak, B., K. Nagpal, S. P. Bottomley, D. T. Golenbock, P. J. Hertzog, and A. Mansell. 2009. MyD88 adapter-like (Mal)/TIRAP interaction with TRAF6 is critical for TLR2- and TLR4mediated NF-κB proinflammatory responses. J. Biol. Chem. 284:24192–24203. Werling, D., O. C. Jann, V. Offord, E. J. Glass, and T. J. Coffey. 2009. Variation matters: TLR structure and species-specific pathogen recognition. Trends Immunol. 30:124–130. Wigley, P. 2004. Genetic resistance to Salmonella infection in domestic animals. Res. Vet. Sci. 76:165–169. Wurfel, M. M., A. C. Gordon, T. D. Holden, F. Radella, J. Strout, O. Kajikawa, J. T. Ruzinski, G. Rona, R. A. Black, S. Stratton, G. P. Jarvik, A. M. Hajjar, D. A. Nickerson, M. Rieder, J. Sevransky, J. P. Maloney, M. Moss, G. Martin, C. Shanholtz, J. G. Garcia, L. Gao, R. Brower, K. C. Barnes, K. R. Walley, J. A. Russell, and T. R. Martin. 2008. Toll-like receptor 1 polymorphisms affect innate immune responses and outcomes in sepsis. Am. J. Respir. Crit. Care Med. 178:710–720. Yilmaz, A., S. Shen, D. L. Adelson, S. Xavier, and J. J. Zhu. 2005. Identification and sequence analysis of chicken toll-like receptors. Immunogenetics 56:743–753.
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on June 14, 2015
Beutler, B., and M. Rehli. 2002. Evolution of the TIR, tolls and TLRs: Functional inferences from computational biology. Curr. Top. Microbiol. Immunol. 270:1–21. Buwitt-Beckmann, U., H. Heine, K. H. Wiesmuller, G. Jung, R. Brock, S. Akira, and A. J. Ulmer. 2006. TLR1- and TLR6-independent recognition of bacterial lipopeptides. J. Biol. Chem. 281:9049–9057. Fukui, A., N. Inoue, M. Matsumoto, M. Nomura, K. Yamada, Y. Matsuda, K. Toyoshima, and T. Seya. 2001. Molecular cloning and functional characterization of chicken toll-like receptors. A single chicken toll covers multiple molecular patterns. J. Biol. Chem. 276:47143–47149. Hawn, T. R., E. A. Misch, S. J. Dunstan, G. E. Thwaites, N. T. Lan, H. T. Quy, T. T. Chau, S. Rodrigues, A. Nachman, M. Janer, T. T. Hien, J. J. Farrar, and A. Aderem. 2007. A common human TLR1 polymorphism regulates the innate immune response to lipopeptides. Eur. J. Immunol. 37:2280–2289. Hawn, T. R., D. Scholes, S. S. Li, H. Wang, Y. Yang, P. L. Roberts, A. E. Stapleton, M. Janer, A. Aderem, W. E. Stamm, L. P. Zhao, and T. M. Hooton. 2009. Toll-like receptor polymorphisms and susceptibility to urinary tract infections in adult women. PLoS ONE 4:e5990. Higuchi, M., A. Matsuo, M. Shingai, K. Shida, A. Ishii, K. Funami, Y. Suzuki, H. Oshiumi, M. Matsumoto, and T. Seya. 2008. Combinational recognition of bacterial lipoproteins and peptidoglycan by chicken toll-like receptor 2 subfamily. Dev. Comp. Immunol. 32:147–155. Jin, M. S., S. E. Kim, J. Y. Heo, M. E. Lee, H. M. Kim, S. G. Paik, H. Lee, and J. O. Lee. 2007. Crystal structure of the TLR1– TLR2 heterodimer induced by binding of a tri-acylated lipopeptide. Cell 130:1071–1082. Jin, M. S., and J. O. Lee. 2008. Structures of the toll-like receptor family and its ligand complexes. Immunity 29:182–191. Johnson, C. M., E. A. Lyle, K. O. Omueti, V. A. Stepensky, O. Yegin, E. Alpsoy, L. Hamann, R. R. Schumann, and R. I. Tapping. 2007. Cutting edge: A common polymorphism impairs cell surface trafficking and functional responses of TLR1 but protects against leprosy. J. Immunol. 178:7520–7524. Keestra, A. M., M. R. de Zoete, R. A. van Aubel, and J. P. van Putten. 2007. The central leucine-rich repeat region of chicken TLR16 dictates unique ligand specificity and species-specific interaction with TLR2. J. Immunol. 178:7110–7119. Leveque, G., V. Forgetta, S. Morroll, A. L. Smith, N. Bumstead, P. Barrow, J. C. Loredo-Osti, K. Morgan, and D. Malo. 2003. Allelic variation in TLR4 is linked to susceptibility to Salmonella enterica serovar Typhimurium infection in chickens. Infect. Immun. 71:1116–1124.
