Sequence diversity
To assess allelic diversity in the CjCATH mature peptide–encoding regions, PCR products were amplified from 99 quail specimens representing six strains. A total of 10 SNPs were detected, 5 each at intron3 and exon4 between partial exon3 and partial exon4 of the CjCATH3 locus (325 bp) (Additional file 1). However, any SNPs did not exist in the mature peptide region. Six alleles (CjCATH3*01~*06: GenBank accession nos. LC426739-LC426744) were identified based on these 10 SNPs of CjCATH3 (Table 1). These six alleles included three major alleles (CjCATH3*01 [35.9%], CjCATH3*02 [26.8%], and CjCATH3*03 [16.2%]) and three minor alleles (CjCATH1*04 [9.1%], CjCATH3*05 [8.1%], and CjCATH3*06 [4.0%]). CjCATH3*04 and CjCATH3*06 were specifically detected in strains K and B, respectively (Table 1).
A total of 11 SNPs were detected, 1 at intron3 and 10 at exon4 (mature peptide region) between partial exon3 and partial exon4 of the CjCATH2 locus (464 bp) (Additional file 2). Four alleles (CjCATH2*01~*04: GenBank accession nos. LC426735-LC426738) were identified based on these 11 SNPs of CjCATH2 (Table 1). Ten SNPs in the mature peptide–encoding region were identified, including six that were non-synonymous. This resulted in the detection of six amino acid substitutions: Ile140Val (I140V), Ile145Phe (I145F), Ala147Thr (A147T), Gln148His (Q148H), Ile149Val (I149V), and Gly150Ala (G150A) (Figure 1). These four alleles included two major alleles (CjCATH2*01 [61.1%] and CjCATH2*02 [25.8%[) and two minor alleles (CjCATH2*03 [4.0%] and CjCATH2*04 [9.1%]). CjCATH2*03 and CjCATH2*04 were specifically detected in strains B and K, respectively (Table 1).
A total of five SNPs were detected, three at intron3 and two at exon4 (mature peptide–encoding region) between partial exon3 and partial exon4 of the CjCATHB1 locus (509 bp) (Additional file 3). Four alleles (CjCATHB1*01~*04: GenBank accession nos. LC426745-LC426748) were identified based on these five SNPs of CjCATHB1 (Table 1).
non-synonymous. These four alleles included three major alleles (CjCATHB1*01 [34.3%], CjCATHB1*02 [25.8%], and CjCATHB1*03 [30.8%]) and one minor allele (CjCATHB1*04 [9.1%]). CjCATHB1*04 was specifically detected in strain K (Table 1).
A total of seven SNPs were detected, five at intron3 and two at exon4 between partial exon3 and partial exon4 of the CjCATH1 locus (517 bp) (Additional file 4). One SNP with synonymous substitution was detected in the mature peptide–encoding region. Six alleles (CjCATH1*01~*06: GenBank accession nos. LC426729-LC426734) were identified based on these seven SNPs of CjCATH1 (Table 1). These six alleles included three major alleles (CjCATH1*01 [36.4%], CjCATH1*02 [25.8%], and CjCATH1*03 [18.7%]) and three minor alleles (CjCATH1*04 [9.1%], CjCATH1*05 [6.1%], and CjCATH1*06 [4.0%]). CjCATH1*04 and CjCATH1*06 were specifically detected in strains K and B, respectively (Table 1).
A comparison of the CATH3, CATH2, CATHB1, and CATH1 genes in the 99 quail specimens revealed six, four, four, and six alleles, respectively. We identified nine haplotypes (HT1-HT9) based on combinations of the CATH3, CATH2, CATHB1, and CATH1 alleles (Table 1). HT1 was the primary haplotype in strains ND and P, whereas HT2 was the primary haplotype in strain B. Haplotypes HT3 and HT4 were detected only in strain ND, and HT9 was found only in strain B. HT7 was a major haplotype specific to strain K.
