Involvement of a pattern recognition receptor C-type lectin 7 in enhancing cellular encapsulation and melanization due to its carboxyl-terminal CRD domain in the cotton bollworm, Helicoverpa armigera

Highlights

• Both HaCTL7 and CRD2 own the agglutinate ability against various bacteria.

• Both HaCTL7 and CRD2 enhance encapsulation and melanization processes.

• Endogenous HaCTL7 binds to granular cells, plasmatocytes and oenocytoids.

• Recombinant HaCTL7 and rCRD2 could bind to both granular cells and oenocytoids.

• HaCTL7 enhances encapsulation and melanization likely through its C-terminal CRD2.

Abstract

C-type lectins play important roles in innate immunity as pattern recognition receptors (PRRs). We have previously reported a novel C-type lectin HaCTL7 from the cotton bollworm (Helicoverpa armigera) which contains two carbohydrate-recognition domains (CRDs), namely N-terminal CRD1 and C-terminal CRD2. Interestingly, there are four but not six of conserved cysteine residues in CRD2 of HaCTL7, which is different from that of other dual CRD C-type lectins. In the current study, we expressed and purified recombinant HaCTL7 (rHaCTL7) as well as rCRD1 and rCRD2, and demonstrated that both rHaCTL7 and rCRD2, but not rCRD1, owned the agglutinate ability against both Gram-negative and Gram-positive bacteria in a calcium dependent manner. In addition, both rHaCTL7 and rCRD2, but not rCRD1, could bind to various bacteria, and enhanced haemocytes mediated encapsulation and melanization processes. HaCTL7 secreted from fat bodies is able to bind to granulocytes, plasmatocytes and oenocytoids, but not to spherulocytes. Recombinant HaCTL7 and rCRD2 are capable of binding to both granulocytes and oenocytoids, while rCRD1 can only bind to granulocytes. Our data suggest that as a PRR HaCTL7 enhances encapsulation and melanization likely through its C-terminal CRD2, but not N-terminal CRD1, which imply that the characteristic four cysteine structure of CRD2 plays key roles in innate immunity.

Introduction

Insects rely on innate immunity to defend against pathogen infection (Aggrawal and Silverman, 2007). Recognition and differentiation of nonself from self is the first and critical step. Host proteins termed pattern recognition receptors (PRRs) are responsible for recognition of conserved surface determinants of microorganisms, like lipopolysaccharide (LPS), peptidoglycan (PGN), lipoteichoic acid, fungal β-1,3-glucan and mannan (Medzhitov and Janeway, 2002, Pal and Wu, 2009). Innate immunity including cellular and humoral responses is initiated rapidly following recognition. Cellular responses include phagocytosis, encapsulation and nodule formation while humoral responses are composed of the synthesis of antimicrobial peptides and prophenoloxidase activation (Lemaitre and Hoffmann, 2007, Williams, 2007).

It was demonstrated that there were five kinds of circulating haemocytes (namely prohaemocytes, plasmatocytes, granulocytes, spherulocytes and oenocytoids) which could be classified by morphological characters in lepidopteran insects including the cotton bollworm Helicoverpa armigera (Lavine and Strand, 2002, Ribeiro and Brehélin, 2006, Zhai and Zhao, 2012). For example, granulocytes with the main function of phagocytosis comprise numerous granules, while plasmatocytes spread rapidly when cultured in vitro and appear as spindle-shape cells (Ribeiro and Brehélin, 2006, Zhai and Zhao, 2012). Oenocytoids as large round cells with low nucleus-to-cytoplasm ratio can synthesize prophenoloxidase (Jiang et al., 1997, Ribeiro and Brehélin, 2006, Zhai and Zhao, 2012).

Encapsulation is a process in which haemocytes attach to the invader and it was reported that granulocytes and plasmatocytes are involved (Pech and Strand, 1996). Melanization requires converting of prophenoloxidase to active phenoloxidase which generates quinones that polymerize to form melanin (Nappi and Christensen, 2005). Extracellular serine proteinase cascades are involved in the phenoloxidase activation (Cerenius and Söderhäll, 2004). Encapsulation followed by melanization is an important defense mechanism and usually employed to killing metazoan parasites which are too large to be phagocytosed (Strand and Pech, 1995, Nappi and Vass, 1993). The process also called melanotic encapsulation requires participation and coordination of a large number of cellular and humoral factors, especially those responsible for pathogen recognition. Wang et al. (2005) demonstrated that RNA interference of β 1,3-glucan recognition proteins (GRP) gene could inhibits melanotic encapsulation of the filarial worm Dirofilaria immitis strongly in the mosquito. C-type (calcium-dependent) lectins belong to one major family of PRRs have multiple functions. To date, C-type lectins identified in insects contain one or two carbohydrate recognition domains (CRDs). Two Drosophila C-type lectins DL2 and DL3 with a single CRD could enhance haemocytes mediated encapsulation and melanization (Ao et al., 2007). There were four dual CRD C-type lectins named immulectins (MsIML-1, 2, 3, and 4) reported in the hornworm Manduca sexta (Yu et al., 2006). MsIML-2 and its carboxyl-terminal CRD could enhance haemocytes mediated encapsulation of beads in vitro and melanization of either beads or the nematode in vivo (Yu and Kanost, 2004). MsIML-4 functions in enhancing encapsulation and melanization (Yu et al., 2006), whereas MsIML-1 and -3 are only involved in enhancing encapsulation (Ling and Yu, 2006a, Yu et al., 2005).

In H. armigera, Chai et al. (2008) cloned and characterized the first C-type lectin (here renamed HaCTL1) in the cotton bollworm. Tian et al. (2009) further studied the function of HaCTL1 and its two CRDs in agglutinating, sugar binding and haemocyte mediated phagocytosis. C-type lectin 2 of H. armigera (GenBank accession No. ACI32834) was identified from the curdlan pull-down (Pauchet et al., 2009). Recently, we isolated another six novel C-type lectin genes (HaCTL3-8) from H. armigera, and analyzed the expression profiles of HaCTL3-8 in response to bacterial and 20-Hydroxyecdysone (20E) injection (Wang et al., 2012). Interestingly, two C-type lectins HaCTL7 and HaCTL8 own a characteristic four but not six cysteine structure at the C-terminal CRD which is different from that of all dual CRD C-type lectins identified previously (Wang et al., 2012).

To determine the function of HaCTL7 and its individual CRDs, we investigated the probable involvement of the proteins in bacterial agglutinating, cellular encapsulation and melanization. We also assayed the binding ability of HaCTL7 and its individual CRDs to various bacteria and haemocytes. Our results indicated HaCTL7 could agglutinate both Gram-negative and Gram-positive bacteria in the presence of calcium. HaCTL7 also bound to various bacteria. HaCTL7 bound to haemocytes could stimulate cellular encapsulation and melanization. Taken together, we suggest that HaCTL7 functions in agglutination, enhancing encapsulation and melanization through its C-terminal CRD2 which has four conserved cysteine residues.