1947
Key words: chicken, leucine-rich repeat, polymorphism, toll–interleukin-1 receptor, toll-like receptor 2011 Poultry Science 90:1941–1947 doi:10.3382/ps.2011-01489
INTRODUCTION Infectious diseases are major threats to the modern poultry industry. In addition to viral diseases, bacterial diseases such as salmonellosis, colibacillosis, and fowl cholera, which are associated with causative organisms, lead to heavy economic losses (Leveque et al., 2003). The causative agents for these diseases, such as Salmonella, Escherichia coli, and Pasteurella multocida, are resident opportunistic microorganisms that are difficult to eradicate from flocks. Therefore, the genetic improvement chickens’ resistance to these opportunistic pathogens may be considered as an alternative control measure against these bacterial infections. Toll-like receptors (TLR) play a crucial role in host immune response via recognition of pathogen-associated molecular patterns (PAMP), such as lipopolysaccharides, lipopeptides, flagellins, double-stranded RNA, or CpG DNA motifs. So far, at least 10 members of the chicken TLR family have been identified, includ-
©2011 Poultry Science Association Inc. Received March 17, 2011. Accepted May 8, 2011. 1 Corresponding author: [email protected]
ing TLR1 type 1 and type 2, TLR2 type 1 and type 2, TLR3, TLR4, TLR5, TLR7, TLR15, and TLR21 (Fukui et al., 2001; Yilmaz et al., 2005; Temperley et al., 2008). Because the amino acid sequences of 2 identified chicken TLR (ChTLR) are almost equally homologous to sequences of human TLR1 and TLR6, it is difficult to determine which of these 2 receptors is ChTLR1 or ChTLR6. Thus, they were named ChTLR1 type 1 (long form) and ChTLR1 type 2 (short form; Yilmaz et al., 2005). Chicken TLR1 type 1 and type 2 are expressed in almost all tissues (Yilmaz et al., 2005). The ChTLR1 cluster is located on chromosome 4 and is believed to result from gene duplication (Beutler and Rehli, 2002). In humans, TLR1 interacts with TLR2 in a heterodimeric complex, which increases their capability to recognize ligands, such as lipids or proteins of extracellular pathogens as well as several synthetic peptides such as N-palmitoyl-S-dipalmitoylglyceryl and Cys-Ser-(Lys)4 (Takeuchi et al., 2002; Buwitt-Beckmann et al., 2006; Jin et al., 2007; Jin and Lee, 2008). In addition, TLR1 family cluster might be involved in the recognition of other unknown ligands (Jin and Lee, 2008). A few studies focused on the identification of ChTLR1 type 1 and type 2 (Yilmaz et al., 2005; Keestra et al., 2007; Higuchi et al., 2008), and it was found that ChTLR1 type 1 and type 2 cooperate with ChTLR2 to recognize pepti-
1941
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on June 14, 2015
ABSTRACT Toll-like receptors (TLR) mediate immune responses via recognition of pathogen-associated molecular patterns, thus playing important roles in the host defense. Polymorphisms of TLR may affect their recognition of pathogen-associated molecular patterns, leading to varied host resistance to pathogenic infections. However, little is known regarding the polymorphisms of chicken TLR (ChTLR) among breeds. In this study, we cloned ChTLR1 type 1 and type 2 genes from 9 chicken breeds (2 commercial breeds and 7 Chinese native breeds) and analyzed their sequences. We found
1942
Ruan and Zheng
MATERIALS AND METHODS Source of Chicken Breeds Chickens used in this study were obtained from commercial breeders and were from the same genetic background. Nine breeds were chosen (see Introduction). Four individual repeats were arranged in each breed.
