An essential early step in an immune response is recognition of an invading pathogen as dangerous non-self (Janeway and Medzhitov, 2002; Matzinger, 2002; Sansonetti, 2006). Surface molecules of potential pathogens such as bacteria and fungi include ß-1,3-glucan, peptidoglycan, lipopolysaccharide (LPS), and lipoteichoic acid (LTA), which can be recognized by insect pattern-recognition proteins (Yu et al, 2002; Kurata et al, 2006). Binding of these pattern-recognition proteins in turn leads to activation of other immune responses. Endogenous danger signals, molecules that are only released from cells under conditions like injury or infection (Matzinger, 2002), are starting to be recognized in insects. Fragments of collagen and nucleic acids can elicit immune responses in G. mellonella (Altincicek and Vilcinskas, 2006; Altincicek et al, 2008). Our discussion will focus on known pattern-recognition receptors.
Hemolin is a 48 kDa protein found so far only in lepidopteran species (Faye and Kanost, 1998). It contains four immunoglobulin domains, which form a horseshoe-like three-dimensional structure (Su et al., 1998). The sequence of hemolin is most similar to the four immunoglobulin-domains at the N-terminal end of the cell-adhesion protein, neuroglian, which is expressed in neural cells and also in a population of plasmatocytes in M. sexta (Zhuang et al, 2007). M. sexta hemolin can bind to many different bacterial surface components including LPS and LTA (Yu and Kanost, 2002), as well as to haemocytes (Ladendorff and Kanost,
1991). We speculate that the effects of hemolin on haemocyte adhesion (Ladendorff and Kanost, 1991; Bettencourt et al, 1997) may be through binding to neuroglian, or to neuroglian ligands such as integ-rin, on haemocyte surfaces (Zhuang et al, 2007). A haemocyte membrane-associated form of hemo-lin has also been observed in Hyalophora cecropia (Bettencourt et al, 1997). Hemolin is present at low levels in naïve haemolymph, but is upregulated in response to bacterial challenge. In naïve insects, hemolin expression increases in both fat body and midgut during the wandering stage (Yu and Kanost, 1999). Recent RNA interference (RNAi) studies showed that knockdown of hemolin expression led to decreased phagocytosis and nodulation of Escherichia coli (Eleftherianos et al, 2007). Hemolin has also been proposed to be involved in antiviral defence (Terenius, 2008).
Another class of proteins that binds both haemo-cytes and microbial polysaccharides are calcium-dependent (C-type) lectins. In M. sexta, four soluble C-type lectins with two carbohydrate-recognition domains have been characterized and named immulectins 1-4 (IML-1-4) (Yu et al, 1999, 2005, 2006; Yu and Kanost, 2000). Similar tandem-domain C-type lectins are found in other lepidopterans and are involved in binding bacteria and haemocyte aggregation (Koizumi et al, 1999; Shin et al., 2000; Watanabe et al., 2006; Chai et al., 2008). Expression of all four M. sexta IMLs is upregulated in the fat body upon immune challenge (Yu et al., 2002, 2005, 2006). IML-1 and IML-4 agglutinate Gram-positive and Gram-negative bacteria and yeast (Yu et al., 1999, 2006). IML-2 causes aggregation of Gramnegative bacteria (Yu and Kanost, 2000) and binds to haemocytes (granular cells and oenocytoids), as well as nematodes (Yu and Kanost, 2004). IML-2 can bind to a wide range of microbial surface molecules, including LTA, laminarin (branched ß-1,3-glucan), mannose, and LPS (Yu and Ma, 2006). Depletion of IML-2 by addition of IML-2 antibodies decreases bacterial clearance and makes larvae more susceptible to bacterial infection with Serratia marcesens (Yu and Kanost, 2003). The importance of IML-2 during an immune response was also shown by RNAi against IML-2, which led to a decrease in the length of M. sexta survival after infection by either of two Photorhabdus species (Eleftherianos et al, 2006a, 2006b).
