- Email: [email protected]
FOXO genes in channel catfish and their response after bacterial infection
Developmental and Comparative Immunology 97 (2019) 38–44
Contents lists available at ScienceDirect
Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/devcompimm
Short communication
FOXO genes in channel catfish and their response after bacterial infection Lei Gao
a,b
a
a
a
a
a
, Zihao Yuan , Tao Zhou , Yujia Yang , Dongya Gao , Rex Dunham , Zhanjiang Liu
T
c,∗
a
The Fish Molecular Genetics and Biotechnology Laboratory, Aquatic Genomics Unit, School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, 36849, USA Key Laboratory of Marine Fishery Molecular Biology of Liaoning Province, Liaoning Ocean and Fisheries Science Research Institute, Dalian, Liaoning, 116023, China c Department of Biology, College of Art and Sciences, Syracuse University, Syracuse, NY, 13244, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: FOXO Fish Gene expression Bacterial infection Immune response
FOXO proteins are a subgroup of the forkhead family of transcription factors that play crucial roles in lifespan regulation. In addition, FOXO proteins are also involved in immune responses. After a systematic study of FOXO genes in channel catfish, Ictalurus punctatus, seven FOXO genes were identified and characterized, including FOXO1a, FOXO1b, FOXO3a, FOXO3b, FOXO4, FOXO6a and FOXO6b. Through phylogenetic and syntenic analyses, it was found that FOXO1, FOXO3 and FOXO6 were duplicated in the catfish genome, as in the zebrafish genome. Analysis of the relative rates of nonsynonymous (dN) and synonymous (dS) substitutions revealed that the FOXO genes were globally strongly constrained by negative selection. Differential expression patterns were observed in the majority of FOXO genes after Edwardsiella ictaluri and Flavobacterium columnare infections. After E. ictaluri infection, four FOXO genes with orthologs in mammal species were significantly upregulated, where FOXO6b was the most dramatically upregulated. However, after F. columnare infection, the expression levels of almost all FOXO genes were not significantly affected. These results suggested that either a pathogenesis-specific pattern or tissue-specific pattern existed in catfish after these two bacterial infections. Taken together, these findings indicated that FOXO genes may play important roles in immune responses to bacterial infections in catfish.
1. Introduction FOXO proteins are a subgroup of the forkhead family of transcription factors, which are characterized by a winged-helix DNA-binding domain known as “forkhead box”. Multiple FOX genes have been identified, from FOXA to FOXR, based on their sequence similarities (Carter and Brunet, 2007; Webb and Brunet, 2014). To date, four FOXO genes have been identified from mammals: FOXO1 (FKHR, FKH1), FOXO3 (FKHRL1), FOXO4 (AFX, AFX1 and MLLT7) and FOXO6, all of which are orthologs of DAF16, an insulin-responsive transcription factor found in worms and flies (Eijkelenboom and Burgering, 2013; Schuff et al., 2010; Wang et al., 2009). In addition, FOXO2 was found to be a paralog of FOXO3, and FOXO5 is only expressed in zebrafish (Danio rerio) and is also known as FOXO3b (Eijkelenboom and Burgering, 2013). These four FOXO members are highly homologous, especially in the forkhead domain, which contains consensus the motif 5′-TTGTTTAC-3′ in helix three of the DNA-binding domain (Van Der Horst and Burgering, 2007; Wang et al., 2009). FOXO proteins exhibit similar physiological functions as transcriptional activators by either binding to
∗
the same core DNA sequence or interacting with other transcription factors to regulate the transcription of target genes (Lin et al., 2017). Another distinguishing feature of FOXO proteins is their highly conserved sites for phosphorylation, initially found to be phosphorylated in DAF-16 by kinase Akt (Alessi et al., 1996; Brunet et al., 1999) and were conserved in all members of the mammalian FOXO family (Huang and Tindall, 2007). In addition, there are two signaling pathways regulating FOXO activity: the canonical insulin signaling pathway, through PI3K and protein kinase B, regulates FOXO proteins negatively in the presence of growth factors, and the Jun N-terminal kinase (JNK) signaling pathway works in the presence of oxidative stress (Eijkelenboom and Burgering, 2013). FOXO genes were reported to be involved in the regulation of many molecular, cellular and physiological processes. One line of interesting research is the involvement of DAF-16 in the regulation of lifespan (Lin et al., 1997). In addition, members of the FOXO group were found to be involved in oxidative stress resistance (Balaban et al., 2005; Kops et al., 2002; Nemoto and Finkel, 2002), DNA damage repair (Tran et al., 2002), cell metabolism (Hall et al., 2000; Puigserver et al., 2003), cell cycle arrest (Rathbone et al., 2008) and apoptosis (Lam et al., 2006;
Corresponding author. E-mail address: [email protected] (Z. Liu).
https://doi.org/10.1016/j.dci.2019.03.010 Received 22 January 2019; Received in revised form 18 March 2019; Accepted 18 March 2019 Available online 21 March 2019 0145-305X/ © 2019 Elsevier Ltd. All rights reserved.
Developmental and Comparative Immunology 97 (2019) 38–44
L. Gao, et al.
were performed to test the phylogenetic tree, and gaps were removed by pairwise deletion.
Stahl et al., 2002). Another important physiological function of FOXO genes is immune modulation, such as induction of antimicrobial peptides (Becker et al., 2010; Zou et al., 2013), T-cell tolerance, glucose and lipid metabolism in the liver, and cellular quality control in muscle, cardiomyocytes and neurons (Haeusler et al., 2014; Kim et al., 2013; Matsumoto et al., 2007; Ochiai et al., 2012; Ouyang et al., 2012; Webb and Brunet, 2014). Channel catfish (Ictalurus punctatus) is the most important aquaculture species in the United States. However, in recent years, outbreaks of bacterial diseases have caused huge economic losses to the catfish industry. In particular, enteric septicemia of catfish (ESC) caused by Edwardsiella ictaluri and columnaris disease caused by Flavobacterium columnare are the most severe and the most frequently occurring diseases, respectively, in the catfish industry (Shoemaker et al., 2008; Wagner et al., 2002; Zhao et al., 2015). A large number of studies have been conducted to understand the response of immune-related genes to bacterial infection, including chemokines (Bao et al., 2006a), claudins (Sun et al., 2015), serpins (Li et al., 2015), NOD-like receptors (Rajendran et al., 2012; Sha et al., 2009), antimicrobial peptides (Bao et al., 2006b), lysozymes (Wang et al., 2013) and apolipoproteins (Yang et al., 2017). However, no comprehensive study of FOXO genes has been conducted, and there is very limited information in terms of their responses after bacterial infection. In this study, seven FOXO genes were identified and characterized, and their expression profiles were determined. These results provided new insights into the roles of FOXO genes in the immune responses of teleost fish.
2.3. Syntenic analysis Syntenic analysis was performed to provide support for orthologies based on the comparison of neighboring genes of FOXO in catfish with those in zebrafish and humans. The FOXO gene sequences of catfish were used as queries to search against the catfish genome sequence database (Liu et al., 2016), with a cutoff E-value of 1e−10. The neighboring genes were identified from the genomic scaffolds by the Fgenesh program and validated using BLASTP against the NCBI Nr database (Salamov and Solovyev, 2000). The conserved syntenic blocks of zebrafish and humans were identified from NCBI and Ensembl and then compared to those of catfish.
2.4. dN/dS analysis Nonsynonymous (dN) and synonymous (dS) substitution rates were determined at each codon site using Datamonkey (Delport et al., 2010). Positive selection and negative selection was calculated with a default p-value threshold of 0.1.
2.5. Expression analysis
2. Materials and methods
Meta-analysis of RNA-Seq datasets was performed with CLC Genomics Workbench (version 6.5.2; CLC bio, Aarhus, Denmark). The RNA-Seq datasets (SRA accession number: SRP009069 and SRP012586) were obtained from two previous bacterial infection experiments (E. ictaluri and F. columnare) in catfish (Li et al., 2012; Sun et al., 2012). Briefly, in the experiment involving E. ictaluri infection, fish were randomly divided into three control groups and three treatment groups, and were challenged using the MS-S97-773 isolate of E. ictaluri bacteria with 200 mL bacterial culture (4 × 108 CFU/mL) per aquaria (20 L water). At 3 h, 24 h and 72 h after the challenge, ten fish from each of the control and treatment groups were taken, and the entire intestinal tracts were sampled because intestine was considered as the primary route of bacterial entry for E. ictaluri into catfish host (Baldwin and Newton, 1993; Menanteau-Ledouble et al., 2011; Newton et al., 1989). The samples were stored at −80 °C until RNA extraction (Li et al., 2012). In the experiment involving F. columnare infection, fish were randomly divided into three control groups and three treatment groups, and were challenged using the BGFS-27 isolate of F. columnare bacteria with a final concentration of 3 × 106 CFU/mL. At 4 h, 24 h and 48 h after challenge, 18 fish from each of the control and treatment groups were taken, and the gill tissues were sampled because gill is the major site of damage and the first barrier of bacterial adhesion in F. columnare infection (Farkas and Oláh, 1986; Song et al., 2016). The samples were stored at −80 °C until RNA extraction (Sun et al., 2012). Total RNA was extracted from the tissues in the two experiments using the RNeasy Plus Kit (Qiagen). RNA-seq library was prepared and sequenced by HudsonAlpha Genomic Services Lab (Huntsville, AL, USA) using an Illumina HiSeq 2000 instrument with 100 bp paired end (PE) reads. The trimmed high-quality RNA-Seq reads were mapped to both of the assembled transcripts, which were used as reference sequences for expression analysis. Mapping parameters were set as 95% or greater of sequence identity with a maximum of two mismatches allowed. The number of total mapped reads for each FOXO gene was determined, and the RPKM (reads per kilobase of exon model per million mapped reads) was calculated. The expression fold change of each FOXO gene was determined based on the normalized RPKM using proportions-based Kal's test. The FOXO genes with p-value < 0.05, absolute fold change value ≥ 1.5 and total reads number≥5 were regarded as differentially expressed genes (DEGs).
