Recognition of microbial molecular patterns and stimulation of prophenoloxidase activation by a β-1,3-glucanase-related protein in Manduca sexta larval plasma

Abstract

Detection of pathogenic invaders is the essential first step of a successful defense response in multicellular organisms. In this study, we have identified a new member of the β-1,3-glucanase-related protein superfamily from the tobacco hornworm Manduca sexta. This protein, designated microbe binding protein (MBP), is 61% identical in sequence to Bombyx mori Gram-negative bacteria binding protein, but only 34–36% identical to M. sexta β-1,3-glucan recognition protein-1 and 2. Its mRNA levels were strongly up-regulated in hemocytes and fat body of immune challenged larvae, along with an increase in concentration of the plasma protein. We expressed M. sexta MBP in a baculovirus-insect cell system. The purified protein associated with intact bacteria and fungi. It specifically bound to lipoteichoic acid, lipopolysaccharide, diaminopimelic acid-type peptidoglycans (DAP-PGs) from Escherichia coli and Bacillus subtilis, but less so to laminarin or Lys-type PG from Staphylococcus aureus. The complex binding pattern was influenced by other plasma factors and additional microbial surface molecules. After different amounts of MBP had been incubated with larval plasma on ice, a concentration-dependent increase in phenoloxidase (PO) activity occurred in the absence of any microbial elicitor. The activity increase was also observed in the mixture of plasma and a bacterial or fungal cell wall component. The prophenoloxidase (proPO) activation became more prominent when DAP-PGs, Micrococcus luteus Lys-PG, or lipoteichoic acid was included in the mixture of MBP and plasma. Statistic analysis suggested that a synergistic enhancement of proPO activation was caused by an interaction between MBP and these elicitors, but not S. aureus Lys-PG, lipopolysaccharide, curdlan, or laminarin. These data indicate that M. sexta MBP is a component of the surveillance mechanism and, by working together with other pattern recognition molecules and serine proteinases, triggers the proPO activation system.

Introduction

Innate immunity is the first line of defense against pathogen infection in all animals including insects (Gillespie et al., 1997, Strand, 2008). It is triggered by host proteins that recognize conserved surface determinants of microbes, such as lipopolysaccharide (LPS) or peptidoglycans (PGs) from bacteria and β-1,3-glucan from fungi. These pattern recognition receptors (PRRs) bind invading cells and initiate rapid cellular and humoral responses. Complex signaling pathways become active and induce the production of antimicrobial peptides and other defense molecules (Ragan et al., 2009, Lemaitre and Hoffmann, 2007). Extracellular serine proteinase cascades coordinate multiple immune responses: one leading to spätzle processing and Toll pathway activation; another causing proteolytic activation of prophenoloxidase (proPO). Active phenoloxidase (PO) then generates quinones that polymerize to form melanin (Nappi and Christensen, 2005). Reactive compounds such as 5,6-dihydroxyindole directly kill pathogenic invaders (Zhao et al., 2007, Kohler et al., 2007).

Biochemical and genetic studies have revealed more than 10 groups of PRRs in invertebrates, including hemolin, peptidoglycan recognition proteins (PGRPs), β-1,3-glucan recognition proteins (βGRPs), C-type lectins, galectins, fibrinogen-related proteins, thioester-containing proteins, Nimrods, scavenger receptors, and Down syndrome cell adhesion molecules (Yu et al., 2002, Lemaitre and Hoffmann, 2007). These proteins bind to surface moieties of pathogens or parasites and initiate a coordinated defense reaction. One of these groups is composed of proteins with a region similar in sequence to β-glucanases from bacteria, fungi, sea urchins, and insects. Some of its members, due to differences in binding properties, are designated βGRPs, LPS- and β-1,3-glucan-binding protein, or Gram-negative bacteria-binding proteins (GNBPs) (Lee et al., 1996, Ochiai and Ashida, 2000, Ma and Kanost, 2000, Lee et al., 2000, Kim et al., 2000, Jiang et al., 2004). Other members of the family have similar names but their binding specificities have not yet been experimentally established.

To better understand the molecular basis of innate immunity in Manduca sexta, we did a pyrosequence analysis of expressed sequence tags for hemolymph proteins (Zou et al., 2008). Of 218 newly discovered cDNA contigs, six encoded polypeptides similar to regions of Bombyx mori GNBP (Lee et al., 1996). In this work, we isolated cDNA clones of the GNBP homolog, characterized the recombinant protein from a baculovirus expression system, and explored its immunological functions. We studied its transcriptional changes in larval hemocytes and fat body after an immune challenge. This protein, named M. sexta microbe binding protein (MBP), specifically bound to microbial cell wall components and triggered proPO activation. Together with other PRRs discovered in the same insect, it contributes to a surveillance mechanism by which the host can detect microbial incursions and stimulate defense reactions.