Molecular Cloning of Chicken TLR1 Type 1 and Type 2 The total RNA was obtained from the spleen using TRIzol (Invitrogen Corp., Carlsbad, CA) per the manufacturer’s instructions and was used for cDNA synthesis. The 20 μL of reaction mixture contained 5 μg of total RNA, 0.5 μg of oligo dT primer (16–18 mer), 40 U of RNasin, 1,000 μM of deoxyribonucleotide triphosphate mix, 10 mM of dithiothreitol, and 5 U of M-MLV reverse transcriptase (Promega, Madison, WI) in 1× reverse transcriptase buffer. The reaction mixture was incubated at 37°C for 1 h. The cDNA synthesis was confirmed by amplifying the β-actin amplicon in PCR. One pair of primers used to amplify the full open reading frame was designed based on the consensus TLR1 mRNA sequences of Gallus gallus TLR1LA (NM_001007488.3) and Gallus gallus TLR1LB (NM_001081709.2) (type 1 primer: forward, 5′-GGATCCCTTACAAGTATCTACGTTT-3′; reverse, 5′-CACTTAATCAGACGTTGGGTTATAC-3′; type 2 primer: forward, 5′-TACTTCAGGTTGTATGGCACTCA-3′; reverse, 5′-CAAGGAGAAGAAGAAACTGCATTA-3′). The ChTLR1 type 1 and type 2 genes were amplified in PCR using the synthesized cDNA. The reaction conditions for amplification in PCR were the same for both types. The 25 μL of PCR reaction contained 50 pmol for each forward and reverse primer, 2 μL of Template cDNA, 200 μM each of deoxyribonucleotide triphosphate mix, and 2.5 U of Pfu DNA polymerase (Promega) in 1× Pfu DNA polymerase buffer. Amplification conditions were as follows: initial denaturation at 94°C for 2 min; 35 cycles at 94°C for 30 s, annealing at 56 to 60°C for 30 s, and extension at 72°C for 5 min; and a final extension at 72°C for 10 min. The PCR amplicons verified by 1% agarose gel electrophoresis were purified and ligated into pEASY-Blunt simple cloning vector (TransGen, Beijing, China) following the manufacturer’s instructions. Recombinant plasmids were characterized by PCR using gene-specific and vector primer pairs. The ChTLR1 type 1 and type 2 gene inserts in recombinant plasmids were sequenced from both ends by ABI 3730XL sequencer from SinoGenoMax Co. Ltd. (Beijing, China).
Sequence Analysis Nucleotide and amino acid sequences of ChTLR type 1 and type 2 were aligned with the analysis software MegAlign (DNAstar Inc., Madison, WI). The extracellular, transmembrane, and cytoplasmic domains of these protein sequences were detected with the analysis tools provided at http://smart.embl-heidelberg.de and http://split.pmfst.hr.
Nucleotide Sequence Accession Numbers The coding sequences for the avian TLR1 type 1 and type 2 have been deposited in GenBank. The acces-
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on June 14, 2015
doglycan and lipoprotein with high efficiency (Higuchi et al., 2008). Polymorphisms of TLR may profoundly influence the response of a host to a wide range of pathogens and are associated with resistance and susceptibility to diseases (Hawn et al., 2007, 2009; Misch and Hawn, 2008). The TLR1 gene family cluster including TLR1, TLR6, and TLR10 is associated with the susceptibility of bacterial infections in human and mice. It was found that variation in the inflammatory responses to bacterial lipopeptides is regulated by a common TLR1 transmembrane domain polymorphism that could potentially affect the innate immune response and clinical susceptibility to a wide spectrum of pathogens (Hawn et al., 2007). Consistently, the hypermorphic genetic variation in TLR1 is associated with increased susceptibility to organ dysfunction, death, and gram-positive bacterial infections in sepsis (Wurfel et al., 2008). In addition, human TLR1 I602S variant is associated with a decreased incidence of leprosy (Johnson et al., 2007) and the response to N-palmitoyl-S-dipalmitoylglyceryl, a synthetic ligand for TLR1 (Hawn et al., 2007; Misch and Hawn, 2008). Furthermore, human TLR6 S249P polymorphism was associated with lower left ventricular wall thickness and inflammatory response in hypertensive women (Sales et al., 2010). These findings suggest that TLR polymorphisms are associated with host susceptibility to diseases. Although it is well known that the susceptibility of chickens with different genetic backgrounds to pathogenic infection varies, little is known regarding the genetic factors contributing to the susceptibility of chickens to diseases. Based on the above information and our clinical observations, we hypothesize that ChTLR1 type 1 and type 2 genes may have polymorphisms that affect chicken susceptibility. In this study, we used 2 commercial breeds (White Leghorn chicken and Hy-Line Brown chicken, which was used for eggs) and 7 Chinese native breeds (Beijing White 939 chicken, which was used for brown eggs; Nongda no. 3 chicken, which is a dwarf and was used for eggs; Luhua chicken, which was raised for meat and eggs; White-Feather Silky chicken, which had the characteristics of white feathers and black bones, used for dual purposes; and Beijing Fatty chicken, Royal chicken, and Laiwu Black chicken, which were characterized by their shape and good meat quality). We cloned and sequenced the full length of ChTLR1 type 1 and type 2 genes from different breeds of chickens with an objective to understand the genetic contributions to the polymorphisms of ChTLR1 type 1 and type 2.