2.1. Identification of channel catfish FOXO genes The FOXO genes in catfish were identified by searching the RNA-Seq database and the whole genome sequence database of catfish, using all available FOXO genes from selected fish species such as zebrafish (D. rerio), tilapia (Oreochromis niloticus), medaka (Oryzias latipes), fugu (Takifugu rubripes), rainbow trout (Oncorhynchus mykiss), red drum (Sciaenops ocellatus), goldfish (Carassius auratus), common carp (Cyprinus carpio), grass carp (Ctenopharyngodon idella), and FOXO genes from Xenopus tropicalis, chicken, mouse, pig, and human as query sequences retrieved from NCBI (http://www.ncbi.nlm.nih.gov) and Ensembl (http://www.ensembl.org) databases. The E-value was set at 1e−5 to collect as more potential FOXO sequences as possible. The retrieved sequences were translated into amino acid sequences using ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The predicted ORFs were verified by BLASTX against the NCBI nonredundant (Nr) protein sequence database. The Fgenesh program of Molquest software (Softberry Int.) (Solovyev et al., 2006) was used to predict genes from genomic sequences. Simple modular architecture research tool (SMART) (Letunic et al., 2012) and MEME (Bailey et al., 2009) was used to identify conserved domains. 2.2. Phylogenetic analysis The FOXO amino acid sequences identified from catfish, along with those from other representative vertebrates, including human, mouse, chicken, frog, and several fish species (zebrafish, fugu and medaka), were used to conduct the phylogenetic analysis (Table S1). An alignment of multiple amino acid sequences was performed using ClustalW (Thompson et al., 2002) with default parameters. The ProtTest program was used to obtain the best-fit model for FOXO evolution according to the Bayesian information criterion (Darriba et al., 2011). Based on the alignment results, the JTT (Jones-Taylor-Thornton) + I (invariant sites) + G (gamma distribution for modeling rate heterogeneity) model was selected. The maximum likelihood method was used to conduct phylogenetic and molecular evolutionary analyses using MEGA 7.0 software (Kumar et al., 2016). Bootstrap tests with 1,000 replications 39
Developmental and Comparative Immunology 97 (2019) 38–44
L. Gao, et al.
3. Results and discussion
event after the whole genome duplication.
3.1. FOXO genes in channel catfish
3.3. Ratios of dN/dS
Seven FOXO genes were identified in catfish, including FOXO1a, FOXO1b, FOXO3a, FOXO3b, FOXO4, FOXO6a and FOXO6b. FOXO1, FOXO3 and FOXO6 each had two copies, while a single copy was identified for FOXO4. The characteristics of the seven FOXO transcripts, including sizes of the transcripts, coding sequences, chromosomal locations, gene organizations, 5′- and 3′-untranslated regions and accession numbers, are summarized in Table S2. The seven FOXO genes were located on different chromosomes. The number of exons of the FOXO genes varied between two to four. All FOXO proteins shared the conserved FH, FOXO_KIX_bdg and FOXO-TAD domains (Fig. S1). A carboxyl-terminal minimal activation domain was also found in each of the FOXO proteins. In addition to the three highly conserved sites for phosphorylation, other phosphorylation sites and three lysines acetylated in mammalian homologs were also identified (Fig. S1). The gene copy numbers of FOXO genes were determined in representative vertebrates. All orthologs of FOXO genes in zebrafish and mammals were also found in the catfish genome (Table S3). In the catfish and zebrafish genomes, FOXO1, FOXO3 and FOXO6 had two duplicated copies, while there was only one copy in humans, cow, mouse, chicken, lizard and frog, indicating a teleost-specific gene duplication. For several other teleost fish species, the FOXO genes were not included for comparison due to lack of annotation consistency. More details are given in Table S4.
To measure selective pressure and delineate evolution dynamics, dN/dS analysis was conducted for FOXO genes. The global dN/dS ratios of all genes ranged from 0.0265 to 0.18, which were much lower than 1.0 (the theoretical boundary for positive and negative selection). In addition, no positive selection site was found in fish FOXO genes, indicating that strong negative selection appeared to be the major selection in the evolution of fish FOXO genes (Table S5). This was probably due to the essential roles of FOXO genes in various biological processes. Consistent with this conclusion, Wang et al. (2009) reported that molecular adaptation played an important role in the evolution of the FOXO gene family and that relaxed selection contributed to the process of functional differentiation evolution through gene duplications. FOXO6a showed the highest global dN/dS ratio. It was reported that FOXO6 was a unique member that was constrained by both relaxed selection in general and positive selection in four sites of the C-terminal part (Wang et al., 2009). This point, at least in part, can be supported by our results that FOXO6a had the highest dN/dS ratio of 0.18 among all FOXO members. 3.4. Expression of FOXO genes after bacterial infection The expression profiles of catfish FOXO genes were determined after bacterial infections (intestine tissues infected by E. ictaluri and gill tissues infected by F. columnare) using RNA-Seq datasets. After E. ictaluri infection, four FOXO genes, FOXO1a, FOXO3b, FOXO4 and FOXO6b, were significantly upregulated, suggesting their involvement in responses to ESC infection. FOXO genes were reported to play essential roles in immune responses, such as maintaining cellular homeostasis and regulating antioxidant defenses and lung cancer development (Chen et al., 2016; Hu et al., 2015; Kim et al., 2015). Classically, the intestine has been considered as the primary route of entry for E. ictaluri into the catfish host (Baldwin and Newton, 1993; Menanteau-Ledouble et al., 2011; Newton et al., 1989), and E. ictaluri is believed to have the ability to rapidly cross the intestinal mucosal barrier. The FOXO genes were reported to be a series of direct regulators of immune responses and were associated with pathogenic infection in the epidermis by inducing the expression of a variety of antimicrobial peptides, such as drosomycin and defensins (Becker et al., 2010; Zou et al., 2013). Combined with the results of this study, we deduced that FOXO genes played an important role in ESC disease by defending against E. ictaluri crossing through the intestinal mucosal barrier. FOXO1b and FOXO3a were not upregulated after ESC infection (Table 1). FOXO6a was not included in the expression analysis because its expressed reads were less than five. It was interesting that only one copy of the duplicated copies of FOXO1, FOXO3, and FOXO6 was upregulated, but the paralogous copy was not upregulated after ESC infection, suggesting that the expression, and thereby possibly function as well, was differentially regulated between the duplicated paralogs. The molecular mechanisms of regulation of the catfish FOXO genes and their involvement in responses to bacterial infection are of great interest for future studies. Among these significantly regulated FOXO genes, the expression of FOXO6b was the most dramatically upregulated after ESC infection, approximately 6.6-fold at 24 h after infection, while the expression of the remaining genes was induced 1.52–2.66-folds. Previous studies indicated that FOXO6 had a faster evolution rate, unique shuttling dynamics and positive selection (Van Der Heide et al., 2004; Wang et al., 2009). The specific functional mechanism of FOXO6 in the immune response to bacterial infection is still unknown at present, but future studies are warranted to determine its functions in bacterial disease interactions. The ESC infection-induced expression of almost all FOXO genes was most apparent early at 3 h and 24 h after infection, indicating the important roles of FOXO genes in disease defenses against acute
3.2. Phylogenetic and syntenic analyses of catfish FOXO genes Phylogenetic analysis was conducted to provide understanding of evolutionary relationships among FOXO proteins. Overall, the results of the phylogenetic analysis supported the annotations of catfish FOXO genes (Fig. 1A). In the phylogenetic tree, almost all members of the catfish FOXO proteins were well placed into four distinct clades and were grouped with those of other fishes with strong bootstrap support, and most members were closely related with their respective counterparts of zebrafish. However, catfish FOXO6a was more similar to FOXO6 of medaka than to that of zebrafish (Fig. 1A). Syntenic analysis was conducted to provide information for orthology of the FOXO genes. Conserved syntenic blocks and their positions were identified for all FOXO genes from catfish, zebrafish and humans (Fig. 1B). The syntenic analysis of FOXO1a, FOXO1b, FOXO3a, FOXO3b, FOXO4 and FOXO6b provided more evidence for their annotations in channel catfish. Meanwhile, from the results of both syntenic analysis and phylogenetic analysis, it was found that FOXO1a in catfish and zebrafish were highly orthologous to FOXO1 in mammals, and FOXO3b in catfish and zebrafish were highly orthologous to FOXO3 in mammals. For FOXO6a, bootstrap support was relatively low from the results of phylogenetic analysis. However, the syntenic analysis provided strong support for the orthology of FOXO6a (Fig. 1B). The syntenic regions were well conserved between the catfish and zebrafish genomic sequences containing the FOXO6a gene, sharing the same neighboring genes, BVES, POPDC3, STX12 and MED18. Meanwhile, FOXO6b in catfish and zebrafish were predicted to be highly orthologous to FOXO6 in mammals according to the results of the syntenic analysis. The phylogenetic analysis and syntenic analysis provided strong evidence that the duplicated copies of FOXO genes in catfish and zebrafish were derived from the whole genome duplication of the teleost genomes (Hoegg et al., 2004; Thornton and DeSalle, 2000). For instance, catfish chromosome 16 and chromosome 17, and catfish chromosome 1 and chromosome 12 are ohnologous, and the teleost-specific whole genome duplication event led to the formation of these paralogs. Although FOXO3a and FOXO3b were likely also duplicated copies derived from the whole genome duplication event, catfish chromosome 2 and chromosome 9 are not ohnologous chromosomes (Liu et al., 2016), suggesting a translocation 40
Developmental and Comparative Immunology 97 (2019) 38–44
L. Gao, et al.
Fig. 1. Identification and annotation of FOXO genes. (A) Phylogenetic analysis of FOXO family members. The phylogenetic tree was constructed with amino acid sequences from selected fish and other species using the maximum likelihood method under the JTT + I + G model with MEGA 7.0 software. The bootstrap consensus tree inferred from 1000 replicates was produced. Bootstrap values are indicated by numbers at the nodes. Black dots indicate the channel catfish FOXO family. The accession numbers of FOXO proteins used in the phylogenetic analysis are provided in Appendix: Table S1. (B) Syntenic analysis of channel catfish FOXO genes with those of zebrafish and humans. Dashed lines show orthologous relationships. Full gene names are provided in Appendix: Table S6.
