It was pretty lame but i really like the fact that the Insects encounter several microbes during different stages of growth and development. Incidental entry of these microbes elicits a host defense response, which mainly consists of aphenol oxidase pathway. The invasion of the microbe is recognised by a specific protein which activates the prophenol oxidase activating enzyme (PPAE). The activated PPAE in turn switches "on" the phenol oxidase, which leads to encapsulation of the invading organism. Understanding the mechanisms of insect immunity will allow us to discover potential targets for inactivating the mechanism of immune action in insects and make them vulnerable to natural microbial infection. We have identified PPAE and phenol oxidase encoding genes from major agricultural pests, H. armigera and Spodoptera litura. A database search for homology to other phenol oxidase of other pests classified S. litura enzymes as phenol oxidase class II. The transcription of gene is induced upon microbial challenge of sixth instar larvae with E. coli and is unresponsive to wound injury. The gene has been cloned and expressed in an E. coli expression vector, pET43. Induction with IPTG resulted in its accumulation into a soluble fraction of cells. The expressed enzyme is catalytically active on dopamine upon activation by cetyl pyridinium chloride. The regulatory role played by this enzyme in insect immunity is being examined.
Insecticidal proteins from Xenorhabdus and Photorhabdus
We have been working on the identification of alternate molecules produced by soil organisms like Xenorhabdus and Photorhabdus with insecticidal activity to enhance the action of Bt toxins and study their mode of action to plan strategies for resistance management in the insects. Xenorhabdus nematophilus is a Gram-negative bacterium belonging to the family enterobacteriaceae. It lives in symbiotic association with entomopathogenic nematodes of the family Steinernematidae. X. nematophilus is pathogenic to a wide range of insects and is currently being used as a biological control agent against Lepidoptera, Coleoptera and Diptera as spray formulations. We have been investigating the insecticidal potential of the proteins secreted by X. nematophilus in the culture medium. We have isolated large protein complexes from the culture medium with oral larvicidal activity. These outer membrane vesicle (OMV) associated proteins, when incorporated in the diet, were found to be active against common lepidopteran pests Helicoverpa armigera and Spodoptera litura. Some of the individual proteins present in the large complex have been characterised. Strong chitinase activity and bacteriocin-like proteins were found to be associated with the protein complex. A 17 kDa pilin protein was isolated, purified and cloned. Pilin protein constitutes hair-like appendages on the surface of a bacterial cell, that mediate host cell recognition and adherence. Interestingly, the evaluation of biological activity of the pilin protein from X. nematophilus, revealed their larvicidal nature for common agricultural pests.
Comparison of the native pilin isolated from the bacteria with the recombinant protein showed that oligomerisation of the protein is critical for larvicidal activity. To understand the basis of oligomerisation, we produced point mutations in the N-terminal region of the pilin gene, and analysed the behaviour of the mutants. Three conserved hydrophobic residues in the N-terminus of the protein were altered individually and together to yield four mutant polypeptides. Comparison of the far UV CD spectrum of native protein with the recombinant protein showed the presence of a negative peak at 215 nm characteristic of an ordered structure comprised of <-sheets, while the CD spectra of the mutant proteins displayed a progressive decrease in the peak intensity