FOXO genes in channel catfish and their response after bacterial infection

Highlights

• Seven FOXO genes were identified in channel catfish.

• The seven FOXO genes were annotated by phylogenetic and syntenic analysis.

• Distinct expression patterns were observed for FOXOs after bacterial infections.

Abstract

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.

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; 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.