Beschreibung

• Acylurea compounds are highly potent insecticides that selectively inhibit chitin synthesis. Insecticide potency derives mainly from defects in chitin formation and its consequences of an abnormal endocuticular deposition and abortive molting. Today, acylureas play an important role in integrated pest management. Although it is well established that acylureas interfere in chitin synthesis, the precise mechanism of action is unknown. Several lines of experiments argue that acylurea compounds do not directly inhibit catalysis as evinced in cell-free chitin synthesizing insect systems. Rather than inhibiting the chitin synthase directly, acylureas may alter either vesicle transport or fusion, block the substrate supply for the chitin synthase, inhibit translocation of chitin polymers across the membrane or interfere in the hormonal regulation of chitin synthesis by influencing ecdysteroid production. Our previous studies revealed that the stored product pest Tribolium castaneum (red flour beetle) is a good model organism for investigating the mode of action of bioactive materials such as acylurea and other insecticides. We propose a comprehensive study utilizing genomic, proteomics, biochemical and immunological approaches to clarify the mode of action of insecticidal acylurea compounds.

Projektlaufzeit

• 01.01.2011 - 30.09.2016

Ergebniszusammenfassung

• We aimed to provide insights into the molecular mechanisms of chitin synthesis, a process which is essential to growth and development of numerous organisms including fungi and insects. Chitin synthesis, transport and deposition are complex processes, in which single chitin chains are produced in the cytoplasm and translocated across the membrane to reach the extracellular space, where they assemble into microfibrils and get cross-linked with components of the extracellular matrix. Apart from the fascination with the underlying molecular mechanisms, there are also agricultural and economical dimensions to chitin synthesis as far as they relate to the control of fungal pathogens and arthropod pests, and the application of chitinous polymers and scaffolds in medicine. In the two past funding periods we investigated the structure and function of the insect chitin synthase, and the enzymes’ zymogenic nature including peptidases that are involved in maturation. We found that the chitin synthase exists in an active oligomeric state and identified chymotrypsin-like peptidases that bind to the chitin synthase and stimulate chitin synthesis. We further showed that chitin synthesis inhibitors, which are in wide use as insecticides, do not inhibit the catalytic activity of the purified chitin synthase complex. Rather, we obtained evidence that chitin synthesis inhibitors act on a pre- or post-catalytic step involving a conserved transmembrane helix of the C-terminal domain of the chitin synthase. In a comprehensive genomic and proteomic study, we further analysed the insecticidal effects of the chitin synthesis inhibitor diflubenzuron in the genomic model of Tribolium castaneum, illustrating that diflubenzuron treatment causes a wide range of effects at the molecular level, but does not alter the expression of genes directly involved in chitin metabolism. As the chitinous peritrophic matrix has important anti-infectious functions in the insect midgut, we examined the role of chitin-binding peritrophic matrix proteins (PMPs) in T. castaneum, and identified two PMPs that help to maintain structural integrity and barrier function. In the genetic model of Saccharomyces cerevisiae, we investigated the chitin synthase III complex, particularly its regulatory subunit Chs4, a CaaX protein, which gets cleaved by the CaaX peptidase Ste24 after being prenylated at the cysteine residue. We showed that membrane association of Chs4 depends on prenylation but not on subsequent steps of CaaX processing, and that Ste24 is required for proper localization of Chs3 at the bud neck. Thrombin accessibility assays using mutagenized versions of Chs3 with cleavage sites strategically placed at different positions helped to determine the topology of Chs3. As sulfonylurea receptors belonging to the ABC superfamily have been suggested to be target sites for diflubenzuron, we became interested in the function of ABC transporters in insects, not least because several of them have been implicated in insecticide resistance. We performed an RNA interference (RNAi) screen to study the function of ABC transporters during T. castaneum development. In ten cases, injection of double-stranded RNA into larvae caused developmental phenotypes. We identified ABC genes that are involved in the transport of ecdysteroids, cuticular lipids and eye-pigments. In addition, two genes that encode ABC proteins known to control protein biosynthesis may be suitable targets for RNAi-based strategies of future pest control regimes.