41
Developmental and Comparative Immunology 97 (2019) 38–44
L. Gao, et al.
Fig. 1. (continued)
15 min following exposure (Baldwin and Newton, 1993). In contrast to the significant upregulation of FOXO genes after ESC infection, the expression of FOXO genes after F. columnare infection was not upregulated. Modestly increased expression of FOXO1b and FOXO4 was observed (∼1.5 × ) (Table 1). This result indicated that the involvement of FOXO genes in bacterial infection is quite specific. While ESC often causes systemic infections, columnaris disease is limited to local infections. Differential expression patterns between ESC infection and F. columnare infection were also observed in the studies of several other immune related genes, such as Rab GTPases, serpins and complement regulatory proteins (Jiang et al., 2015; Li et al., 2015; Wang et al., 2014).
Table 1 Fold change of catfish FOXO gene expression in the intestine after E. ictaluri infection and in the gill after F. columnare infection. The fold change of significant DEGs (p-value < 0.05, fold change≥1.5, reads number≥5) is in bold. E. ictaluri infection
FOXO1a FOXO1b FOXO3a FOXO3b FOXO4 FOXO6b
F. columnare infection
3h
24h
72h
4h
24h
48h
2.41 1.46 1.26 2.66 2.22 5.51
2.56 1.25 1.39 1.54 2.09 6.57
1.86 −1.03 −1.01 1.59 1.52 3.80
1.40 1.50 1.01 1.32 1.28 1.19
1.18 1.36 1.20 1.22 1.33 1.19
1.11 −1.12 1.03 −1.13 1.53 1.38
4. Conclusion bacterial infections. The reason why innate immune responses of FOXO genes were induced very early during the infection was also because E. ictaluri is known to invade the intestinal mucosal barrier as early as
A total of seven FOXO genes were identified and characterized in catfish, including two FOXO1 genes, two FOXO3 genes, one FOXO4 gene and two FOXO6 genes. The results of the phylogenetic and 42
Developmental and Comparative Immunology 97 (2019) 38–44
L. Gao, et al.
syntenic analyses provided strong evidence that the duplicated FOXO genes were derived from teleost-specific whole genome duplication events. The FOXO genes were specifically induced with ESC infection but not with columnaris infection. These results suggested that the infection-induced expression is either pathogenesis-specific or tissuespecific.
carcinoma through up-regulation of USP7. Mol. Med. Rep. 12, 575–580. Huang, H., Tindall, D.J., 2007. Dynamic FoxO transcription factors. J. Cell Sci. 120, 2479–2487. Jiang, C., Zhang, J., Yao, J., Liu, S., Li, Y., Song, L., Li, C., Wang, X., Liu, Z., 2015. Complement regulatory protein genes in channel catfish and their involvement in disease defense response. Dev. Comp. Immunol. 53, 33–41. Kim, D.H., Park, M.H., Chung, K.W., Kim, M.J., Park, D., Lee, B., Lee, E.K., Choi, Y.J., Kim, N.D., Yu, B.P., 2015. Suppression of FoxO6 by lipopolysaccharide in aged rat liver. Oncotarget 6, 34143. Kim, M.V., Ouyang, W., Liao, W., Zhang, M.Q., Li, M.O., 2013. The transcription factor Foxo1 controls central-memory CD8+ T cell responses to infection. Immunity 39, 286–297. Kops, G.J.P.L., Dansen, T.B., Polderman, P.E., Saarloos, I., Wirtz, K.W.A., Coffer, P.J., Huang, T.-T., Bos, J.L., Medema, R.H., Burgering, B.M.T., 2002. Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress. Nature 419, 316. Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874. Lam, E.W.F., Francis, R.E., Petkovic, M., 2006. FOXO transcription factors: key regulators of cell fate. Biochem. Soc. Trans. 34, 722–726. Letunic, I., Doerks, T., Bork, P., 2012. SMART 7: recent updates to the protein domain annotation resource. Nucleic Acids Res. 40, D302–D305. Li, C., Zhang, Y., Wang, R., Lu, J., Nandi, S., Mohanty, S., Terhune, J., Liu, Z., Peatman, E., 2012. RNA-seq analysis of mucosal immune responses reveals signatures of intestinal barrier disruption and pathogen entry following Edwardsiella ictaluri infection in channel catfish, Ictalurus punctatus. Fish Shellfish Immunol. 32, 816–827. Li, Y., Liu, S., Qin, Z., Yao, J., Jiang, C., Song, L., Dunham, R., Liu, Z., 2015. The serpin superfamily in channel catfish: identification, phylogenetic analysis and expression profiling in mucosal tissues after bacterial infections. Dev. Comp. Immunol. 49, 267–277. Lin, K., Dorman, J.B., Rodan, A., Kenyon, C., 1997. daf-16: an HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegans. Science 278, 1319–1322. Lin, S., Chiang, M., Shih, H., Chiang, K., Cheng, Y., 2017. Spatiotemporal expression of foxo4, foxo6a, and foxo6b in the developing brain and retina are transcriptionally regulated by PI3K signaling in zebrafish. Dev. Gene. Evol. 227, 219–230. Liu, Z., Liu, S., Yao, J., Bao, L., Zhang, J., Li, Y., Jiang, C., Sun, L., Wang, R., Zhang, Y., et al., 2016. The channel catfish genome sequence provides insights into the evolution of scale formation in teleosts. Nat. Commun. 7, 11757. Matsumoto, M., Pocai, A., Rossetti, L., DePinho, R.A., Accili, D., 2007. Impaired regulation of hepatic glucose production in mice lacking the forkhead transcription factor Foxo1 in liver. Cell Metabol. 6, 208–216. Menanteau-Ledouble, S., Karsi, A., Lawrence, M.L., 2011. Importance of skin abrasion as a primary site of adhesion for Edwardsiella ictaluri and impact on invasion and systematic infection in channel catfish Ictalurus punctatus. Vet. Microbiol. 148, 425–430. Nemoto, S., Finkel, T., 2002. Redox regulation of forkhead proteins through a p66shcdependent signaling pathway. Science 295, 2450–2452. Newton, J., Wolfe, L., Grizzle, J., Plumb, J., 1989. Pathology of experimental enteric septicaemia in channel catfish, Ictalurus punctatus (rafinesque), following immersion‐exposure to Edwardsiella ictaluri. J. Fish Dis. 12, 335–347. Ochiai, K., Maienschein-Cline, M., Mandal, M., Triggs, J.R., Bertolino, E., Sciammas, R., Dinner, A.R., Clark, M.R., Singh, H., 2012. A self-reinforcing regulatory network triggered by limiting IL-7 activates pre-BCR signaling and differentiation. Nat. Immunol. 13, 300–307. Ouyang, W., Liao, W., Luo, C.T., Yin, N., Huse, M., Kim, M.V., Peng, M., Chan, P., Ma, Q., Mo, Y., 2012. Novel Foxo1-dependent transcriptional programs control Treg cell function. Nature 491, 554–559. Puigserver, P., Rhee, J., Donovan, J., Walkey, C.J., Yoon, J.C., Oriente, F., Kitamura, Y., Altomonte, J., Dong, H., Accili, D., Spiegelman, B.M., 2003. Insulin-regulated hepatic gluconeogenesis through FOXO1–PGC-1α interaction. Nature 423, 550. Rajendran, K.V., Zhang, J., Liu, S., Kucuktas, H., Wang, X., Liu, H., Sha, Z., Terhune, J., Peatman, E., Liu, Z., 2012. Pathogen recognition receptors in channel catfish: I. Identification, phylogeny and expression of NOD-like receptors. Dev. Comp. Immunol. 