1943
POLYMORPHISMS OF CHICKEN TOLL-LIKE RECEPTORS 1
Figure 1. Chicken toll-like receptor TLR (ChTLR) type 1 and type 2 structures. Dark rectangles represent the transmembrane domain and gray bands indicate leucine-rich repeats (LRR) involved in pathogen-associated molecular pattern recognition. Gray circles represent C terminal LRR (CTLRR). TIR = toll–IL-1 receptor. Analysis tool provided at http://smart.embl-heidelberg.de.
RESULTS AND DISCUSSION Chicken TLR1 type 1 and type 2 are located on chromosome 4 71561343–71566597 and 513–2199 (Table 1). The full-length open reading frame of type 1 contains 2,457 nucleotides encoding 818 amino acids, and type 2 contains 1,590 nucleotides encoding 516 amino acids. The predicted results also show that 593 amino acids of type 1 and 317 amino acids of type 2 are located in the extracellular domain whereas 177 amino acids of type 1 and type 2 are located in the cytoplasmic domain. Using analysis software, we predicted that ChTLR type 1 and type 2 are type I transmembrane proteins, with a large extracellular domain comprising leucine-rich repeats (LRR) flanked by C-terminal cap motifs, a transmembrane domain, and a cytoplasmic toll–IL-1 receptor (TIR) domain (Figure 1). Similar to human or mouse LRR, the LRR of chickens are motifs of 20 to 30 amino acids in length that fold into a horseshoe shape and are involved in recognition of PAMP, mostly via interaction with other proteins (Jin and Lee, 2008; Werling et al., 2009). The TIR domain is similar to that of the IL-1 receptor, which is highly conserved and interacts with adapter proteins such as
myeloid differentiation factor (MyD88) and TIR domain containing adaptor protein (TIRAP, also known as Mal; Verstak et al., 2009). Ligand binding triggers homo- or heterodimerization of TLR and subsequent recruitment of intracellular adapter proteins (Higuchi et al., 2008; Jin and Lee, 2008). Multiple intermediates such as tumor necrosis factor receptor associated factor 6, IL-1 receptor-associated kinase 4 family proteins, Ik-kinase family proteins, mitogen-activated protein kinase, and transcription factors including nuclear factor κB family proteins are involved in TLR-mediated signal transduction, leading to the expression of inflammatory cytokines, chemokines, and adhesion molecules (Beutler, 2005; Vandewalle, 2008). The structural analysis of ChTLR1 type 1 reveals that it contains 5 LRR motifs, 1 C terminal LRR (LRRCT), 2 low compositional complexities, 1 transmembrane domain, and 1 TIR domain. The ChTLR1 type 2 structure includes 3 LRR, 1 LRRCT, 1 low compositional complexity, 1 transmembrane domain, and 1 TIR domain (Figure 1). It also shows transmembrane helix preference, β preference, and modified hydrophobic moment index as determined by online software SPLIT server (http://split. pmfst.hr; Figure 2). There is intensive hydrophobicity in ChTLR1 type 1 structures in the N terminal, which is predicted as a signal peptide domain, and other intensive hydrophobicity in the middle, which is predicted as a transmembrane domain. The sequences of ChTLR type 1 and type 2 from the 9 breeds showed varied sequence patterns (Tables 2 and 3). The amino acid sequence analysis of ChTLR1 type 1 and type 2 from 9 chicken breeds indicates that there are 27 sites of amino acid sequence with varied amino acids (Figure 3), including 14 sites in type 1 and 13 sites in type 2. Eight polymorphic sites are located
Table 1. Chicken toll-like receptor 1 (ChTLR1) type 1 and type 2 details1 Name
Chromosome
Location
ChTLR1 type 1 ChTLR1 type 2
4 4
71561343–71566597 513–2199
1AA
= amino acids.
Length (nucleotides)
Length (AA)
Extracellular AA
Cytoplasmic AA
2,457 1,590
818 516
593 317
177 177
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on June 14, 2015
sion numbers for type 1 and type 2 are, respectively, JF7823968 and JF7823977 (Royal chicken), JF823969 and JF823978 (Beijing White 939 chicken), JF823970 and JF823979 (White Leghorn chicken), JF823971 and JF823980 (Laiwu Black chicken), JF823972 and JF823981 (Hy-Line Brown chicken), JF823973 and JF823982 (Luhua chicken), JF823974 and JF823983 (Nongda no. 3 chicken), JF823975 and JF823984 (White-Feather Silky chicken), and JF823976 and JF823985 (Beijing Fatty chicken).
