The Zebrafish Antiapoptotic Protein BIRC2 Promotes Edwardsiella piscicida Infection by Inhibiting Caspases and Accumulating p53 in a p53 Transcription-Dependent and -Independent Manner.

Lu Cao,Jun Xiao,Ming Xian Chang,Dong Yan,Hao Feng

doi:10.3389/fimmu.2021.781680

Abstract

IAPs (inhibitors of apoptosis) are endogenous caspase inhibitors with multiple biological activities. In the present study, we show functional characteristics of antiapoptotic protein BIRC2 (cIAP1) in response to Edwardsiella piscicida infection. Overexpression of BIRC2 in zebrafish larvae promoted the proliferation of E. piscicida, leading to a decreased larvae survival. The expression levels of caspases including casp3, casp8, and casp9 were significantly inhibited by BIRC2 overexpression in the case of E. piscicida infection. Treatment of zebrafish larvae microinjected with BIRC2 with the caspase activator PAC-1 completely blocked the negative regulation of BIRC2 on the E. piscicida infection, with the reduced inhibition on the casp3 and without inhibition on casp8 and casp9. In contrast to the regulation of BIRC2 on the caspases, BIRC2 overexpression significantly induced the expression of p53, especially at 24 hpi. In addition to the cytoplasmic p53 expression, BIRC2 overexpression also induced the expression of the nuclear p53 protein. Further analysis demonstrated that BIRC2 could interact and colocalize with p53 in the cytoplasm. The numbers of E. piscicida in larvae overexpressed with BIRC2 and treated with pifithrin-μ (an inhibitor of mitochondrial p53) or pifithrin-α (an inhibitor of p53 transactivation) were lower than those of larvae without pifithrin-μ or pifithrin-α treatment. Critically, the p53 inactivators pifithrin-μ and pifithrin-α had no significant effect on larval survival, but completely rescued larval survival for zebrafish microinjected with BIRC2 in the case of E. piscicida infection. Collectively, the present study suggest that piscine BIRC2 is a negative regulator for antibacterial immune response in response to the E. piscicida infection via inhibiting caspases, and accumulating p53 in a p53 transcription-dependent and -independent manner.

Highlights

The inhibitor of apoptosis proteins (IAPs), known as baculovirus IAP repeat (BIR)-containing proteins (BIRCs), are a family of proteins that are evolutionarily conserved in plants, animals and microorganisms [1–3]
The results show that zebrafish BIRC2 is grouped with common carp and channel catfish BIRC2 proteins, with bootstrap value 100% (Figure 1B)
The results from quantivative RT-PCR (qRT-PCR) showed that the transcript level of zebrafish BIRC2 sharply increased from 6 to 12 hpf, decreased from 12 hpf to 2 dpf, maintained a relatively stable level from 2 to 5 dpf, and increased at 7 dpf (Figure 2A)

Summary

Introduction

The inhibitor of apoptosis proteins (IAPs), known as baculovirus IAP repeat (BIR)-containing proteins (BIRCs), are a family of proteins that are evolutionarily conserved in plants, animals and microorganisms [1–3]. BIRC2, BIRC3, and BIRC4, which own three tandem BIR domains, one ubiquitin associated (UBA) domain and one C-term RING domain, can bind caspases and IAP antagonists through the BIR domains [4]. Different from BIRC4, while BIRC2 and BIRC3 bind to caspase-3, caspase-7, and caspase-9, they do not inhibit proteolytic activities of caspases [5, 6]. BIRC2 and BIRC3 were shown to protect cells from apoptosis by acting as E3 ubiquitin ligases for effector caspases and receptor-interacting protein 1 (RIP1), whose E3 ligase activity was conferred by their Cterminal RING domain [6, 7]. BIRC2 and BIRC3 contain a central caspase-associated recruitment domain (CARD). The E3 ligase activity of BIRC2 mediated by the RING domain is inhibited by the CARD of BIRC2 [9]

Methods

Results

Conclusion