37, 77–86. Rathbone, C.R., Booth, F.W., Lees, S.J., 2008. FoxO3a preferentially induces p27Kip1 expression while impairing muscle precursor cell‐cycle progression. Muscle Nerve 37, 84–89. Salamov, A.A., Solovyev, V.V., 2000. Ab initio gene finding in Drosophila genomic DNA. Genome Res. 10, 516–522. Schuff, M., Siegel, D., Bardine, N., Oswald, F., Donow, C., Knöchel, W., 2010. FoxO genes are dispensable during gastrulation but required for late embryogenesis in Xenopus laevis. Dev. Biol. 337, 259–273. Sha, Z., Abernathy, J.W., Wang, S., Li, P., Kucuktas, H., Liu, H., Peatman, E., Liu, Z., 2009. NOD-like subfamily of the nucleotide-binding domain and leucine-rich repeat containing family receptors and their expression in channel catfish. Dev. Comp. Immunol. 33, 991–999. Shoemaker, C.A., Olivares-Fuster, O., Arias, C.R., Klesius, P.H., 2008. Flavobacterium columnare genomovar influences mortality in channel catfish (Ictalurus punctatus). Vet. Microbiol. 127, 353–359. Solovyev, V., Kosarev, P., Seledsov, I., Vorobyev, D., 2006. Automatic annotation of eukaryotic genes, pseudogenes and promoters. Genome Biol. 7, S10. Song, L., Li, C., Xie, Y., Liu, S., Zhang, J., Yao, J., Jiang, C., Li, Y., Liu, Z., 2016. Genomewide identification of Hsp70 genes in channel catfish and their regulated expression after bacterial infection. Fish Shellfish Immunol. 49, 154–162. Stahl, M., Dijkers, P.F., Kops, G.J., Lens, S.M., Coffer, P.J., Burgering, B.M., Medema, R.H., 2002. The forkhead transcription factor FoxO regulates transcription of p27Kip1
Conflicts of interest The authors declare that they have no competing interests. Acknowledgements This project was supported by USDA National Institute of Food and Agriculture (NIFA) through competitive grants from the Animal Disease Program (2015-67015-22975) and the Animal Genomics, Genetics and Breeding Program (2017-67015-26295), and the National Natural Science Foundation of China (31602155). We are grateful to the availability of RNA-Seq datasets produced by our group. L. Gao was supported by a scholarship from the China Scholarship Council (CSC201604180028). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.dci.2019.03.010. References Alessi, D.R., Barry Caudwell, F., Andjelkovic, M., Hemmings, B.A., Cohen, P., 1996. Molecular basis for the substrate specificity of protein kinase B; comparison with MAPKAP kinase-1 and p70 S6 kinase. FEBS Lett. 399, 333–338. Bailey, T.L., Boden, M., Buske, F.A., Frith, M., Grant, C.E., Clementi, L., Ren, J., Li, W.W., Noble, W.S., 2009. MEME Suite: tools for motif discovery and searching. Nucleic Acids Res. 37, W202–W208. Balaban, R.S., Nemoto, S., Finkel, T., 2005. Mitochondria, oxidants, and aging. Cell 120, 483–495. Baldwin, T.J., Newton, J.C., 1993. Pathogenesis of enteric septicemia of channel catfish, caused by Edwardsiella ictaluri: bacteriologic and light and electron microscopic findings. J. Aquat. Anim. Health 5, 189–198. Bao, B., Peatman, E., Peng, X., Baoprasertkul, P., Wang, G., Liu, Z., 2006a. Characterization of 23 CC chemokine genes and analysis of their expression in channel catfish (Ictalurus punctatus). Dev. Comp. Immunol. 30, 783–796. Bao, B., Peatman, E., Xu, P., Li, P., Zeng, H., He, C., Liu, Z., 2006b. The catfish liverexpressed antimicrobial peptide 2 (LEAP-2) gene is expressed in a wide range of tissues and developmentally regulated. Mol. Immunol. 43, 367–377. Becker, T., Loch, G., Beyer, M., Zinke, I., Aschenbrenner, A.C., Carrera, P., Inhester, T., Schultze, J.L., Hoch, M., 2010. FOXO-dependent regulation of innate immune homeostasis. Nature 463, 369–373. Brunet, A., Bonni, A., Zigmond, M.J., Lin, M.Z., Juo, P., Hu, L.S., Anderson, M.J., Arden, K.C., Blenis, J., Greenberg, M.E., 1999. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96, 857–868. Carter, M.E., Brunet, A., 2007. FOXO transcription factors. Curr. Biol. 17, R113–R114. Chen, H., Chen, Y., Wu, J., Yang, F., Lv, Z., Xu, X., Zheng, S., 2016. Expression of FOXO6 is associated with oxidative stress level and predicts the prognosis in hepatocellular cancer: a comparative study. Medicine 95 e3708. Darriba, D., Taboada, G.L., Doallo, R., Posada, D., 2011. ProtTest 3: fast selection of bestfit models of protein evolution. Bioinformatics 27, 1164–1165. Delport, W., Poon, A.F.Y., Frost, S.D.W., Kosakovsky Pond, S.L., 2010. Datamonkey 2010: a suite of phylogenetic analysis tools for evolutionary biology. Bioinformatics 26, 2455–2457. Eijkelenboom, A., Burgering, B.M.T., 2013. FOXOs: signalling integrators for homeostasis maintenance. Nat. Rev. Mol. Cell Biol. 14, 83. Farkas, J., Oláh, J., 1986. Gill necrosis - a complex disease of carp. Aquaculture 58, 17–26. Haeusler, R.A., Hartil, K., Vaitheesvaran, B., Arrieta–Cruz, I., Knight, C.M., Cook, J.R., Kammoun, H.L., Febbraio, M.A., Gutierrez–Juarez, R., Kurland, I.J., 2014. Integrated control of hepatic lipogenesis versus glucose production requires FoxO transcription factors. Nat. Commun. 5, 5190. Hall, R.K., Yamasaki, T., Kucera, T., Waltner-Law, M., O'Brien, R., Granner, D.K., 2000. Regulation of phosphoenolpyruvate carboxykinase and insulin-like growth factorbinding protein-1 gene expression by insulin the role of winged helix/forkhead proteins. J. Biol. Chem. 275, 30169–30175. Hoegg, S., Brinkmann, H., Taylor, J.S., Meyer, A., 2004. Phylogenetic timing of the fishspecific genome duplication correlates with the diversification of teleost fish. J. Mol. Evol. 59, 190–203. Hu, H., Zhang, L., Wang, Z., Guo, X., 2015. FoxO6 inhibits cell proliferation in lung
43
Developmental and Comparative Immunology 97 (2019) 38–44
L. Gao, et al.
diseases in food-size channel catfish. J. Aquat. Anim. Health 14, 263–272. Wang, M., Zhang, X., Zhao, H., Wang, Q., Pan, Y., 2009. FoxO gene family evolution in vertebrates. BMC Evol. Biol. 9, 222. Wang, R., Feng, J., Li, C., Liu, S., Zhang, Y., Liu, Z., 2013. Four lysozymes (one c-type and three g-type) in catfish are drastically but differentially induced after bacterial infection. Fish Shellfish Immunol. 35, 136–145. Wang, R., Zhang, Y., Liu, S., Li, C., Sun, L., Bao, L., Feng, J., Liu, Z., 2014. Analysis of 52 Rab GTPases from channel catfish and their involvement in immune responses after bacterial infections. Dev. Comp. Immunol. 45, 21–34. Webb, A.E., Brunet, A., 2014. FOXO transcription factors: key regulators of cellular quality control. Trends Biochem. Sci. 39, 159–169. Yang, Y., Fu, Q., Zhou, T., Li, Y., Liu, S., Zeng, Q., Wang, X., Jin, Y., Tian, C., Qin, Z., Dunham, R., Liu, Z., 2017. Analysis of apolipoprotein genes and their involvement in disease response of channel catfish after bacterial infection. Dev. Comp. Immunol. 67, 464–470. Zhao, H., Li, C., Beck, B.H., Zhang, R., Thongda, W., Davis, D.A., Peatman, E., 2015. Impact of feed additives on surface mucosal health and columnaris susceptibility in channel catfish fingerlings, Ictalurus punctatus. Fish Shellfish Immunol. 46, 624–637. Zou, C., Tu, Q., Niu, J., Ji, X., Zhang, K., 2013. The DAF-16/FOXO transcription factor functions as a regulator of epidermal innate immunity. PLoS Pathog. 9 e1003660.
