The chemokinome superfamily in channel catfish: I. CXC subfamily and their involvement in disease defense and hypoxia responses

Highlights

• 17 CXC chemokine genes were identified in channel catfish genome.

• The fish-specific and the expansion of CXC chemokines in the teleost genomes were revealed.

• The expression of CXC chemokines showed a gene-specific manner after bacterial infections.

• The expression of CXC chemokine genes showed a gene-specific pattern under hypoxia.

Abstract

Chemokines are a superfamily of structurally related chemotactic cytokines exerting significant roles in regulating cell migration and activation. They are defined by the presence of four conserved cysteine residues and are divided into four subfamilies depending on the arrangement of the first two conserved cysteines residues: CXC, CC, C and CX3C. In this study, a complete set of 17 CXC chemokine ligand (CXCL) genes was systematically identified and characterized from channel catfish genome through data mining of existing genomic resources. Phylogenetic analysis allowed annotation of the 17 CXC chemokines. Extensive comparative genomic analyses supported their annotations and orthologies, revealing the existence of fish-specific CXC chemokines and the expansion of CXC chemokines in the teleost genomes. The analysis of gene expression after bacterial infection indicated the CXC chemokines were expressed in a gene-specific manner. CXCL11.3 and CXCL20.3 were expressed significantly higher in resistant fish than in susceptible fish after ESC infection, while CXCL20.2 were expressed significantly higher in resistant fish than in susceptible fish after columnaris infection. The expression of those CXC chemokines, therefore can be a useful indicator of disease resistance. A similar pattern of expression was observed between resistant and susceptible fish with biotic and abiotic stresses, ESC, columnaris and hypoxia, suggesting that high levels of expression of the majority of CXC chemokines, with exception of CXC11 and CXC20, are detrimental to the host.

Introduction

Chemokines are a superfamily of chemotactic cytokines playing significant roles in regulating cell migration and activation under inflammatory conditions [1], [2], [3]. They are key immune regulators, acting as a bridge between innate and adaptive immunity [4]. Specifically, chemokines promote leukocyte mobilization and regulate the immune responses and differentiation of the recruited cells [4], [5]. In addition to their roles in immunity, chemokines also participate in angiogenesis [6], [7], neurological development [8], [9], organogenesis and germ cell migration [10], [11], and hypoxia responses [12], [13]. Chemokines are structurally related small peptides, typically 8–15 kDa, with majority of them containing four conserved cysteine residues [3], [14]. Based on the arrangement of the first two of the cysteine residues, chemokines are divided into four subfamilies, CXC, CC, C and CX3C [15]. The CXC subfamily is one of the largest subfamilies.

Initially identified as effective mediators of neutrophil chemotaxis [16], [17], CXC chemokines are also known to function in chemotaxis of monocytes and lymphocytes [18], [19], acting by binding to G-protein-coupled cell surface receptors that can activate an array of signaling pathways [20], [21]. In mammals, the CXC subfamily consists of 17 members, but each species has slightly variable numbers of genes, with 16 members for human (lacking CXCL15), 15 for mouse (lacking CXCL6 and CXCL8) and a smaller number identified from other mammalian species [22]. CXC chemokines are further classified into two subgroups, ELR⁺ and ELR⁻, based on the presence or absence of a tri-amino acid motif (Glu-Leu-Arg or E-L-R) preceding the first conserved cysteine at the N-terminus [23], [24], [25]. In mammals, the ELR⁺ subgroup includes CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8 and CXCL15. These chemokines play major roles in promoting the adherence of neutrophils to endothelial cells and subsequent migration along a gradient of chemokines associated with matrix proteins and cell surfaces towards inflammatory sites [26]. In addition, the ELR⁺ group exhibits angiogenic properties [19]. The ELR⁻ chemokine group includes CXCL4, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14 and CXCL16. They have functions to attract lymphocytes and monocytes, while with little or no ability for neutrophils [27], [28]. In contrast to ELR⁺ groups, most ELR⁻ CXC chemokines are angiostatic and have anti-angiogentic characteristics [27]. In teleost fish, the ELR motif is sometimes replaced by a defective DLR motif (Asp-Leu-Arg). Initially, this conservative change from E to D was assumed not to affect function [29]. However, the DLR motif has been demonstrated unnecessary for the attraction of neutrophils by fish CXC chemokines [30].

To date, six different CXC clades, including CXCa, CXCb, CXCc, CXCd, CXCL12 and CXCL14 have been identified from teleost fish [31], [32]. However, not all fish contained the six clades of CXC chemokine genes. Moreover, several fish-specific members were observed in CXC chemokines subfamily such as CXCa and CXCb in carp (Cyprinus carpio) [31], and CXCd in rainbow trout (Oncorhynchus mykiss) [33]. In zebrafish, over 100 chemokine genes were initially reported [1], but the latest annotation included fewer number of chemokine genes, with 23 CXC chemokine genes (http://zfin.org/).

Channel catfish (Ictalurus punctatus) is a primary aquaculture species in the United States (http://www.fao.org/fishery/culturedspecies/Ictalurus_punctatus/en), but its sustainable production is threatened by severe disease outbreaks. In particular, the enteric septicemia of catfish (ESC) caused by Edwardsiella ictaluri, columnaris disease caused by Flavobacterium columnare, and the emerging Aeromonas disease caused by Aeromonas hydrophila, all bacterial diseases, cause major economic losses to the catfish industry [34], [35]. The disease incidents were increased with exposures to environmental stresses, especially when exposed to hypoxia [36]. Understanding of molecular linkage between disease and hypoxia responses should help strategic thinking of solving these problems in genetic enhancing programs. With catfish, many innate immune genes have been characterized including pathogen recognition receptors [37], [38], [39], [40], [41], [42], [43], antimicrobial peptides [44], [45], [46], [47], [48], lysozymes [49], lectins [50], [51], [52], NOS genes [53], protease inhibitors [54], [55] and septins [56]. With chemokines, our previous studies [57], [58], [59] reported five members of CXC chemokines (CXCL2-like, CXCL8, CXCL10, CXCL12 and CXCL14). However, the entire chemokinome of CXC subfamily has not been conducted, and their involvement in disease and hypoxia responses has not been fully understood. The objective of this work was to determine all possible members of the CXC chemokine subfamily in channel catfish, and their expression after bacterial infections, and under hypoxia stress.