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Multidrug resistance and the evolutionary ecology of insect immunity

Final Report Summary - EVORESIN (Multidrug resistance and the evolutionary ecology of insect immunity)

The main hypothesis of this project was that antimicrobial peptides in insects deal with persistent infections and/or prevent the evolution of resistant bacterial mutants. The research conducted here combined two hitherto unrelated areas within a single conceptual and experimental framework:
(a) How to prevent the evolution of bacterial resistance against antimicrobials?
(b) Why is it adaptive for insects to exhibit long lasting costly immune responses?

In this project we first established the temporal dynamics of immune effectors with a special focus on antimicrobial peptides (AMPs). It was found that a gram-positive immune challenge resulted in a broad and long lasting up-regulation, both on the transcriptomic and the proteomic level, of AMPs mediated by Toll- and Imd pathways. The long-lasting up-regulation of AMPs was correlated with a long-lasting suppression of metabolic genes, consistent with a high physiological cost of immune responses.

Using this information we designed RNAi to knock down the most abundant AMPs either individually or in combinations. This revealed that reducing complexity of the suite of AMPs lead to higher bacterial persistence in the host. Moreover, AMPs that have no in vitro function against our model pathogen S. aureus displayed strong synergistic inter-actions in vivo. Double-knockdowns resulted in much higher bacterial resistance and also significantly increased mortality around day five. This demonstrates an adaptive value of co-expression of AMPs.
Persistent bacteria did not show a genomic signature of selection for resistance against the host immune defense. This opens new questions and opportunities to understand the formation of persister cells in vivo.

In a parallel experiment we carried out experimental evolution of S. aureus in the presence of Tenecin 1, a beetle defensin strongly active against S. aureus. In addition we carried out selection against Tenecin 1 and 2 combined (Tenecin 2 does not kill S. aureus), reasoning that they might display synergisms. Using genome re-sequencing we found that bacteria selected against Tenecin 1 carry significantly lower numbers of mutation than strains selected against Ten 1 in the presence of Tenecin 2.

Antibiotics have been shown to increase mutagenesis of bacteria and hence fuel resistance evolution. We showed that this does not apply to AMPs, as the stress response against AMPs do not result in the activation of error-prone alternative polymerase.

The results from this research program can antibiotic strategies in medical and veterinary science and change the way we understand the evolution of insect immunity.