and Bim in response to IL-2. J. Immunol. 168, 5024–5031. Sun, F., Peatman, E., Li, C., Liu, S., Jiang, Y., Zhou, Z., Liu, Z., 2012. Transcriptomic signatures of attachment, NF-κB suppression and IFN stimulation in the catfish gill following columnaris bacterial infection. Dev. Comp. Immunol. 38, 169–180. Sun, L., Liu, S., Bao, L., Li, Y., Feng, J., Liu, Z., 2015. Claudin multigene family in channel catfish and their expression profiles in response to bacterial infection and hypoxia as revealed by meta-analysis of RNA-Seq datasets. Comp. Biochem. Physiol. Genom. Proteonom. 13, 60–69. Thompson, J.D., Gibson, T., Higgins, D.G., 2002. Multiple sequence alignment using ClustalW and ClustalX. Curr. Protoc. Bioinformatics 2 (3) 1-2.3.22. Thornton, J.W., DeSalle, R., 2000. Gene family evolution and homology: genomics meets phylogenetics. Annu. Rev. Genom. Hum. 1, 41–73. Tran, H., Brunet, A., Grenier, J.M., Datta, S.R., Fornace, A.J., DiStefano, P.S., Chiang, L.W., Greenberg, M.E., 2002. DNA repair pathway stimulated by the forkhead transcription factor FOXO3a through the Gadd45 protein. Science 296, 530–534. Van Der Heide, L.P., Hoekman, M.F., Smidt, M.P., 2004. The ins and outs of FoxO shuttling: mechanisms of FoxO translocation and transcriptional regulation. Biochem. J. 380, 297–309. Van Der Horst, A., Burgering, B.M.T., 2007. Stressing the role of FoxO proteins in lifespan and disease. Nat. Rev. Mol. Cell Biol. 8, 440. Wagner, B.A., Wise, D.J., Khoo, L.H., Terhune, J.S., 2002. The epidemiology of bacterial
44
Contents lists available at ScienceDirect
Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/devcompimm
Short communication
FOXO genes in channel catfish and their response after bacterial infection Lei Gao
a,b
a
a
a
a
a
, Zihao Yuan , Tao Zhou , Yujia Yang , Dongya Gao , Rex Dunham , Zhanjiang Liu
T
c,∗
a
The Fish Molecular Genetics and Biotechnology Laboratory, Aquatic Genomics Unit, School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, 36849, USA Key Laboratory of Marine Fishery Molecular Biology of Liaoning Province, Liaoning Ocean and Fisheries Science Research Institute, Dalian, Liaoning, 116023, China c Department of Biology, College of Art and Sciences, Syracuse University, Syracuse, NY, 13244, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: FOXO Fish Gene expression Bacterial infection Immune response
FOXO proteins are a subgroup of the forkhead family of transcription factors that play crucial roles in lifespan regulation. In addition, FOXO proteins are also involved in immune responses. After a systematic study of FOXO genes in channel catfish, Ictalurus punctatus, seven FOXO genes were identified and characterized, including FOXO1a, FOXO1b, FOXO3a, FOXO3b, FOXO4, FOXO6a and FOXO6b. Through phylogenetic and syntenic analyses, it was found that FOXO1, FOXO3 and FOXO6 were duplicated in the catfish genome, as in the zebrafish genome. Analysis of the relative rates of nonsynonymous (dN) and synonymous (dS) substitutions revealed that the FOXO genes were globally strongly constrained by negative selection. Differential expression patterns were observed in the majority of FOXO genes after Edwardsiella ictaluri and Flavobacterium columnare infections. After E. ictaluri infection, four FOXO genes with orthologs in mammal species were significantly upregulated, where FOXO6b was the most dramatically upregulated. However, after F. columnare infection, the expression levels of almost all FOXO genes were not significantly affected. These results suggested that either a pathogenesis-specific pattern or tissue-specific pattern existed in catfish after these two bacterial infections. Taken together, these findings indicated that FOXO genes may play important roles in immune responses to bacterial infections in catfish.
1. Introduction FOXO proteins are a subgroup of the forkhead family of transcription factors, which are characterized by a winged-helix DNA-binding domain known as “forkhead box”. Multiple FOX genes have been identified, from FOXA to FOXR, based on their sequence similarities (Carter and Brunet, 2007; Webb and Brunet, 2014). To date, four FOXO genes have been identified from mammals: FOXO1 (FKHR, FKH1), FOXO3 (FKHRL1), FOXO4 (AFX, AFX1 and MLLT7) and FOXO6, all of which are orthologs of DAF16, an insulin-responsive transcription factor found in worms and flies (Eijkelenboom and Burgering, 2013; Schuff et al., 2010; Wang et al., 2009). In addition, FOXO2 was found to be a paralog of FOXO3, and FOXO5 is only expressed in zebrafish (Danio rerio) and is also known as FOXO3b (Eijkelenboom and Burgering, 2013). These four FOXO members are highly homologous, especially in the forkhead domain, which contains consensus the motif 5′-TTGTTTAC-3′ in helix three of the DNA-binding domain (Van Der Horst and Burgering, 2007; Wang et al., 2009). FOXO proteins exhibit similar physiological functions as transcriptional activators by either binding to
∗
the same core DNA sequence or interacting with other transcription factors to regulate the transcription of target genes (Lin et al., 2017). Another distinguishing feature of FOXO proteins is their highly conserved sites for phosphorylation, initially found to be phosphorylated in DAF-16 by kinase Akt (Alessi et al., 1996; Brunet et al., 1999) and were conserved in all members of the mammalian FOXO family (Huang and Tindall, 2007). In addition, there are two signaling pathways regulating FOXO activity: the canonical insulin signaling pathway, through PI3K and protein kinase B, regulates FOXO proteins negatively in the presence of growth factors, and the Jun N-terminal kinase (JNK) signaling pathway works in the presence of oxidative stress (Eijkelenboom and Burgering, 2013). FOXO genes were reported to be involved in the regulation of many molecular, cellular and physiological processes. One line of interesting research is the involvement of DAF-16 in the regulation of lifespan (Lin et al., 1997). In addition, members of the FOXO group were found to be involved in oxidative stress resistance (Balaban et al., 2005; Kops et al., 2002; Nemoto and Finkel, 2002), DNA damage repair (Tran et al., 2002), cell metabolism (Hall et al., 2000; Puigserver et al., 2003), cell cycle arrest (Rathbone et al., 2008) and apoptosis (Lam et al., 2006;
Corresponding author. E-mail address: [email protected] (Z. Liu).
https://doi.org/10.1016/j.dci.2019.03.010 Received 22 January 2019; Received in revised form 18 March 2019; Accepted 18 March 2019 Available online 21 March 2019 0145-305X/ © 2019 Elsevier Ltd. All rights reserved.
Developmental and Comparative Immunology 97 (2019) 38–44
L. Gao, et al.
were performed to test the phylogenetic tree, and gaps were removed by pairwise deletion.
Stahl et al., 2002). Another important physiological function of FOXO genes is immune modulation, such as induction of antimicrobial peptides (Becker et al., 2010; Zou et al., 2013), T-cell tolerance, glucose and lipid metabolism in the liver, and cellular quality control in muscle, cardiomyocytes and neurons (Haeusler et al., 2014; Kim et al., 2013; Matsumoto et al., 2007; Ochiai et al., 2012; Ouyang et al., 2012; Webb and Brunet, 2014). Channel catfish (Ictalurus punctatus) is the most important aquaculture species in the United States. However, in recent years, outbreaks of bacterial diseases have caused huge economic losses to the catfish industry. In particular, enteric septicemia of catfish (ESC) caused by Edwardsiella ictaluri and columnaris disease caused by Flavobacterium columnare are the most severe and the most frequently occurring diseases, respectively, in the catfish industry (Shoemaker et al., 2008; Wagner et al., 2002; Zhao et al., 2015). A large number of studies have been conducted to understand the response of immune-related genes to bacterial infection, including chemokines (Bao et al., 2006a), claudins (Sun et al., 2015), serpins (Li et al., 2015), NOD-like receptors (Rajendran et al., 2012; Sha et al., 2009), antimicrobial peptides (Bao et al., 2006b), lysozymes (Wang et al., 2013) and apolipoproteins (Yang et al., 2017). However, no comprehensive study of FOXO genes has been conducted, and there is very limited information in terms of their responses after bacterial infection. In this study, seven FOXO genes were identified and characterized, and their expression profiles were determined. These results provided new insights into the roles of FOXO genes in the immune responses of teleost fish.
2.3. Syntenic analysis Syntenic analysis was performed to provide support for orthologies based on the comparison of neighboring genes of FOXO in catfish with those in zebrafish and humans. The FOXO gene sequences of catfish were used as queries to search against the catfish genome sequence database (Liu et al., 2016), with a cutoff E-value of 1e−10. The neighboring genes were identified from the genomic scaffolds by the Fgenesh program and validated using BLASTP against the NCBI Nr database (Salamov and Solovyev, 2000). The conserved syntenic blocks of zebrafish and humans were identified from NCBI and Ensembl and then compared to those of catfish.
2.4. dN/dS analysis Nonsynonymous (dN) and synonymous (dS) substitution rates were determined at each codon site using Datamonkey (Delport et al., 2010). Positive selection and negative selection was calculated with a default p-value threshold of 0.1.
2.5. Expression analysis
2. Materials and methods
Meta-analysis of RNA-Seq datasets was performed with CLC Genomics Workbench (version 6.5.2; CLC bio, Aarhus, Denmark). The RNA-Seq datasets (SRA accession number: SRP009069 and SRP012586) were obtained from two previous bacterial infection experiments (E. ictaluri and F. columnare) in catfish (Li et al., 2012; Sun et al., 2012). Briefly, in the experiment involving E. ictaluri infection, fish were randomly divided into three control groups and three treatment groups, and were challenged using the MS-S97-773 isolate of E. ictaluri bacteria with 200 mL bacterial culture (4 × 108 CFU/mL) per aquaria (20 L water). At 3 h, 24 h and 72 h after the challenge, ten fish from each of the control and treatment groups were taken, and the entire intestinal tracts were sampled because intestine was considered as the primary route of bacterial entry for E. ictaluri into catfish host (Baldwin and Newton, 1993; Menanteau-Ledouble et al., 2011; Newton et al., 1989). The samples were stored at −80 °C until RNA extraction (Li et al., 2012). In the experiment involving F. columnare infection, fish were randomly divided into three control groups and three treatment groups, and were challenged using the BGFS-27 isolate of F. columnare bacteria with a final concentration of 3 × 106 CFU/mL. At 4 h, 24 h and 48 h after challenge, 18 fish from each of the control and treatment groups were taken, and the gill tissues were sampled because gill is the major site of damage and the first barrier of bacterial adhesion in F. columnare infection (Farkas and Oláh, 1986; Song et al., 2016). The samples were stored at −80 °C until RNA extraction (Sun et al., 2012). Total RNA was extracted from the tissues in the two experiments using the RNeasy Plus Kit (Qiagen). RNA-seq library was prepared and sequenced by HudsonAlpha Genomic Services Lab (Huntsville, AL, USA) using an Illumina HiSeq 2000 instrument with 100 bp paired end (PE) reads. The trimmed high-quality RNA-Seq reads were mapped to both of the assembled transcripts, which were used as reference sequences for expression analysis. Mapping parameters were set as 95% or greater of sequence identity with a maximum of two mismatches allowed. The number of total mapped reads for each FOXO gene was determined, and the RPKM (reads per kilobase of exon model per million mapped reads) was calculated. The expression fold change of each FOXO gene was determined based on the normalized RPKM using proportions-based Kal's test. The FOXO genes with p-value < 0.05, absolute fold change value ≥ 1.5 and total reads number≥5 were regarded as differentially expressed genes (DEGs).