Properties (Figure 1)
The amino acid sequence (29 amino acid residues [AA]) of the mature CjCATH3 peptide matched that of GgCATH3. Nine of the 32 AA of the sequence of the mature CjCATH2 peptide differed from the sequence of GgCATH2 (K134R, V136I, I/V140R, I144T, A146T, A/T147I, V/I149G, G/A150S, and S151A). The net charge of the C-terminal 15 AA of CjCATH2 was lower than that of GgCATH2. Conversely, the hydropathy index of CjCATH2 was higher than that of GgCATH2. No significant inter-allelic differences were found in terms of net charge or hydropathy index among the four alleles of CjCATH2. The PROVEAN and PolyPhen-2 software programs were used to evaluate the possibility that six amino acid substitutions among the four alleles of CjCATH2 constituted missense mutations that could adversely affect the function of the peptide. These analyses suggested that the I145F and Q148H substitutions detected in CjCATH2*02 would have an adverse effect on the function of the peptide (Additional file 5).
Of the 40 AA of the mature CjCATHB1 peptide, nine were different from the sequence of GgCATHB1 (T251I, R254W, W257L, D258N, R262K, H271Y, R275H, I278V, and S279T). Although no significant difference in net charge between the 15 N-terminal AA of both CjCATHB1s and GgCATHB1 was detected, the N-terminal hydropathy index of CjCATHB1 was lower than that of GgCATHB1. The net charge of the C-terminal 15 AA of CjCATHB1s was higher than that of the C-terminal 15 amino acid residues GgCATHB1, but there was no significant difference in the C-terminal hydropathy index between CjCATHB1s and GgCATHB1. No significant inter-allelic differences in terms of net charge and hydropathy index of the mature peptides were found among the four alleles of CjCATHB1. However, the PROVEAN and PolyPhen-2 analyses suggested that an amino acid substitution detected in CjCATHB1*04 (Pro245His [P245H]) could affect the function of the CjCATHB1 peptide (Additional file 5).
Among the 26 AA of the sequence of the mature CjCATH1 peptide, two differed from the sequence of GgCATH1 (L128W and R147K). However, no significant differences in net charge or hydropathy index were observed between CjCATH1 and GgCATH1.
Antimicrobial activity (Table 2)
Synthetic peptides representing all quail and chicken CATHs exhibited concentration-dependent antibacterial activity against E. coli. The amino acid sequence of the antimicrobial region of both chicken and Japanese quail CATH3 was the same. However, the amino acid sequences of CATH2, CATHB1 and CATH1 in the quail and chicken differed from each other.
At a concentration of 0.1 to 1 µM, the antimicrobial activity of CjCATH2s against E. coli tended to be higher than that of GgCATH2. The antimicrobial activity of CjCATH2*01*03 and CjCATH2*02 in particular was significantly more potent than that of GgCATH2. However, there were minimal differences in the antimicrobial activity of CjCATH2 and GgCATH2 against E. coli at concentrations of 10 and 100 µM.
At concentrations of 0.1 to 1 µM, the antimicrobial activity of CjCATHB1s against E. coli tended to be higher than that of GgCATHB1. The antimicrobial activity of CjCATHB1*01*02*03 at a concentration of 0.5 µM was significantly more potent than that of GgCATHB1. However, there were minimal differences in the antimicrobial activity of CjCATHB1 and GgCATHB1 against E. coli at concentrations of 10 and 100 µM. In addition, there were minimal differences in the antimicrobial activity of CjCATH1 and GgCATH1 against E. coli at all concentrations examined.
At a peptide concentration of 0.1 µM, CjCATH3 (=GgCATH3), CjCATH1, and GgCATH1 exhibited significantly more-potent antimicrobial activity against E. coli than GgCATH2, CjCATHB1, and GgCATHB1. At a peptide concentration of 0.5 μM, CjCATH3, CjCATH2s, CjCATHB1*01*02*03, CjCATH1, and GgCATH1 exhibited significantly more-potent antimicrobial activity against E. coli than GgCATH2 and GgCATHB1. At a peptide concentration of 1 μM, CjCATH2*01*03 and CjCATH1 exhibited significantly more-potent antimicrobial activity against E. coli than GgCATH2 and GgCATHB1. In contrast, at a peptide concentration of 10 μM, no differences between CATH-derived peptides were observed in terms of antimicrobial activity against E. coli. At a CATH-derived peptide concentration of 100 µM, no survival of E. coli was observed.