Abstract
The ‘danger model’ is an alternative concept for immune response postulating that the immune system reacts to entities that do damage (danger associated molecular patterns, DAMP) and not only to entities that are foreign (pathogen-associated molecular patterns, PAMP) as proposed by classical immunology concepts. In this study we used Galleria mellonella to validate the danger model in insects. Hemolymph of G. mellonella was digested with thermolysin (as a representative for virulence-associated metalloproteinases produced by humanpathogens) followed by chromatographic fractionation. Immune-stimulatory activity was tested by measuring lysozyme activity with the lytic zone assays against Micrococcus luteus cell wall components. Peptides were analyzed by nano-scale liquid chromatography coupled to high-resolution Fourier transform mass spectrometers. Addressing the lack of a genome sequence we complemented the rudimentary NCBI protein database with a recently established transcriptome and de novo sequencing methods for peptide identification. This approach led to identification of 127 peptides, 9 of which were identified in bioactive fractions. Detailed MS/MS experiments in comparison with synthetic analogues confirmed the amino acid sequence of all 9 peptides. To test the potential of these putative danger signals to induce immune responses we injected the synthetic analogues into G. mellonella and monitored the anti-bacterial activity against living Micrococcus luteus. Six out of 9 peptides identified in the bioactive fractions exhibited immune-stimulatory activity when injected. Hence, we provide evidence that small peptides resulting from thermolysin-mediated digestion of hemolymph proteins function as endogenous danger signals which can set the immune system into alarm. Consequently, our study indicates that the danger model also plays a role in insect immunity.
Highlights
A prerequisite for the evolution of Metazoa was the ability to discriminate between self and nonself
We have identified peptides which are generated in G. mellonella under presence of microbial metalloproteinases, and which mediate the activation of immune responses encompassing the synthesis and release into the hemolymph of both antimicrobial peptides and the insect metalloproteinase inhibitor (IMPI)
In this study we used high-resolution mass spectrometry to identify peptidic hemolymph protein fragments which are generated in the hemolymph of the model host G. mellonella when harmful microbial metalloproteinases belonging to the M4 family, with thermolysin as the prototype, are present
Summary
A prerequisite for the evolution of Metazoa was the ability to discriminate between self and nonself. Vertebrate and invertebrate animals share the ancient innate immunity mediating discrimination between infectious nonself and noninfectious self by a limited number of germ line encoded receptors which bind to molecules common among pathogens, but absent from the host such as microbial cell wall components, bacterial lipopolysaccharides (LPS), peptidoglycans, and fungal ß-1,3-glucans [1]. This binding of receptor proteins to pathogen-associated molecular patterns (PAMPs) differs from the highly specific recognition of a wide range of antigens by a large and diverse spectrum of somatically rearranged receptors of T and B cells which represent an evolutionary novelty of vertebrates. Binding of PAMPs to corresponding pattern recognition receptors results in activation of immune responses including both cellular mechanisms such as phagocytosis or multicellular encapsulation of microbes, and humoral defences such as the rapid synthesis of antimicrobial peptides and proteins [4]
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