1944
Ruan and Zheng
Table 2. Chicken toll-like receptor 1 type 1 polymorphism details1 Position (AA)
Structural characteristic
Majority AA (codon)
Polymorphic AA (codon)
Chicken breed2
Synonymous substitution
36 38 130 144 149 207 245 249 359 431 447 465 471 477 499 502 519 527 556 560 611 642 645 650 711 730 735 746 747 749 760 761 790
Leucine-rich repeats Leucine-rich repeats Leucine-rich repeats Leucine-rich repeats C terminal leucine-rich repeats C terminal leucine-rich repeats Transmembrane domain Toll–IL-1 receptor Toll–IL-1 receptor Toll–IL-1 receptor Toll–IL-1 receptor Toll–IL-1 receptor Toll–IL-1 receptor Toll–IL-1 receptor Toll–IL-1 receptor Toll–IL-1 receptor Toll–IL-1 receptor
G (GGA) L (TTA) T (ACT) L (TTA) L (TTA) L (CTC) T (ACA) T (ACG) R (CGA) C (TGC) L (CTG) Q (CAA) N (AAT) T (ACT) R (AGG) T (ACA) A (GCT) S (AGC) G (GGC) G (GGC) P (CCG) R (CGG) A (GCT) A (GCC) S (TCG) K (AAG) N (AAC) P (CCG) I (ATC) P (CCA) K (AAG) A (GCT) N (AAC)
G (GGG) L (CTA) A (GCT) L (TTG) L (CTA) L (CTT) A (GCA) M (ATG) P (CCA) C (TGT) L (CTA) E (GAA) S (AGT) A (GCT) R (AGA) T (ACG) A (GCC) S (AGT) G (GGA) S (AGC) L (CTG) Q (CAG) T (ACT) T (ACC) S (TCC) R (AGG) N (AAT) P (CCA) I (ATT) S (TCA) K (AAA) A (GCC) N (AAT)
LH HL, LB RY LB BF LB LB LB BF NN3, WS, HL, LB BF BF NN3, BF HL, LB WS, HL, LB LH NN3, WS NN3 NN3 WS WS BF WL LB WS WS, HL, LB NN3, WS, HL, LB BW, WL, LH, RY BF NN3, WS, HL, LB LH NN3 NN3, BF, HL
Yes Yes No Yes Yes Yes No No No Yes Yes No No No Yes Yes Yes Yes Yes No No No No No Yes No Yes Yes Yes No Yes Yes Yes
1AA
= amino acid. = Beijing Fatty; BW = Beijing White 939; HL = Hy-Line Brown; LB = Laiwu Black; LH = Luhua; NN3 = Nongda no. 3; RY = Royal; WL = White Leghorn; WS = White-Feather Silky. 2BF
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on June 14, 2015
Figure 2. The structure of chicken toll-like receptor 1 (ChTLR1) type 1 and type 2 predicted results. Line A: transmembrane helix preference. Line B: modified hydrophobic moment index. Line C: beta preference. Gray boxes indicate predicted transmembrane helix position. Analysis tool provided at http://split.pmfst.hr.
POLYMORPHISMS OF CHICKEN TOLL-LIKE RECEPTORS 1
brane of type 1 and the polymorphic site L336P exists in the transmembrane of type 2; this might regulate signaling in response to the ligands for ChTLR1 type 1 and type 2. The variant sites in ChTLR may be involved in the resistance or susceptibility of chickens to infectious diseases. Although we found amino acid variation in T245A, T249M, R359P, Q465E, N471S, T477A, R642Q, and A645T in type 1 and in T27A, K33N, V56I, S83P, Q190E, S196N, T202A, G228S, and R367W in type 2, how these variations affect chicken response to pathogenic infections require further investigation. Among the chicken breeds we used in this study, White Leghorn chickens are well known for their resistance to salmonellosis (Wigley, 2004); however, the cause for the resistance of this breed to salmonellosis is unknown. Because innate immunity is the first line of host defense, it is possible that the allelic variation in ChTLR1 type 1 and type 2 may be connected with host defense. Therefore, we compared the amino acid sequences of ChTLR1 type 1 and type 2 with those of other chicken lines (JF7823968, JF823969, JF823971JF823978, and JF823980-JF823985). We found that there is 1 distinctive polymorphic site (A645T) in ChTLR1 type 1 and 1 distinctive polymorphic site (L275P) in ChTLR1 type 2. The L275P in LRRCT may functionally affect the recognizing ligands in ChTLR1 type 2 because the LRRCT domain is responsible for binding ligands. These findings may provide some clues toward the understanding of the resistance of White Leghorn chickens to salmonellosis. In addition, the polymorphic sites exist in other breeds of chickens. For example, Laiwu Black chickens
Figure 3. Polymorphism in chicken toll-like receptor 1 (ChTLR1) type 1 and type 2 proteins detected in 9 kinds of chicken. Rectangles indicate TLR. Gray boxes indicate putative signal peptides, transmembrane domains, leucine-rich repeats (LRR) and toll–IL-1 receptor (TIR) domains (predicted by SMART service; http://smart.embl-heidelberg.de). Positions of polymorphisms are indicated by the number of amino acids from the start in each protein.
