Bacterial priming against antimicrobials and the evolution primability

Principal Investigator: Prof. Dr. Jens Rolff

Priming in microbiology is often referred to as acquired stress resistance, cross-protection or predictive response strategy and is generally understood to be the improved reaction of populations or cultures to an external stressor as a function of a preceding stressor. In phase I of CRC 973 we tested whether resistance of Escherichia coli to antimicrobials is primable when the bacteria were exposed to antimicrobial peptides (AMPs) of animal origin, their synthetic mimics, conventional antibiotics or reactive oxygen.

Our results from phase I demonstrate for the first time that exposure of bacteria to AMPs primes bacterial resistance to antimicrobial peptides. We found that sub-lethal doses of the synthetic AMP pexiganan prime Escherichia coli to survive a secondary exposure to a dosage that is lethal to naïve cells. These results were also repeated with other priming stressors than pexiganan, such as reactive oxygen and ciprofloxacin, a potent synthetic antibiotic. Surprisingly, the RpoS-pathway is not elicited by AMPs though this pathway is involved in other bacterial priming phenomena. We have also established a microfluidic set-up, allowing the investigation of primed bacteria at single cell level (ongoing research). We are currently carrying out RNAseq experiments to understand the genetic mechanisms underlying priming.

In phase II we will study the evolution of primability in E. coli in different environments. We will expose bacteria to different antimicrobials using the same framework as in phase I of the project. The new project has two objectives:

1. To experimentally evolve populations of E. coli for ‘primability’: i.e. the ability to be primed. As in the current project, we are going to use immune effectors, mostly AMPs, antibiotics and H2O2 as stressors. The environmental fluctuations (i.e. the sequence and predictability of the stressors) will be designed to capture the parameter space described in theoretical work and hence allow experimental testing of those models.

2. The resulting E. coli strains will be sequenced to analyse their genomes for potentially differential gene sequences of primable and non-primable strains. We will further sequence the transcriptome of these strains to study the expected differential expression of genes in primed and non-primed bacteria. To establish the function of candidate genes, we will use a microfluidic device studying the functional aspects at the level of single cells using mutant strains.
These experiments will have three main outcomes. (a) They will provide a test of current theory on the evolution of primability in bacteria (also in collaboration with project A1). (b) These experiments will unravel the underlying molecular mechanisms of priming. (c) And finally, this study will advance our understanding of

References

• Dobson, A.J., Purves, J. and Rolff, J. 2014. Increased survival of experimentally evolved antimicrobial peptide-resistant S. aureus in an animal host. Evol. Appl. 7: 905-912.
• Dobson, A.J., Purves, J., Kamyzs, W. and Rolff, J. 2013. Comparing selection on S. aureus between antimicrobial peptides and common antibiotics. PLoS One 8: e76521.
• Dorling, J., Moraes, C. and Rolff, J. 2014. Recognition, survival and persistence of Staphylococcus aureus in Tenebrio molitor. Develop. Comp. Immunol. 48: 284-290.
• Hilker, M., Schwachtje, J., Baier, M. Balazadeh, S.; Bäurle, I., Geiselhardt, S., Hincha, D., Kunze, R., Mueller-Roeber, B., Rillig, M., Rolff, J., Romeis, T., Schmülling, T., Steppuhn, A., van Dongen, J., Withcomb, S., Wurst, S., Zuther, E. and Kopka, J. 2015. Priming and memory of stress responses in organisms without a nervous system, Biol. Rev. doi: 10.1111/brv.12215.
• Johnston, P., Makarova, O. and Rolff, J. 2014. Inducible defenses stay up late: temporal patterns of immune gene expression in Tenebrio molitor. G3: Genes, Genom. Genet. 4: 947-955.
• Rillig, M.C., Rolff, J., Tietjen, B., Wehner, J. and Andrade-Linares, D. 2015. Community priming – effects of sequential stressors on microbial assemblages. FEMS Microbiol. Ecol. doi: 10.1093/femsec/fiv040.
• Rodríguez-Rojas, A., Makarova, O. and Rolff, J. 2014 Antimicrobials, stress and mutagenesis. PLoS Pathog. 10: e1004445.