High-Resolution Mass Spectrometry Driven Discovery of Peptidic Danger Signals in Insect Immunity

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.

Reprograming of epigenetic mechanisms controlling host insect immunity and development in response to egg-laying by a parasitoid wasp

“…Interestingly, IMPI is part of an immunity-related signalling cascade that does not require microbial pathogen-associated molecular patterns (PAMPs) to elicit the synthesis of antimicrobial peptides, but senses the presence of danger-associated molecular patterns (DAMPs) [39,40]. Microbial metalloproteinases can digest targets such as collagen type IV in insects [41], generating peptide fragments that operate as DAMPs [42], which in turn activate immune responses including the synthesis of IMPI, galiomycin and cecropin A [43]. IMPI is the only known defence peptide that can inhibit virulenceassociated metalloproteinases [44].…”

Thermal and physical stresses induce a short-term immune priming effect in Galleria mellonella larvae

“…PPO is the inactive storage form of the enzyme which may increase for an immune investment, whereas PO reflects the active enzyme used in active components of the immune response (Bocher et al, 2007). An increase in PPO could occur as part of priming for an immune response to a potential infection within the larvae whereas the activated PO would be required for dealing with an active infection within the larvae (Berisha et al, 2013).…”

Damage signals in the insect immune response

“…The underlying scenario appears to resemble the one described above for the protein Persephone (see Figure 1 ), which also acts as a detector for a proteolytic activity that is indispensable for host invasion ( Altincicek et al, 2009 ). Additional peptides that are produced upon exposure to the protease thermolysine and have immune-stimulatory activity have been isolated from Galleria hemolymph and may also act as damage signals ( Berisha et al, 2013 ).…”