2.1. Identification of channel catfish FOXO genes The FOXO genes in catfish were identified by searching the RNA-Seq database and the whole genome sequence database of catfish, using all available FOXO genes from selected fish species such as zebrafish (D. rerio), tilapia (Oreochromis niloticus), medaka (Oryzias latipes), fugu (Takifugu rubripes), rainbow trout (Oncorhynchus mykiss), red drum (Sciaenops ocellatus), goldfish (Carassius auratus), common carp (Cyprinus carpio), grass carp (Ctenopharyngodon idella), and FOXO genes from Xenopus tropicalis, chicken, mouse, pig, and human as query sequences retrieved from NCBI (http://www.ncbi.nlm.nih.gov) and Ensembl (http://www.ensembl.org) databases. The E-value was set at 1e−5 to collect as more potential FOXO sequences as possible. The retrieved sequences were translated into amino acid sequences using ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The predicted ORFs were verified by BLASTX against the NCBI nonredundant (Nr) protein sequence database. The Fgenesh program of Molquest software (Softberry Int.) (Solovyev et al., 2006) was used to predict genes from genomic sequences. Simple modular architecture research tool (SMART) (Letunic et al., 2012) and MEME (Bailey et al., 2009) was used to identify conserved domains. 2.2. Phylogenetic analysis The FOXO amino acid sequences identified from catfish, along with those from other representative vertebrates, including human, mouse, chicken, frog, and several fish species (zebrafish, fugu and medaka), were used to conduct the phylogenetic analysis (Table S1). An alignment of multiple amino acid sequences was performed using ClustalW (Thompson et al., 2002) with default parameters. The ProtTest program was used to obtain the best-fit model for FOXO evolution according to the Bayesian information criterion (Darriba et al., 2011). Based on the alignment results, the JTT (Jones-Taylor-Thornton) + I (invariant sites) + G (gamma distribution for modeling rate heterogeneity) model was selected. The maximum likelihood method was used to conduct phylogenetic and molecular evolutionary analyses using MEGA 7.0 software (Kumar et al., 2016). Bootstrap tests with 1,000 replications 39
Developmental and Comparative Immunology 97 (2019) 38–44
L. Gao, et al.
3. Results and discussion
event after the whole genome duplication.
3.1. FOXO genes in channel catfish
3.3. Ratios of dN/dS
Seven FOXO genes were identified in catfish, including FOXO1a, FOXO1b, FOXO3a, FOXO3b, FOXO4, FOXO6a and FOXO6b. FOXO1, FOXO3 and FOXO6 each had two copies, while a single copy was identified for FOXO4. The characteristics of the seven FOXO transcripts, including sizes of the transcripts, coding sequences, chromosomal locations, gene organizations, 5′- and 3′-untranslated regions and accession numbers, are summarized in Table S2. The seven FOXO genes were located on different chromosomes. The number of exons of the FOXO genes varied between two to four. All FOXO proteins shared the conserved FH, FOXO_KIX_bdg and FOXO-TAD domains (Fig. S1). A carboxyl-terminal minimal activation domain was also found in each of the FOXO proteins. In addition to the three highly conserved sites for phosphorylation, other phosphorylation sites and three lysines acetylated in mammalian homologs were also identified (Fig. S1). The gene copy numbers of FOXO genes were determined in representative vertebrates. All orthologs of FOXO genes in zebrafish and mammals were also found in the catfish genome (Table S3). In the catfish and zebrafish genomes, FOXO1, FOXO3 and FOXO6 had two duplicated copies, while there was only one copy in humans, cow, mouse, chicken, lizard and frog, indicating a teleost-specific gene duplication. For several other teleost fish species, the FOXO genes were not included for comparison due to lack of annotation consistency. More details are given in Table S4.
To measure selective pressure and delineate evolution dynamics, dN/dS analysis was conducted for FOXO genes. The global dN/dS ratios of all genes ranged from 0.0265 to 0.18, which were much lower than 1.0 (the theoretical boundary for positive and negative selection). In addition, no positive selection site was found in fish FOXO genes, indicating that strong negative selection appeared to be the major selection in the evolution of fish FOXO genes (Table S5). This was probably due to the essential roles of FOXO genes in various biological processes. Consistent with this conclusion, Wang et al. (2009) reported that molecular adaptation played an important role in the evolution of the FOXO gene family and that relaxed selection contributed to the process of functional differentiation evolution through gene duplications. FOXO6a showed the highest global dN/dS ratio. It was reported that FOXO6 was a unique member that was constrained by both relaxed selection in general and positive selection in four sites of the C-terminal part (Wang et al., 2009). This point, at least in part, can be supported by our results that FOXO6a had the highest dN/dS ratio of 0.18 among all FOXO members. 3.4. Expression of FOXO genes after bacterial infection The expression profiles of catfish FOXO genes were determined after bacterial infections (intestine tissues infected by E. ictaluri and gill tissues infected by F. columnare) using RNA-Seq datasets. After E. ictaluri infection, four FOXO genes, FOXO1a, FOXO3b, FOXO4 and FOXO6b, were significantly upregulated, suggesting their involvement in responses to ESC infection. FOXO genes were reported to play essential roles in immune responses, such as maintaining cellular homeostasis and regulating antioxidant defenses and lung cancer development (Chen et al., 2016; Hu et al., 2015; Kim et al., 2015). Classically, the intestine has been considered as the primary route of entry for E. ictaluri into the catfish host (Baldwin and Newton, 1993; Menanteau-Ledouble et al., 2011; Newton et al., 1989), and E. ictaluri is believed to have the ability to rapidly cross the intestinal mucosal barrier. The FOXO genes were reported to be a series of direct regulators of immune responses and were associated with pathogenic infection in the epidermis by inducing the expression of a variety of antimicrobial peptides, such as drosomycin and defensins (Becker et al., 2010; Zou et al., 2013). Combined with the results of this study, we deduced that FOXO genes played an important role in ESC disease by defending against E. ictaluri crossing through the intestinal mucosal barrier. FOXO1b and FOXO3a were not upregulated after ESC infection (Table 1). FOXO6a was not included in the expression analysis because its expressed reads were less than five. It was interesting that only one copy of the duplicated copies of FOXO1, FOXO3, and FOXO6 was upregulated, but the paralogous copy was not upregulated after ESC infection, suggesting that the expression, and thereby possibly function as well, was differentially regulated between the duplicated paralogs. The molecular mechanisms of regulation of the catfish FOXO genes and their involvement in responses to bacterial infection are of great interest for future studies. Among these significantly regulated FOXO genes, the expression of FOXO6b was the most dramatically upregulated after ESC infection, approximately 6.6-fold at 24 h after infection, while the expression of the remaining genes was induced 1.52–2.66-folds. Previous studies indicated that FOXO6 had a faster evolution rate, unique shuttling dynamics and positive selection (Van Der Heide et al., 2004; Wang et al., 2009). The specific functional mechanism of FOXO6 in the immune response to bacterial infection is still unknown at present, but future studies are warranted to determine its functions in bacterial disease interactions. The ESC infection-induced expression of almost all FOXO genes was most apparent early at 3 h and 24 h after infection, indicating the important roles of FOXO genes in disease defenses against acute
3.2. Phylogenetic and syntenic analyses of catfish FOXO genes Phylogenetic analysis was conducted to provide understanding of evolutionary relationships among FOXO proteins. Overall, the results of the phylogenetic analysis supported the annotations of catfish FOXO genes (Fig. 1A). In the phylogenetic tree, almost all members of the catfish FOXO proteins were well placed into four distinct clades and were grouped with those of other fishes with strong bootstrap support, and most members were closely related with their respective counterparts of zebrafish. However, catfish FOXO6a was more similar to FOXO6 of medaka than to that of zebrafish (Fig. 1A). Syntenic analysis was conducted to provide information for orthology of the FOXO genes. Conserved syntenic blocks and their positions were identified for all FOXO genes from catfish, zebrafish and humans (Fig. 1B). The syntenic analysis of FOXO1a, FOXO1b, FOXO3a, FOXO3b, FOXO4 and FOXO6b provided more evidence for their annotations in channel catfish. Meanwhile, from the results of both syntenic analysis and phylogenetic analysis, it was found that FOXO1a in catfish and zebrafish were highly orthologous to FOXO1 in mammals, and FOXO3b in catfish and zebrafish were highly orthologous to FOXO3 in mammals. For FOXO6a, bootstrap support was relatively low from the results of phylogenetic analysis. However, the syntenic analysis provided strong support for the orthology of FOXO6a (Fig. 1B). The syntenic regions were well conserved between the catfish and zebrafish genomic sequences containing the FOXO6a gene, sharing the same neighboring genes, BVES, POPDC3, STX12 and MED18. Meanwhile, FOXO6b in catfish and zebrafish were predicted to be highly orthologous to FOXO6 in mammals according to the results of the syntenic analysis. The phylogenetic analysis and syntenic analysis provided strong evidence that the duplicated copies of FOXO genes in catfish and zebrafish were derived from the whole genome duplication of the teleost genomes (Hoegg et al., 2004; Thornton and DeSalle, 2000). For instance, catfish chromosome 16 and chromosome 17, and catfish chromosome 1 and chromosome 12 are ohnologous, and the teleost-specific whole genome duplication event led to the formation of these paralogs. Although FOXO3a and FOXO3b were likely also duplicated copies derived from the whole genome duplication event, catfish chromosome 2 and chromosome 9 are not ohnologous chromosomes (Liu et al., 2016), suggesting a translocation 40
Developmental and Comparative Immunology 97 (2019) 38–44
L. Gao, et al.
Fig. 1. Identification and annotation of FOXO genes. (A) Phylogenetic analysis of FOXO family members. The phylogenetic tree was constructed with amino acid sequences from selected fish and other species using the maximum likelihood method under the JTT + I + G model with MEGA 7.0 software. The bootstrap consensus tree inferred from 1000 replicates was produced. Bootstrap values are indicated by numbers at the nodes. Black dots indicate the channel catfish FOXO family. The accession numbers of FOXO proteins used in the phylogenetic analysis are provided in Appendix: Table S1. (B) Syntenic analysis of channel catfish FOXO genes with those of zebrafish and humans. Dashed lines show orthologous relationships. Full gene names are provided in Appendix: Table S6.
