Lipopolysaccharide‐Binding Proteins and their Involvement in the Bacterial Clearance from the Hemolymph of the Silkworm Bombyx Mori

Abstract

Proteins having the ability to bind to Escherichia coli K12W3110 (rough (R) mutant) were isolated and purified by affinity precipitation from the larval hemolymph of the silkworm Bombyx mori. These proteins were found to consist of two components with molecular masses of 43 kDa and 40 kDa by SDS/ PAGE. They bound to all E. coli R mutants (Ra, Rb,, Rc, Rd, and Re) and Salmonella minnesota R mutants. However, they did not bind to smooth types of the above bacteria. They bound to both lipopolysaccharide(LPS)-coated and lipid-A-coated microtiter plates and have similar dissociation constants for LPS and lipid A. This indicates that the binding proteins recognize the lipid A portion of LPS and thus, we have named these proteins BmLBP (B. mori LPS-binding proteins). We also found that BmLBP participated in the clearance of E. coli cells injected into the body cavity of the silkworm.Keywords: pattern recognition; lipopolysaccharide; insect ; immunity.Insects have not acquired adaptive immunity which is based on clonal selection of individual specific lymphocytes, yet they have an efficient defense mechanism against invading microorganisms [ 1-31, This defense mechanism involves both cellular and humoral responses. The cellular response includes phagocytosis and/or nodule formation of bacteria and encapsulation of larger parasites by blood cells (hemocytes) [4]. The humoral response utilizes various antimicrobial peptides synthesized in the fat body (a functional homologue of the mammalian liver) and some hemocytes upon induction by septic injury and secreted into the hemolymph [S, 61. These responses are called innate immunity. Information is scanty on the initial step of these responses, that is, the recognition mechanism of invading microorgani sms .Microbes have highly conserved and widely distributed features, certain of which are not found on the cells of multicellular organisms. These features are called microbial pattern with a recognition system termed pattern recognition [7, 81. Pattern recognition is an important selfhon-self discrimination system in innate immunity in various animals including mammals, and the molecules that function in recognizing microbial pattern are called pattern-recognition molecules. It is known that gramnegative bacteria have a characteristic structure, lipopolysaccharide (LPS), on the outer membrane. In mammals, two effector molecules, LPS-binding protein (LBP) and CD14 are known as LPS-recognition proteins and play a role in pattern recognition [9]. In insects, the American cockroach Peripluneta americana has two LPS-recognition proteins, LBP [lo, 111 and Periplanteta lectin [12, 131. They recognize the outer core oligosaccharide of Escherichia coli K12 and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), respectively, and function as an opsonin [13, 141. In the Mediterranean fruit fly Ceratitis capitata, a protein known as p47 participates in the binding of LPS to hemocytes and the internalization of the bound LPS [IS]. Recently, gramnegative-bacteria-binding protein (GNBP) which was...

Immulectin-2, a Lipopolysaccharide-specific Lectin from an Insect, Manduca sexta, Is Induced in Response to Gram-negative Bacteria

“…These results are similar to those found with insect proteins related to IML-2. B. mori LPS-binding protein and the individual recombinant CRDs of H. cunea lectin have been shown to bind to bacterial LPS (14,19).…”

“…M. sexta IML-2 is 55% identical in sequence to B. mori lipopolysaccharide-binding protein, 47% identical to H. cunea lectin, and only 27% identical to M. sexta IML-1. These insect C-type lectins, all of which bind to bacterial LPS (14,16,19) and are made up of two CRDs, form a distinct group, differing from most animal C-type lectins that contain a single CRD. It is known that single C-type CRDs have weak affinity for carbohydrates and that multivalent interactions of mammalian Ctype lectin oligomers are responsible for their specific binding at the cell surface of pathogens (5).…”

“…These C-type lectins function in insect innate immune system by participating in hemocyte nodule formation (14,15), activating prophenol oxidase in hemolymph (16), and opsonization (17). Among these insect lectins is a group of C-type lectins that contain two tandem CRDs.…”

Evidence of a bactericidal permeability increasing protein in an invertebrate, theCrassostrea gigas Cg-BPI

“…In invertebrates, LPSbinding proteins (LBP) participate in the transduction of cellular signals from LPS. LBPs have been characterized in the freshwater crayfish Pacifastacus leniusculus (3), the shrimp Litopenaeus stylirostris (4), the earthworm Eisenia foetida (5), and the silkworm Bombyx mori (6). In mammals, LBP is an acute phase plasma protein constitutively secreted by liver that induces cellular responses (7).…”

A proteomics approach for the analysis of hemolymph proteins involved in the immediate defense response of the soft tick, Ornithodoros savignyi, when challenged with Candida albicans

“…The strategy used in our study was based on the method described by Zhu et al (2005) who were able to identify a functional homolog of vertebrate complement 3 in the hemolymph of the horseshoe crab, Carcinoscorpius rotundicauda, using Grampositive Staphylococcus aureus. Using a similar strategy Koizumi et al (1997) isolated a protein involved in the clearance of E. coli from the larval hemolymph of the silkworm Bombyx mori.…”