Abstract
Many members of the nucleotide-binding and oligomerization domain (NACHT)- and leucine-rich-repeat-containing protein (NLR) family play crucial roles in pathogen recognition and innate immune response regulation. In our previous work, a unique and Vibrio splendidus-inducible NLRC4 receptor comprising Ig and NACHT domains was identified from the sea cucumber Apostichopus japonicus, and this receptor lacked the CARD and LRR domains that are typical of common cytoplasmic NLRs. To better understand the functional role of AjNLRC4, we confirmed that AjNLRC4 was a bona fide membrane PRR with two transmembrane structures. AjNLRC4 was able to directly bind microbes and polysaccharides via its extracellular Ig domain and agglutinate a variety of microbes in a Ca2+-dependent manner. Knockdown of AjNLRC4 by RNA interference and blockade of AjNLRC4 by antibodies in coelomocytes both could significantly inhibit the phagocytic activity and elimination of V. splendidus. Conversely, overexpression of AjNLRC4 enhanced the phagocytic activity of V. splendidus, and this effect could be specifically blocked by treatment with the actin-mediated endocytosis inhibitor cytochalasin D but not other endocytosis inhibitors. Moreover, AjNLRC4-mediated phagocytic activity was dependent on the interaction between the intracellular domain of AjNLRC4 and the β-actin protein and further regulated the Arp2/3 complex to mediate the rearrangement of the cytoskeleton and the polymerization of F-actin. V. splendidus was found to be colocalized with lysosomes in coelomocytes, and the bacterial quantities were increased after injection of chloroquine, a lysosome inhibitor. Collectively, these results suggested that AjNLRC4 served as a novel membrane PRR in mediating coelomocyte phagocytosis and further clearing intracellular Vibrio through the AjNLRC4-β-actin-Arp2/3 complex-lysosome pathway.
Highlights
Host innate immunity has emerged as the first line of defense against the invasion of endogenous antigens and foreign pathogens throughout long-term phylogenetic evolution [1]
Phagocytosis is the first step of pathogen clearance and is triggered by specific interactions between host pattern recognition receptors (PRRs) and pathogen-associated molecular patterns (PAMPs) from invasive bacteria
The Ig domain of AjNLRC4 is replaced with a conventional LRR domain to bind V. splendidus, and the intracellular domain of AjNLRC4 interacts with β-actin to mediate V. splendidus endocytosis in an actin-dependent manner
Summary
Host innate immunity has emerged as the first line of defense against the invasion of endogenous antigens and foreign pathogens throughout long-term phylogenetic evolution [1]. Pattern recognition receptors (PRRs) are critical parts of innate immunity and play important roles in initiating innate immune responses by recognizing a variety of pathogen- and dangerassociated molecular patterns (PAMPs and DAMPs, respectively), such as lipopolysaccharide (LPS), peptidoglycan (PGN), flagellin, mannan (MAN) and danger signals, including uric acid, ATP, and HMGB1 [2]. The NACHT domain is the only domain that is common to all NLR family members and is composed of four substructures: neuronal apoptosis inhibitory protein (NAIP), class II transactivator (CIIT), incompatibility locus protein from Podospora anserina (HET-E) and telomerase-associated protein (TP1) This domain enables the activation of the signaling complex via ATP-dependent oligomerization, which is essential for the downstream functions of several NLRs [15,16,17,18]. The NACHT domain can directly interact with other proteins to
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