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on June 14, 2015
in the extracellular domain of type 1, including T130A, T245A, T249M, R359P, Q465E, N471S, T477A, and G560S. Five polymorphic sites are located in cytoplasmic domain of type 1, including R642Q, A645T, A650T, K730R, and P749S. One polymorphic site is located in transmembrane domain of type 1 (P611L). Eleven polymorphic sites are located in the extracellular domain of type 2, including T27A, K33N, V56I, S83P, F129L, Q190E, S196N, T220A, G228S, L275P, and G285S. One polymorphic site (R367W) is located in the cytoplasmic domain of type 2. One polymorphic site is located in the transmembrane domain of type 2 (L336P). It was reported that 5 amino acid variants (G225E, E301D, R343K, Y383H, and Q611R) are located within LRR of the extracellular domain of ChTLR4 and are associated with the susceptibility of chickens to salmonellosis (Leveque et al., 2003). Because LRR or LRRCT are the functional domains of recognizing ligands (Jin and Lee, 2008), the variation of amino acids in T130A and G560S in LRR or LRRCT domain of type 1 and F129L, C228S, L275P, and G285S in LRR or LRRCT domain of type 2 might affect the recognition of PAMP by ChTLR1 type 1 and type 2. It was found that a TIR domain variant (D96N) results in loss of MyD88 binding and reduced TLR2/TLR4 signaling (Nagpal et al., 2009). Because TIR is the functional domain of subsequent recruitment of intracellular adapter proteins (Werling et al., 2009), the variation of amino acids in A650T, K730R, and P749S in TIR domain of type 1 might be connected with the signal transduction mediated by ChTLR1 type 1. Similar to a previous study with human TLR1 (Hawn et al., 2007), the polymorphic site P611L also exists in the transmem-
1945
1946
Ruan and Zheng
Table 3. Chicken toll-like receptor 1 type 2 polymorphism details1 Structural characteristic
Majority AA (codon)
Polymorphic AA (codon)
Chicken breed2
Synonymous substitution
24 27 33 34 56 83 122 129 156 172 173 190 196 202 216 224 228 244 262 266 275 276 285 301 315 317 336 355 367 369 436 447 471 492
Leucine-rich repeats Leucine-rich repeats Leucine-rich repeats Leucine-rich repeats Leucine-rich repeats Leucine-rich repeats C terminal leucine-rich repeats C terminal leucine-rich repeats C terminal leucine-rich repeats C terminal leucine-rich repeats C terminal leucine-rich repeats C terminal leucine-rich repeats Transmembrane domain Toll–IL-1 receptor Toll–IL-1 receptor Toll–IL-1 receptor Toll–IL-1 receptor
V (GTC) T (ACT) K (AAA) T (ACA) V (GTT) S (TCT) E (GAA) F (TTC) C (TGC) L (CTG) T (ACA) Q (CAA) S (AGT) T (ACT) R (CGG) R (AGG) C (GGC) A (GCC) P (CCG) S (TCG) L (CTG) E (GAG) G (GGC) T (ACA) T (ACG) L (CTG) L (CTG) T (ACG) R (CGG) T (ACC) S (TCG) Y (TAC) P (CCA) T (ACG)
V (GTT) A (GCT) N (AAC) T (ACT) I (ATT) P (CCT) E (GAG) L (CTC) C (TGT) L (CTA) T (ACG) E (GAA) N (AAT) A (GCT) R (AGG) R (AGA) S (AGC) A (GCT) P (CCA) S (TCA) P (CCG) E (GAA) S (AGC) T (ACT) T (ACA) L (TTG) P (CCG) T (ACA) W (TGG) T (ACT) S (TCC) Y (TAT) P (CCG) T (ACC)
NN3, LB HL BF, HL BF, HL BW, RY HL BF, HL BF NN3, BF, HL, LB WL HL WS HL HL BF HL NN3, LB WL, HL, RY BF BW, BF, LH WL BF WS WS, HL BW, LH BF, WS HL, RY WL, RY LB NN3, LB BF, WS LH BF WL, BF, NN3, LB
Yes No No Yes No No Yes No Yes Yes Yes No No No Yes Yes No Yes Yes Yes No Yes No Yes Yes Yes No Yes No Yes Yes Yes Yes Yes
1AA
= amino acid. = Beijing Fatty; BW = Beijing White 939; HL = Hy-Line Brown; LB = Laiwu Black; LH = Luhua; NN3 = Nongda no. 3; RY = Royal; WL = White Leghorn; WS = White-Feather Silky. 2BF
