Identification of Aadnr1, a novel gene related to innate immunity and apoptosis in Aedes albopictus

Highlights

• Aadnr1, an ortholog of Drosophila dnr1 from Aedes albopictus C6/36 cells was identified.

• Aadnr1 was expressed during all the developmental and adult stages.

• Transcriptional level of Aadnr1 was decreased under apoptotic and immune stimuli.

• Silence of AaDnr1 enhanced Act D-induced caspase activity.

• RING domain destabilized AaDnr1 protein.

Abstract

Innate immunity and apoptosis play critical roles in defending pathogens in insects. In Drosophila, Dnr1 was reported as a negative regulator of apoptosis and immune deficiency (Imd) pathway which belongs to innate immunity. Aedes albopictus is an important kind of arbovirus vector and becoming a significant threat to public health due to its rapid global expansion. Here we identified an ortholog of dnr1 from A. albopictus, named as Aadnr1. Aadnr1 encoded a putative protein containing an N-terminal FERM domain and a C-terminal RING domain. AaDnr1 shared high identity with dipteran insects Dnr1 orthologs. Phylogenetic analyses showed that the closest relative of AaDnr1 was Aedes aegypti Dnr1. Real-time PCR proved that Aadnr1 mRNA was expressed ubiquitously during developmental and adult stages. Transcriptional levels of Aadnr1 were decreased drastically in C6/36 cells underwent apoptosis induced by Actinomycin D (Act D) treatment. Partial silence of Aadnr1 enhanced Act D-induced caspase activity. When challenged by heat-inactivated E. coli, transcriptional level of Aadnr1 was also decreased dramatically in C6/36 cells. While when C6/36 cells were infected with Sindbis virus TE/GFP, transcriptional level of Aadnr1 was reduced and recovered repeatedly, with an overall decreasing trend. It was also shown in this study that similar to Drosophila Dnr1, RING domain destabilized AaDnr1 protein. Taken together, the study identified an innate immunity and apoptosis related gene Aadnr1 in A. albopictus.

Introduction

Aedes albopictus is an efficient epidemic viral vector to transmit Dengue virus (DENV) and Chikungunya virus (CHIKV). It is native in Southeast Asia, but spreads around the world rapidly and turns to be a serious threat to current public health (Gratz, 2004, Paupy et al., 2009, Wu et al., 2010, Rezza, 2012, Bonizzoni et al., 2013). The current strategy to prevent arbovirus infection is to control the mosquito's population. Therefore, it is important to elucidate the virus transmission and antiviral strategy of A. albopictus.

In Drosophila, innate immune systems including Toll, Imd and Jak-Stat pathways regulate immune response against bacteria, fungi and viruses. Upon Gram-negative bacterial infection, Imd pathway is triggered by binding of microbial diaminopimelic acid (DAP)-type peptidoglycan to membrane receptors then results in a cascade of events involving Imd, Fas-associated death domain protein (FADD), death-related ced-3/Nedd2-like gene (Dredd) and other proteins, finally increases the expression of antimicrobial peptide (AMP) genes (Myllymaki et al., 2014). Imd pathway is negatively regulated by Caspar and Defense repressor 1 (Dnr1) (Foley and O'Farrell, 2004, Kim et al., 2006, Guntermann et al., 2009). Mosquito possesses a similar Imd pathway (Hillyer, 2015, Viljakainen, 2015). In Aedes aegypti and Anopheles gambiae, the core components of Imd pathway including Imd, FADD, Dredd and Rel2 were characterized (Christophides et al., 2002, Meister et al., 2005, Cooper et al., 2007a, Waterhouse et al., 2007, Antonova et al., 2009, Cooper et al., 2009a). Orthologs of negative regulators such as Caspar and Dnr1 were also reported (Waterhouse et al., 2007, Bryant et al., 2008, Erickson et al., 2009, Garver et al., 2009). Recently, there is a growth of knowledge about mosquito Imd pathway which is implicated in antiviral response. (Ruckert et al., 2014, Sim et al., 2014, Cheng et al., 2016). DENV infection up-regulated cecropin, peptidoglycan recognition protein-lc (PGRPLC) and gram-negative bacteria binding protein (GNBP) of the Imd pathway in A. aegypti salivary glands (Luplertlop et al., 2011). Knockdown of Imd resulted in a significant increase in midgut virus load in DENV-refractory A. aegypti Orl strain (Sim et al., 2013). Knockdown of Rel2 in A. gambiae midgut enhanced the infection of O'nyong'nyong virus (ONNV) (Carissimo et al., 2015). Mosquito immunology is getting more attentions.

Apoptosis is also involved in antiviral immunity in insects (Clarke and Clem, 2003, Blair and Olson, 2014). The executioners of apoptosis are cysteine proteases called cysteine-aspartic proteases (caspases), which are stringently regulated by inhibitor of apoptosis proteins (IAPs), IAP antagonists and others (Thornberry and Lazebnik, 1998). Apoptosis is a highly conserved cellular process for metazoan. In Drosophila, Dronc is an initiator caspase required for almost all the apoptosis and is negatively regulated by DIAP1 and Dnr1 (Dorstyn et al., 1999, Meier et al., 2000, Primrose et al., 2007). In mosquitoes, the Dronc orthologs has been characterized in Aedes aeygpti and identified in A. gambiae, but the detailed regulation mechanisms need to be further investigated (Cooper et al., 2007b, Bryant et al., 2008, Cooper et al., 2009b, Liu and Clem, 2011).

As mentioned above, Drosophila Dnr1 is involved in both innate immunity and apoptosis pathway. Dnr1 contains a RING domain, which is important for E3 ligase activity and targeting other proteins or themselves for ubiquitination and degradation (Foley and O'Farrell, 2004, Deshaies and Joazeiro, 2009). Initially Dnr1 was reported as a negative regulator of Imd pathway because suppression of Dnr1 by RNAi led to transcriptional activation of a Dredd responsive reporter construct (Dipt-lacZ) in Drosophila S2 reporter cell line (Foley and O'Farrell, 2004). Dnr1 negatively regulated Imd pathway by depleting Dredd through RING domain-dependent ubiquitination, and Dredd upregulated Dnr1 to form a feedback loop (Foley and O'Farrell, 2004, Guntermann et al., 2009). Dnr1 also depleted Dronc protein level and inhibited apoptosis in Drosophila S2 cells (Deshaies and Joazeiro, 2009). Experimentally, Dnr1 protein was unstable and RING domain of Dnr1 destabilized itself (Foley and O'Farrell, 2004, Guntermann et al., 2009). It has been reported that A. aegypti Dnr1 (AeDnr1) has similar domain architecture with Dnr1 (Bryant et al., 2008).

In this study, a dnr1 ortholog from A. albopictus named as Aadnr1 was identified. The expression profile of Aadnr1 during developmental and adult stages was analyzed. Similar to Drosophila Dnr1, RING domain of AaDnr1 regulated its own stability. Act D treatment decreased transcriptional level of Aadnr1 in C6/36 cells, and partial silence of Aadnr1 increased Act D-induced apoptosis in C6/36 cells. Also, E. coli challenge and Sindbis virus infection decreased Aadnr1 transcription. Taken together, this study provides the foundation for understanding of the function of Aadnr1 in A. albopictus.