Insects were able to develop their rich species diversity during evolution not least due to their efficient immune system, which protects them from natural enemies and allows many species to colonize even extreme habitats. A major task of the insects’ immune system is the defense against microorganisms and parasites, which it meets by the production of numerous antimicrobials.
While the number of bacteria resistant to antibiotics is continuously increasing, only few newly developed antibiotics are available due to the high expenditure of time and money. Antimicrobial peptides (AMPs) from insects with different modes of action might qualify as alternative agents for fighting human-pathogenic microorganisms. Insect AMPs, which exhibit activity against phytopathogenic fungi or insect pests, can be used in plant protection. Another goal would be the discovery of antimicrobials, which are suitable for food preservation.
Our research aims at the identification and characterization of insect AMPs, which can be utilized for the above mentioned areas of application. The recombinant production of selected AMPs allows the subsequent analysis of their activity spectra and modes of action.
Insects produce many antimicrobial peptides when their immune system is challenged. These peptides may show activity against human pathogens, but in order to test them it is necessary to produce these molecules in larger quantities using heterologous expression platforms. Bacterial expression systems usually achieve high yields of recombinant proteins with relatively little effort, but in many cases they are not suitable for the production of antimicrobial peptides because the products are toxic to the host cells, making cultivation impossible. Additional difficulties arise when the peptides require posttranslational modification (e.g. the formation of disulfide bonds). In such cases, insect cells offer an ideal alternative for production.
There are two different insect cell expression systems established in the Fraunhofer Bioresources Project Group, both of which can be used for the production of antimicrobial peptides. In the Drosophila Expression System (DES), the gene of interest is stably integrated into the genome, and when the promoter is activated, the gene is transcribed and the protein is produced. In the Baculovirus Expression Vector System (BEVS), recombinant insect viruses are used to introduce the gene of interest into moth cells. The virus promoter ensures strong transgene expression during the late phase of the infection cycle. In both expression systems, the recombinant proteins are usually secreted into the culture supernatant. from which they can be isolated and purified by chromatography.
The Peptides are isolated and purified from the cell culture media or from bacterial lysate by chromatography. In order to determine the peptides' activity spectra, their effect on the growth of different microorganisms like bacteria, yeast and fungi is investigated.
X-Tox peptides have been isolated from different genera of only Lepidoptera. Structurally, they consist of several cysteine-stabilized alpha beta (CS-αß) motifs ranging from 4 to 11 (as known so far), which refers to as X in X-Tox terminology. Despite the notable induction of X-Tox gene expression upon microbial challenges, apparently these peptides lack antimicrobial properties. In order to characterize these peptides, we are focused in-part on recombinant production of X-Tox peptides in insect cell lines and their purification from the culture supernatant to eventually analyze their biological activities using insect models. Based on X-Tox peptides’ structural similarity to scorpion toxins, we assume that one of the X-Tox biological significance may perhaps be disturbing the function of ion channels in parasitoids of Lepidopterans. This hypothesis is currently under investigation using Apis mellifera, Schistocerca gregaria, and Galleria mellonella models.
Antimicrobial peptides (AMPs) of different kinds contribute to host immunity in microbial challenges based on their specificities to different target proteins. Additionally, they have immune-modulatory functions. Through different modes of action, AMPs affect the susceptible microorganisms by intemperance of trans-membrane electrochemical ion gradients, inhibition of protein and/or DNA syntheses, membrane permeabilization and rupture, induction of reactive oxygen species, etc. Therefore, identification of the target proteins of AMPs in microorganisms helps us better understand the pathophysiology of diseases. On the other hand, elucidation of action modes of AMPs contributes to developing effective pharmaceutical leads in order to deal with multidrug resistance of super-microbes. In our lab, we are using different techniques, e.g. microscopy, biochemical analytical tools, yeast two-hybrid system, etc. to identify target proteins of different AMPs in microorganisms.
Prof. Dr. Andreas Vilcinskas
Aphids take up sieve element sap from plants. This diet has a low content of essential amino acids. For this reason, aphids show a close symbiosis with bacteria. Especially the endosymbiotic bacteria of the species Buchnera aphidicola are of importance in this context, because they metabolize non-essential into essential amino acids. Elimination of these bacteria by application of antibiotics to the aphid shows a reduction of growth, development and reproduction.
The aim is to induce such an effect by feeding of antimicrobial peptides. Reduced aphid fitness leads to lower infestation of crops, potentially below an economically relevant infestation level, resulting in reduced application of insecticides.