have distinctive polymorphic sites in T245A, T249M, and A650T in ChTLR1 type 1 and R367W in ChTLR1 type 2. Beijing Fatty chickens have distinctive polymorphic sites in R359P, Q465E, and R642Q in ChTLR1 type 1 and F129L in ChTLR1 type 2. White-Feather Silky chickens have distinctive polymorphic sites in G560S and P611L in ChTLR1 type 1 and Q190E and G285S in ChTLR1 type 2. Royal chickens have distinctive polymorphic sites in T130A in ChTLR1 type 1. HyLine Brown chickens have distinctive polymorphic sites in T27A, S83P, S196N, and T202A in ChTLR1 type 2. Chinese native Nongda no. 3 chickens and Beijing Fatty chickens carry identical allelic variation sites in N471S in ChTLR1 type 1. Nongda no. 3 chickens and Laiwu Black chickens carry identical allelic variation sites in C228S in ChTLR1 type 2. Furthermore, it was found that each allelic variation site in both TLR1 type 1 and 2 switched between only 2 amino acids; for example, at 749 site in ChTLR1 type 1, the Pro changed to Ser and vice versa. These data demonstrated that the heterogeneity of chicken TLR proteins of various breeds was preserved in chicken breeds associated with poultry improvement (Leveque et al., 2003; Wigley, 2004).
In conclusion, we investigated polymorphic sites of ChTLR1 type 1 and type 2 among various breeds of chickens and demonstrated their variation of amino acids within the ChTLR1 type 1 and type 2 proteins. Based on TLR1 polymorphism and its correlation with the susceptibility of humans and mice to bacterial infections, we propose that ChTLR1 type 1 and type 2 polymorphisms may be associated with the resistance or susceptibility of chickens to infectious diseases. This information may help to further understanding of varied resistance among the breeds of chickens to diseases.
ACKNOWLEDGMENTS We thank the Earmarked Fund for Modern Agro-Industry Technology Research System (#NYCYTX-41, Beijing, China) National Natural Science Foundation of China (no. 30725026 and 31072117, Beijing, China) for financial support.
REFERENCES Beutler, B. 2005. The toll-like receptors: Analysis by forward genetic methods. Immunogenetics 57:385–392.
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on June 14, 2015
Position (AA)
POLYMORPHISMS OF CHICKEN TOLL-LIKE RECEPTORS 1
Misch, E. A., and T. R. Hawn. 2008. Toll-like receptor polymorphisms and susceptibility to human disease. Clin. Sci. (Lond.) 114:347–360. Nagpal, K., T. S. Plantinga, J. Wong, B. G. Monks, N. J. Gay, M. G. Netea, K. A. Fitzgerald, and D. T. Golenbock. 2009. A TIR domain variant of MyD88 adapter-like (Mal)/TIRAP results in loss of MyD88 binding and reduced TLR2/TLR4 signaling. J. Biol. Chem. 284:25742–25748. Sales, M. L., R. Schreiber, M. C. Ferreira-Sae, M. N. Fernandes, C. S. Piveta, J. A. Cipolli, C. C. Cardoso, J. R. Matos-Souza, B. Geloneze, K. G. Franchini, and W. Nadruz Jr. 2010. Toll-like receptor 6 Ser249Pro polymorphism is associated with lower left ventricular wall thickness and inflammatory response in hypertensive women. Am. J. Hypertens. 23:649–654. Takeuchi, O., S. Sato, T. Horiuchi, K. Hoshino, K. Takeda, Z. Dong, R. L. Modlin, and S. Akira. 2002. Cutting edge: Role of toll-like receptor 1 in mediating immune response to microbial lipoproteins. J. Immunol. 169:10–14. Temperley, N. D., S. Berlin, I. R. Paton, D. K. Griffin, and D. W. Burt. 2008. Evolution of the chicken toll-like receptor gene family: A story of gene gain and gene loss. BMC Genomics 9:62. Vandewalle, A. 2008. Toll-like receptors and renal bacterial infections. Chang Gung Med. J. 31:525–537. Verstak, B., K. Nagpal, S. P. Bottomley, D. T. Golenbock, P. J. Hertzog, and A. Mansell. 2009. MyD88 adapter-like (Mal)/TIRAP interaction with TRAF6 is critical for TLR2- and TLR4mediated NF-κB proinflammatory responses. J. Biol. Chem. 284:24192–24203. Werling, D., O. C. Jann, V. Offord, E. J. Glass, and T. J. Coffey. 2009. Variation matters: TLR structure and species-specific pathogen recognition. Trends Immunol. 30:124–130. Wigley, P. 2004. Genetic resistance to Salmonella infection in domestic animals. Res. Vet. Sci. 76:165–169. Wurfel, M. M., A. C. Gordon, T. D. Holden, F. Radella, J. Strout, O. Kajikawa, J. T. Ruzinski, G. Rona, R. A. Black, S. Stratton, G. P. Jarvik, A. M. Hajjar, D. A. Nickerson, M. Rieder, J. Sevransky, J. P. Maloney, M. Moss, G. Martin, C. Shanholtz, J. G. Garcia, L. Gao, R. Brower, K. C. Barnes, K. R. Walley, J. A. Russell, and T. R. Martin. 2008. Toll-like receptor 1 polymorphisms affect innate immune responses and outcomes in sepsis. Am. J. Respir. Crit. Care Med. 178:710–720. Yilmaz, A., S. Shen, D. L. Adelson, S. Xavier, and J. J. Zhu. 2005. Identification and sequence analysis of chicken toll-like receptors. Immunogenetics 56:743–753.