41
Developmental and Comparative Immunology 97 (2019) 38–44
L. Gao, et al.
Fig. 1. (continued)
15 min following exposure (Baldwin and Newton, 1993). In contrast to the significant upregulation of FOXO genes after ESC infection, the expression of FOXO genes after F. columnare infection was not upregulated. Modestly increased expression of FOXO1b and FOXO4 was observed (∼1.5 × ) (Table 1). This result indicated that the involvement of FOXO genes in bacterial infection is quite specific. While ESC often causes systemic infections, columnaris disease is limited to local infections. Differential expression patterns between ESC infection and F. columnare infection were also observed in the studies of several other immune related genes, such as Rab GTPases, serpins and complement regulatory proteins (Jiang et al., 2015; Li et al., 2015; Wang et al., 2014).
Table 1 Fold change of catfish FOXO gene expression in the intestine after E. ictaluri infection and in the gill after F. columnare infection. The fold change of significant DEGs (p-value < 0.05, fold change≥1.5, reads number≥5) is in bold. E. ictaluri infection
FOXO1a FOXO1b FOXO3a FOXO3b FOXO4 FOXO6b
F. columnare infection
3h
24h
72h
4h
24h
48h
2.41 1.46 1.26 2.66 2.22 5.51
2.56 1.25 1.39 1.54 2.09 6.57
1.86 −1.03 −1.01 1.59 1.52 3.80
1.40 1.50 1.01 1.32 1.28 1.19
1.18 1.36 1.20 1.22 1.33 1.19
1.11 −1.12 1.03 −1.13 1.53 1.38
4. Conclusion bacterial infections. The reason why innate immune responses of FOXO genes were induced very early during the infection was also because E. ictaluri is known to invade the intestinal mucosal barrier as early as
A total of seven FOXO genes were identified and characterized in catfish, including two FOXO1 genes, two FOXO3 genes, one FOXO4 gene and two FOXO6 genes. The results of the phylogenetic and 42
Developmental and Comparative Immunology 97 (2019) 38–44
L. Gao, et al.
syntenic analyses provided strong evidence that the duplicated FOXO genes were derived from teleost-specific whole genome duplication events. The FOXO genes were specifically induced with ESC infection but not with columnaris infection. These results suggested that the infection-induced expression is either pathogenesis-specific or tissuespecific.
carcinoma through up-regulation of USP7. Mol. Med. Rep. 12, 575–580. Huang, H., Tindall, D.J., 2007. Dynamic FoxO transcription factors. J. Cell Sci. 120, 2479–2487. Jiang, C., Zhang, J., Yao, J., Liu, S., Li, Y., Song, L., Li, C., Wang, X., Liu, Z., 2015. Complement regulatory protein genes in channel catfish and their involvement in disease defense response. Dev. Comp. Immunol. 53, 33–41. Kim, D.H., Park, M.H., Chung, K.W., Kim, M.J., Park, D., Lee, B., Lee, E.K., Choi, Y.J., Kim, N.D., Yu, B.P., 2015. Suppression of FoxO6 by lipopolysaccharide in aged rat liver. Oncotarget 6, 34143. Kim, M.V., Ouyang, W., Liao, W., Zhang, M.Q., Li, M.O., 2013. The transcription factor Foxo1 controls central-memory CD8+ T cell responses to infection. Immunity 39, 286–297. Kops, G.J.P.L., Dansen, T.B., Polderman, P.E., Saarloos, I., Wirtz, K.W.A., Coffer, P.J., Huang, T.-T., Bos, J.L., Medema, R.H., Burgering, B.M.T., 2002. Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress. Nature 419, 316. Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874. Lam, E.W.F., Francis, R.E., Petkovic, M., 2006. FOXO transcription factors: key regulators of cell fate. Biochem. Soc. Trans. 34, 722–726. Letunic, I., Doerks, T., Bork, P., 2012. SMART 7: recent updates to the protein domain annotation resource. Nucleic Acids Res. 40, D302–D305. Li, C., Zhang, Y., Wang, R., Lu, J., Nandi, S., Mohanty, S., Terhune, J., Liu, Z., Peatman, E., 2012. RNA-seq analysis of mucosal immune responses reveals signatures of intestinal barrier disruption and pathogen entry following Edwardsiella ictaluri infection in channel catfish, Ictalurus punctatus. Fish Shellfish Immunol. 32, 816–827. Li, Y., Liu, S., Qin, Z., Yao, J., Jiang, C., Song, L., Dunham, R., Liu, Z., 2015. The serpin superfamily in channel catfish: identification, phylogenetic analysis and expression profiling in mucosal tissues after bacterial infections. Dev. Comp. Immunol. 49, 267–277. Lin, K., Dorman, J.B., Rodan, A., Kenyon, C., 1997. daf-16: an HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegans. Science 278, 1319–1322. Lin, S., Chiang, M., Shih, H., Chiang, K., Cheng, Y., 2017. Spatiotemporal expression of foxo4, foxo6a, and foxo6b in the developing brain and retina are transcriptionally regulated by PI3K signaling in zebrafish. Dev. Gene. Evol. 227, 219–230. Liu, Z., Liu, S., Yao, J., Bao, L., Zhang, J., Li, Y., Jiang, C., Sun, L., Wang, R., Zhang, Y., et al., 2016. The channel catfish genome sequence provides insights into the evolution of scale formation in teleosts. Nat. Commun. 7, 11757. Matsumoto, M., Pocai, A., Rossetti, L., DePinho, R.A., Accili, D., 2007. Impaired regulation of hepatic glucose production in mice lacking the forkhead transcription factor Foxo1 in liver. Cell Metabol. 6, 208–216. Menanteau-Ledouble, S., Karsi, A., Lawrence, M.L., 2011. Importance of skin abrasion as a primary site of adhesion for Edwardsiella ictaluri and impact on invasion and systematic infection in channel catfish Ictalurus punctatus. Vet. Microbiol. 148, 425–430. Nemoto, S., Finkel, T., 2002. Redox regulation of forkhead proteins through a p66shcdependent signaling pathway. Science 295, 2450–2452. Newton, J., Wolfe, L., Grizzle, J., Plumb, J., 1989. Pathology of experimental enteric septicaemia in channel catfish, Ictalurus punctatus (rafinesque), following immersion‐exposure to Edwardsiella ictaluri. J. Fish Dis. 12, 335–347. Ochiai, K., Maienschein-Cline, M., Mandal, M., Triggs, J.R., Bertolino, E., Sciammas, R., Dinner, A.R., Clark, M.R., Singh, H., 2012. A self-reinforcing regulatory network triggered by limiting IL-7 activates pre-BCR signaling and differentiation. Nat. Immunol. 13, 300–307. Ouyang, W., Liao, W., Luo, C.T., Yin, N., Huse, M., Kim, M.V., Peng, M., Chan, P., Ma, Q., Mo, Y., 2012. Novel Foxo1-dependent transcriptional programs control Treg cell function. Nature 491, 554–559. Puigserver, P., Rhee, J., Donovan, J., Walkey, C.J., Yoon, J.C., Oriente, F., Kitamura, Y., Altomonte, J., Dong, H., Accili, D., Spiegelman, B.M., 2003. Insulin-regulated hepatic gluconeogenesis through FOXO1–PGC-1α interaction. Nature 423, 550. Rajendran, K.V., Zhang, J., Liu, S., Kucuktas, H., Wang, X., Liu, H., Sha, Z., Terhune, J., Peatman, E., Liu, Z., 2012. Pathogen recognition receptors in channel catfish: I. Identification, phylogeny and expression of NOD-like receptors. Dev. Comp. Immunol. 37, 77–86. Rathbone, C.R., Booth, F.W., Lees, S.J., 2008. FoxO3a preferentially induces p27Kip1 expression while impairing muscle precursor cell‐cycle progression. Muscle Nerve 37, 84–89. Salamov, A.A., Solovyev, V.V., 2000. Ab initio gene finding in Drosophila genomic DNA. Genome Res. 10, 516–522. Schuff, M., Siegel, D., Bardine, N., Oswald, F., Donow, C., Knöchel, W., 2010. FoxO genes are dispensable during gastrulation but required for late embryogenesis in Xenopus laevis. Dev. Biol. 337, 259–273. Sha, Z., Abernathy, J.W., Wang, S., Li, P., Kucuktas, H., Liu, H., Peatman, E., Liu, Z., 2009. NOD-like subfamily of the nucleotide-binding domain and leucine-rich repeat containing family receptors and their expression in channel catfish. Dev. Comp. Immunol. 33, 991–999. Shoemaker, C.A., Olivares-Fuster, O., Arias, C.R., Klesius, P.H., 2008. Flavobacterium columnare genomovar influences mortality in channel catfish (Ictalurus punctatus). Vet. Microbiol. 127, 353–359. Solovyev, V., Kosarev, P., Seledsov, I., Vorobyev, D., 2006. Automatic annotation of eukaryotic genes, pseudogenes and promoters. Genome Biol. 7, S10. Song, L., Li, C., Xie, Y., Liu, S., Zhang, J., Yao, J., Jiang, C., Li, Y., Liu, Z., 2016. Genomewide identification of Hsp70 genes in channel catfish and their regulated expression after bacterial infection. Fish Shellfish Immunol. 49, 154–162. Stahl, M., Dijkers, P.F., Kops, G.J., Lens, S.M., Coffer, P.J., Burgering, B.M., Medema, R.H., 2002. The forkhead transcription factor FoxO regulates transcription of p27Kip1