IML-3 and IML-4 both bind to LPS, LTA, and laminarin (Yu et al., 2005, 2006). Coating agarose beads with carbohydrate-recognition domain 2 of IML-2 or full-length IML-3 or IML-4 stimulated increased haemocytic encapsulation of those beads (Yu and Kanost, 2004; Yu et al, 2005, 2006). IML-2 and IML-4 also enhanced melanization of encapsulated beads (Yu and Kanost, 2004; Yu et al., 2006). Immulectins bind microbial surfaces and haemocytes and participate in aggregation and encapsulation responses; this evidence indicates that immulectins are pattern-recognition proteins important for cellular responses. IML-1 and -2 also have a role in the melanization response, as they increase phenoloxidase activation in haemolymph (Yu et al, 1999; Yu and Kanost, 2000), and IML-2 binds to cleaved serine protease homologues (Yu et al, 2003).
3.3.3 P-1,3-Glucan-recognition proteins/ Gram-negative-binding proteins
P-1,3-Glucan-recognition proteins (PGRPs) and Gram-negative-bacteria-binding proteins (GNBPs) form a family of pattern-recognition proteins that bind P-1,3-glucan, LPS, or both, and trigger proPO activation and antimicrobial peptide synthesis (Beschin et al, 1998; Lee et al, 2000; Ma and Kanost, 2000; Ochiai and Ashida, 2000; Kim et al., 2000b; Fabrick et al, 2003; Jiang et al, 2004). PGRPs were first identified in B. mori (Ochiai and Ashida, 1988, 2000) and have an N-terminal glucan-binding domain and a catalytically inactive glucanase domain at the C-terminus (Kanost et al, 2004). PGRPs stimulate proPO activation in the presence of P-1,3-glucans (Jiang et al., 2004; Ma and Kanost, 2000). The functions of the two individual domains from PGRP from the moth Plodia interpunctella have been investigated (Fabrick et al., 2004). The N-terminal domain alone can bind to P-1,3-glucans (curdlan or laminarin), LPS, and LTA, and the C-terminal domain binds to branched P-1,3-glucans (laminarin). However, full-length P. interpunctella PGRP is required for agglutination of bacteria.
Two PGRPs have been characterized in M. sexta. Both bind and aggregate yeast, Gram-negative bacteria, and Gram-positive bacteria. However, their expression patterns differ. PGRP1 is not induced by immune challenges and is constitutively expressed during the feeding and wandering stages (Jiang et al., 2004; Ma and Kanost, 2000). PGRP2 is only expressed in feeding-stage larvae after wounding or immune challenge, yet is strongly expressed in naïve insects starting at the wandering stage (Jiang et al., 2004). PGRP-mediated recognition of fungi stimulates autoactivation of a serine protease that triggers proPO activation in M. sexta (Wang and Jiang, 2006).
Lee et al. (1996) isolated a B. mori GNBP from plasma by taking advantage of its binding to Gramnegative bacteria. Related proteins, which form a branch of the PGRP family, have been identified by DNA sequencing in a number of insect species (Fabrick et al, 2003). The D. melanogaster GNBP1 binds LPS and P-1,3-glucan (Kim et al., 2000b) and is required for Toll activation in response to Gram-positive bacteria (Pili-Floury et al., 2004). D. melanogaster GNBP1 forms complexes with peptidoglycan-recognition proteins PGRP-SA and PGRP-SD during Toll activation (Wang et al., 2006, 2008). GNBP1 from the beetle Tenebrio molitor also interacts with a PGPR-SA and triggers proPO activation (Kim et al., 2008).
3.3.4 Peptidoglycan-recognition proteins (PGRPs)
Peptidoglycan, a component of bacterial cell walls, is a polymer of a repeating disaccharide unit, P-1,4-linked N-acetylglucosamine and N-acetylmuramic acid. These polymer chains are cross-linked by short peptides in which the third residue is a meso-diaminopimelic acid (DAP) group, as in Gramnegative bacteria and Bacillus species, or another amino acid, often a lysine, in many Gram-positive bacteria. Gram-negative bacteria and most Bacillus species have a direct cross-link between the DAP group in position three and a D-Ala in position four of the other chain. However, Gram-positive bacteria show diversity in the length and composition of the interpeptide bridge that links the two peptidoglycan chains (Schleifer and Kandler, 1972).