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on June 14, 2015
Beutler, B., and M. Rehli. 2002. Evolution of the TIR, tolls and TLRs: Functional inferences from computational biology. Curr. Top. Microbiol. Immunol. 270:1–21. Buwitt-Beckmann, U., H. Heine, K. H. Wiesmuller, G. Jung, R. Brock, S. Akira, and A. J. Ulmer. 2006. TLR1- and TLR6-independent recognition of bacterial lipopeptides. J. Biol. Chem. 281:9049–9057. Fukui, A., N. Inoue, M. Matsumoto, M. Nomura, K. Yamada, Y. Matsuda, K. Toyoshima, and T. Seya. 2001. Molecular cloning and functional characterization of chicken toll-like receptors. A single chicken toll covers multiple molecular patterns. J. Biol. Chem. 276:47143–47149. Hawn, T. R., E. A. Misch, S. J. Dunstan, G. E. Thwaites, N. T. Lan, H. T. Quy, T. T. Chau, S. Rodrigues, A. Nachman, M. Janer, T. T. Hien, J. J. Farrar, and A. Aderem. 2007. A common human TLR1 polymorphism regulates the innate immune response to lipopeptides. Eur. J. Immunol. 37:2280–2289. Hawn, T. R., D. Scholes, S. S. Li, H. Wang, Y. Yang, P. L. Roberts, A. E. Stapleton, M. Janer, A. Aderem, W. E. Stamm, L. P. Zhao, and T. M. Hooton. 2009. Toll-like receptor polymorphisms and susceptibility to urinary tract infections in adult women. PLoS ONE 4:e5990. Higuchi, M., A. Matsuo, M. Shingai, K. Shida, A. Ishii, K. Funami, Y. Suzuki, H. Oshiumi, M. Matsumoto, and T. Seya. 2008. Combinational recognition of bacterial lipoproteins and peptidoglycan by chicken toll-like receptor 2 subfamily. Dev. Comp. Immunol. 32:147–155. Jin, M. S., S. E. Kim, J. Y. Heo, M. E. Lee, H. M. Kim, S. G. Paik, H. Lee, and J. O. Lee. 2007. Crystal structure of the TLR1– TLR2 heterodimer induced by binding of a tri-acylated lipopeptide. Cell 130:1071–1082. Jin, M. S., and J. O. Lee. 2008. Structures of the toll-like receptor family and its ligand complexes. Immunity 29:182–191. Johnson, C. M., E. A. Lyle, K. O. Omueti, V. A. Stepensky, O. Yegin, E. Alpsoy, L. Hamann, R. R. Schumann, and R. I. Tapping. 2007. Cutting edge: A common polymorphism impairs cell surface trafficking and functional responses of TLR1 but protects against leprosy. J. Immunol. 178:7520–7524. Keestra, A. M., M. R. de Zoete, R. A. van Aubel, and J. P. van Putten. 2007. The central leucine-rich repeat region of chicken TLR16 dictates unique ligand specificity and species-specific interaction with TLR2. J. Immunol. 178:7110–7119. Leveque, G., V. Forgetta, S. Morroll, A. L. Smith, N. Bumstead, P. Barrow, J. C. Loredo-Osti, K. Morgan, and D. Malo. 2003. Allelic variation in TLR4 is linked to susceptibility to Salmonella enterica serovar Typhimurium infection in chickens. Infect. Immun. 71:1116–1124.
1947