Conflicts of interest The authors declare that they have no competing interests. Acknowledgements This project was supported by USDA National Institute of Food and Agriculture (NIFA) through competitive grants from the Animal Disease Program (2015-67015-22975) and the Animal Genomics, Genetics and Breeding Program (2017-67015-26295), and the National Natural Science Foundation of China (31602155). We are grateful to the availability of RNA-Seq datasets produced by our group. L. Gao was supported by a scholarship from the China Scholarship Council (CSC201604180028). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.dci.2019.03.010. References Alessi, D.R., Barry Caudwell, F., Andjelkovic, M., Hemmings, B.A., Cohen, P., 1996. Molecular basis for the substrate specificity of protein kinase B; comparison with MAPKAP kinase-1 and p70 S6 kinase. FEBS Lett. 399, 333–338. Bailey, T.L., Boden, M., Buske, F.A., Frith, M., Grant, C.E., Clementi, L., Ren, J., Li, W.W., Noble, W.S., 2009. MEME Suite: tools for motif discovery and searching. Nucleic Acids Res. 37, W202–W208. Balaban, R.S., Nemoto, S., Finkel, T., 2005. Mitochondria, oxidants, and aging. Cell 120, 483–495. Baldwin, T.J., Newton, J.C., 1993. Pathogenesis of enteric septicemia of channel catfish, caused by Edwardsiella ictaluri: bacteriologic and light and electron microscopic findings. J. Aquat. Anim. Health 5, 189–198. Bao, B., Peatman, E., Peng, X., Baoprasertkul, P., Wang, G., Liu, Z., 2006a. Characterization of 23 CC chemokine genes and analysis of their expression in channel catfish (Ictalurus punctatus). Dev. Comp. Immunol. 30, 783–796. Bao, B., Peatman, E., Xu, P., Li, P., Zeng, H., He, C., Liu, Z., 2006b. The catfish liverexpressed antimicrobial peptide 2 (LEAP-2) gene is expressed in a wide range of tissues and developmentally regulated. Mol. Immunol. 43, 367–377. Becker, T., Loch, G., Beyer, M., Zinke, I., Aschenbrenner, A.C., Carrera, P., Inhester, T., Schultze, J.L., Hoch, M., 2010. FOXO-dependent regulation of innate immune homeostasis. Nature 463, 369–373. Brunet, A., Bonni, A., Zigmond, M.J., Lin, M.Z., Juo, P., Hu, L.S., Anderson, M.J., Arden, K.C., Blenis, J., Greenberg, M.E., 1999. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96, 857–868. Carter, M.E., Brunet, A., 2007. FOXO transcription factors. Curr. Biol. 17, R113–R114. Chen, H., Chen, Y., Wu, J., Yang, F., Lv, Z., Xu, X., Zheng, S., 2016. Expression of FOXO6 is associated with oxidative stress level and predicts the prognosis in hepatocellular cancer: a comparative study. Medicine 95 e3708. Darriba, D., Taboada, G.L., Doallo, R., Posada, D., 2011. ProtTest 3: fast selection of bestfit models of protein evolution. Bioinformatics 27, 1164–1165. Delport, W., Poon, A.F.Y., Frost, S.D.W., Kosakovsky Pond, S.L., 2010. Datamonkey 2010: a suite of phylogenetic analysis tools for evolutionary biology. Bioinformatics 26, 2455–2457. Eijkelenboom, A., Burgering, B.M.T., 2013. FOXOs: signalling integrators for homeostasis maintenance. Nat. Rev. Mol. Cell Biol. 14, 83. Farkas, J., Oláh, J., 1986. Gill necrosis - a complex disease of carp. Aquaculture 58, 17–26. Haeusler, R.A., Hartil, K., Vaitheesvaran, B., Arrieta–Cruz, I., Knight, C.M., Cook, J.R., Kammoun, H.L., Febbraio, M.A., Gutierrez–Juarez, R., Kurland, I.J., 2014. Integrated control of hepatic lipogenesis versus glucose production requires FoxO transcription factors. Nat. Commun. 5, 5190. Hall, R.K., Yamasaki, T., Kucera, T., Waltner-Law, M., O'Brien, R., Granner, D.K., 2000. Regulation of phosphoenolpyruvate carboxykinase and insulin-like growth factorbinding protein-1 gene expression by insulin the role of winged helix/forkhead proteins. J. Biol. Chem. 275, 30169–30175. Hoegg, S., Brinkmann, H., Taylor, J.S., Meyer, A., 2004. Phylogenetic timing of the fishspecific genome duplication correlates with the diversification of teleost fish. J. Mol. Evol. 59, 190–203. Hu, H., Zhang, L., Wang, Z., Guo, X., 2015. FoxO6 inhibits cell proliferation in lung
43
Developmental and Comparative Immunology 97 (2019) 38–44
L. Gao, et al.
diseases in food-size channel catfish. J. Aquat. Anim. Health 14, 263–272. Wang, M., Zhang, X., Zhao, H., Wang, Q., Pan, Y., 2009. FoxO gene family evolution in vertebrates. BMC Evol. Biol. 9, 222. Wang, R., Feng, J., Li, C., Liu, S., Zhang, Y., Liu, Z., 2013. Four lysozymes (one c-type and three g-type) in catfish are drastically but differentially induced after bacterial infection. Fish Shellfish Immunol. 35, 136–145. Wang, R., Zhang, Y., Liu, S., Li, C., Sun, L., Bao, L., Feng, J., Liu, Z., 2014. Analysis of 52 Rab GTPases from channel catfish and their involvement in immune responses after bacterial infections. Dev. Comp. Immunol. 45, 21–34. Webb, A.E., Brunet, A., 2014. FOXO transcription factors: key regulators of cellular quality control. Trends Biochem. Sci. 39, 159–169. Yang, Y., Fu, Q., Zhou, T., Li, Y., Liu, S., Zeng, Q., Wang, X., Jin, Y., Tian, C., Qin, Z., Dunham, R., Liu, Z., 2017. Analysis of apolipoprotein genes and their involvement in disease response of channel catfish after bacterial infection. Dev. Comp. Immunol. 67, 464–470. Zhao, H., Li, C., Beck, B.H., Zhang, R., Thongda, W., Davis, D.A., Peatman, E., 2015. Impact of feed additives on surface mucosal health and columnaris susceptibility in channel catfish fingerlings, Ictalurus punctatus. Fish Shellfish Immunol. 46, 624–637. Zou, C., Tu, Q., Niu, J., Ji, X., Zhang, K., 2013. The DAF-16/FOXO transcription factor functions as a regulator of epidermal innate immunity. PLoS Pathog. 9 e1003660.
and Bim in response to IL-2. J. Immunol. 168, 5024–5031. Sun, F., Peatman, E., Li, C., Liu, S., Jiang, Y., Zhou, Z., Liu, Z., 2012. Transcriptomic signatures of attachment, NF-κB suppression and IFN stimulation in the catfish gill following columnaris bacterial infection. Dev. Comp. Immunol. 38, 169–180. Sun, L., Liu, S., Bao, L., Li, Y., Feng, J., Liu, Z., 2015. Claudin multigene family in channel catfish and their expression profiles in response to bacterial infection and hypoxia as revealed by meta-analysis of RNA-Seq datasets. Comp. Biochem. Physiol. Genom. Proteonom. 13, 60–69. Thompson, J.D., Gibson, T., Higgins, D.G., 2002. Multiple sequence alignment using ClustalW and ClustalX. Curr. Protoc. Bioinformatics 2 (3) 1-2.3.22. Thornton, J.W., DeSalle, R., 2000. Gene family evolution and homology: genomics meets phylogenetics. Annu. Rev. Genom. Hum. 1, 41–73. Tran, H., Brunet, A., Grenier, J.M., Datta, S.R., Fornace, A.J., DiStefano, P.S., Chiang, L.W., Greenberg, M.E., 2002. DNA repair pathway stimulated by the forkhead transcription factor FOXO3a through the Gadd45 protein. Science 296, 530–534. Van Der Heide, L.P., Hoekman, M.F., Smidt, M.P., 2004. The ins and outs of FoxO shuttling: mechanisms of FoxO translocation and transcriptional regulation. Biochem. J. 380, 297–309. Van Der Horst, A., Burgering, B.M.T., 2007. Stressing the role of FoxO proteins in lifespan and disease. Nat. Rev. Mol. Cell Biol. 8, 440. Wagner, B.A., Wise, D.J., Khoo, L.H., Terhune, J.S., 2002. The epidemiology of bacterial
44