Role of SPH-3 a catalytically inactive serine proteinase homologue protein in insect immune defences

Better knowledge of insect immune systems has also led in recent years to important advances in understanding human immunity. Nevertheless, the signaling systems used in recognising and responding to bacteria remain poorly understood. A protein, SPH-3, present in the hemolymph (blood) of a large experimental model insect, Manduca sexta, plays a key role in this insect's ability to defend itself against infection by pathogenic bacteria. SPH-3 is a member of the serine proteinase familiy of proteins, but is catalytically inactive. It appears to play a signalling role in the insect's immune system, but its precise role is unclear. The proposed work seeks to determine with which other immune system proteins SPH-3 interacts, and how it exerts its downstream effects. The work will lead to enhanced understanding of insect immune systems, and will thereby provide information that may also enable better understanding of immunity in man and other mammals. The work is of wide biological interest in that it concerns the biological functions of an enzyme that has evolved to gain a signaling function, but in doing so has lost its original catalytic activity. Further, since the protein in question is the target of a major virulence gene of a widely used agent of biological insect pest control, there may also be applied spin-off.

We have discovered that SPH-3, an inactive serine proteinase homologue secreted into the hemolymph (blood), is essential for induced immune responses in the insect Manduca sexta. RNAi knockdown of SPH-3 has a seriously adverse effect on the ability of the insect defend itself against this bacterium Photorhabdus luminescens. SPH-3 plays a central role in immune defences, as RNAi knockdown prevents the expression in response to microbial challenge of all the humoral effectors that we have examined. In this work, the immune role of SPH-3 will be elucidated in more detail. We will express recombinant SPH-3 and use this to determine whether SPH-3 recognises and binds to microbial cell surfaces and pattern molecules; whether SPH-3 is a component of one or more multiprotein complexes in Manduca hemolymph; whether SPH-3 directly activates PPO; whether SPH-3 is a substrate for hemolymph proteinases, and the cleavage site(s) for these enzymes; whether SPH-3 itself is an immune activating signal specifically with reference to: i. phagocytosis ii. cell adhesion iii. immune related gene expression; whether SPH-3 cleavage products are immune activators; whether SPH-3 has antimicrobial properties and whether SPH-3 expression depends upon previous recognition of bacterial pattern molecules by other recognition proteins. Better understanding of the central role of this immune-related protein will lead to enhanced understanding of insect immune systems, enabling better understanding of immunity in man and other mammals. Further, since the protein in question is the target of a major virulence gene of a widely used agent of biological insect pest control, there may also be applied spin-off.