Molecular characterization of NLRC3 and its interaction with other inflammasome components and regulation on the bacterial colonization in Qihe crucian carp Carassius auratus

Abstract

The NLRC as a very unique subfamily of Nod like receptors (NLRs), is believed to play an important role in the bacterial recognition of animals. However, the molecular characterization and immunological role of NLRC3 in Carassius auratus is little known. In this study, we identified and achieved a complete cDNA sequence of NLRC3 gene in Qihe crucian carp Carassius auratus (named as CaNLRC3). The full-length cDNA sequence of CaNLRC3 was composed of 3823 bp, which contained a 5′-UTR of 251 bp, a 3′-UTR of 158 bp, and an open reading frame (ORF) of 3414 bp encoded 1137 amino acids with a predicted isoelectric point of 8.25 and a molecular mass of 124.1 kDa, characterized with a caspase recruitment domain (CARD) at N-terminus. The mRNA expression of CaNLRC3 was detected to be constitutive in all the examined tissues, with the high expression levels in spleen, skin and intestine. After challenges with bacteria or pathogenic analogue, expression levels of CaNLRC3 gene were strongly induced. Co-localization and co-immunoprecipitation results found that CaNLRC3 can assemble CaASC through CARD domain interaction, then CaASC associated with CaCaspase-1a, presumably to assemble the NLRC3 inflammasome complex. The overexpression of CaNLRC3 could significantly increase the mRNA expression of IL-1β, and promote the bacterial elimination and result in the decrease of bacterial loading in liver, spleen and kidney after bacterial infection. Vice versa, the knockdown of CaNLRC3 could obviously reduce IL-1β expression at mRNA level, and bacterial loading was significantly increased in tissue. Taken together, CaNLRC3 is proved to be a pivotal cytosolic innate immune receptor in this study, which is acted as the potential component of inflammasome to regulate inflammation reaction, and could modify bacterial loading in tissue and restrict bacterial infection in teleost.