Peptidoglycan can be detected by PGRPs, which are present in invertebrates and vertebrates (Dziarski and Gupta, 2006). PGRP was discovered in B. mori and found to activate the proPO cascade in the presence of peptidoglycan (Yoshida et al, 1996; Ochiai and Ashida, 1999). Studies initiated using plasma proteins from caterpillars also demonstrated that PGRP sequences are related to bacteriophage lyso-zymes and are conserved in mammals, as well as in insects (Kang et al., 1998).
Thirteen PGRP genes are present in the D. mela-nogaster genome. Different PGRPs can recognize different types of peptidoglycan (Kurata et al., 2006; Wang and Ligoxygakis, 2006). Many of the long forms of PGRPs (PGRP-Ls) are transmembrane proteins with an extracellular PGRP domain, whereas short PGRPs (PGRP-Ss) are secreted (Werner et al., 2000). PGRP-LE appears to function in proPO activation (Takehana et al, 2002). PGRP-LC and PGRP-LE stimulate activation of the IMD pathway leading to synthesis of antimicrobial peptides in response to DAP-type peptidoglycan (Choe et al., 2002; Gottar et al, 2002; Takehana et al, 2004). PGRP-SA binds Gram-positive bacteria and activates the Toll pathway (Michel et al., 2001) through a mechanism that involves GNBP1 (Gobert et al, 2003). PGRP-SD also activates the Toll pathway in response to some Gram-positive bacteria (Bischoff et al., 2004) and, to a lesser degree, Gram-negative bacteria (Leone et al., 2008). PGRP-SD interacts with GNBP1 and PGRP-SA (Wang et al, 2008).
Recently it was determined that B. mori contains six short and six long PGRPs (Tanaka et al., 2008). There are at least three short, secreted PGRPs in M. sexta, of which PGRP-1 has been most studied. We expect additional PGRPs will be found in M. sexta, including long PGRPs. PGRP-1 expression is immune-inducible (Zhu et al, 2003a), and it increases from 2 pg/ml in naive larval plasma to 60 pg/ml in plasma from larvae 24 h after injection of bacteria (Yu et al., 2002). RNAi knockdown of M. sexta PGRP-1 increased susceptibility to Photorhabdus species (Eleftherianos et al, 2006a, 2006b). PGRP-1 binds to the surface of Gramnegative bacteria and Bacillus thuringiensis, suggesting that it binds to DAP-type peptidoglycan (Ragan, 2008). PGRP-A from Samia cynthia ricini, a wild silkworm, has high sequence similarity to
M. sexta PGRP-1 and was purified based on binding to Bacillus subtilis cell walls. It binds to DAP-type cross-linked peptidoglycan (from Bacillus cell wall) and to non-cross-linked Lys-type pep-tidoglycan (from Micrococcus luteus) but not very strongly to cross-linked Lys-type peptidoglycan (from M. luteus) (Onoe et al., 2007). Additional studies of M. sexta PGRP-1, -2, and -3 underway have indicated that all three PGRPs bind E. coli and to purified peptidoglycan from M. luteus (H. Jiang and J. Sumathipala, unpublished results). Non-cross-linked peptidoglycan has been shown to be a powerful inducer of lysozyme and bactericidal activity in M. sexta and B. mori (Kanost et al., 1988; Iketani and Morishima, 1993; Iketani et al., 1999). In T. molitor, partial lysozyme digestion of peptido-glycan from M. luteus and Staphyloccocus aureus enhanced binding of PGRP-SA (Park et al., 2007). These results suggest that the interpeptide bridges found in peptidoglycan from many Gram-positive bacteria prevent effective PGRP binding and that M. sexta PGPR-1 and Samia PGPR-A may recognize peptidoglycan regardless of the presence of DAP or Lys in the third position of the peptide.
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