Functional characterization of an immunity-related GTPase gene in immune defense from obscure puffer (Takifugu obscurus)

Highlights

• The immunity-related GTPase from obscure puffer (ToIRG) was identified.

• ToIRG shared highly similar molecular features with mammalian GKS IRGs.

• ToIRG was dramatically upregulated after Aeromonas hydrophila or Edwardsiella tarda infection.

• ToIRG is localized in the cytoplasm and characterized as condensed form around the nucleus.

• ToIRG helped host cell to resist against both intracellular (E.tarda) and extracellular pathogens (A.hydrophila).

Abstract

Immunity-related GTPases (IRGs) are a family of large interferon-inducible GTPases that function in effective host defense against invading pathogens. IRGs have been extensively studied in mammals for their roles in the elimination of intracellular pathogens; however, their homologs in lower vertebrates are not well known. In this study, an IRG from obscure puffer (Takifugu obscurus), ToIRG, was identified and further characterized for its functional activity. The ToIRG gene encodes a protein of 396 amino acids containing a typical N-terminal GTPase domain with three conserved motifs. Phylogenetic analysis revealed that it has a closer evolutionary relationship with mammalian GKS IRGs. Gene expression profile analysis revealed that ToIRG was ubiquitously expressed in all tested healthy tissues of obscure puffer and upregulated in response to Aeromonas hydrophila or Edwardsiella tarda challenge. The subcellular localization of ToIRG is characterized as condensed forms around the nucleus. Importantly, an antimicrobial assay in vitro suggested that ToIRG enhanced the ability of host cells to resist both intracellular (E. tarda) and extracellular pathogens (A. hydrophila). Taken together, these results provide the functional characterization of obscure puffer IRGs in immune defense, which is the first study to reveal the function of IRGs in bony fish and will provide important insights into the evolutionary divergence of IRGs.

Introduction

Interferon (IFN) is a strong inducer of antimicrobial effectors that promotes the expression of IFN-stimulated genes (ISGs) to initiate innate immune signals and enhances clearance of the pathogen or restricts viral, bacterial and parasitic replication in the host cell [1]. Among ISGs, the most studied is the IFN-inducible GTPase superfamily, which comprises the myxoma resistance protein (Mx) family, immunity-related GTPase (IRG) family, guanylate binding protein (GBPs) family and very large inducible GTPase (VLIGs/GVINs) family [2,3].

As a subfamily of IFN-inducible GTPases, IRGs in mammals have been well studied and shown to play significant roles in host defense [4]. In particular, IRGs harbor the exceptional ability to defend against intracellular pathogens in host cells [5]. For example, murine Irgm1 (LRG47), a 47-kDa (p47) GTPase, functions as a crucial trafficking vacuolar component in IFNγ-activated macrophages that controls Mycobacterium tuberculosis infection [6]. IRGM, the human Irgm1 ortholog, plays an analogous role to that of murine Irgm1 [7]. Further studies revealed that the mechanism of action for the elimination of intracellular M. tuberculosis is dependent on the induction of autophagy and the generation of autolysosomal organelles [7]. For clearance of another intracellular pathogen, Toxoplasma gondii, murine Irgm1 disrupts pathogen-containing vacuoles by interacting with proteins such as p62, TRAF6 and GBPs and then targeting the vacuole membrane [8,9]. Evolutionary analysis of IRGs shows that IRGs are widely present in vertebrates, implying that IRGs may exert conserved roles in lower vertebrates [7,8]. Our recent comparative study revealed that IRG5 of the Chinese soft-shell turtle (Pelodiscus sinensis) was involved in autophagy induction, which first unveiled the functional role of IRGs in reptiles [9]. However, the function of IRGs from other lower vertebrates, including fish, remains to be elucidated.

As an important aquaculture species in southeast China, obscure puffers (Takifugu obscurus) are popular among people because of their good taste. However, diseases caused by pathogen infection, such as Aeromonas hydrophila and Edwardsiella tarda, threaten the productivity of the fish. Fish infected with A. hydrophila will show a number of symptoms, such as fin rot, tail rot, hemorrhagic septicemia, scale protrusion disease, and ulcer disease [10]. Diseases such as distended abdomen, prolapsed rectum and fibrinous peritonitis are usually caused by E. tarda in fresh and marine fish [11]. With the increase in infectious diseases, research on immune-related genes and their roles in defense against pathogens may contribute to the breeding industry of obscure puffers. Recent studies have identified a few immune-related genes of obscure puffer, such as B-cell activating factor and interferon-γ-inducible-lysosomal thiol reductase [12,13]. Transcriptomic and proteomic analyses of spleen tissue identified 2339 differentially expressed genes and 537 proteins during the response to A. hydrophila infection [14]. However, few reports have focused on IFN-inducible GTPase superfamily genes.

In the present study, the obscure puffer IRG (ToIRG) was identified and characterized. The expression pattern and subcellular localization of ToIRG were analyzed. Furthermore, the in vitro antibacterial activity of ToIRG against E. tarda and A. hydrophila was investigated. Our data demonstrated the role of ToIRG in the antibacterial defense of fish and provided important insights into the evolutionary divergence of IRGs.