Multiscale Study of Antimicrobial Peptide Activity in E. coli

World-wide multi-resistant pathogenic bacteria gain grounds rapidly, especially in health-care units, representing a global health problem with a strong social and economic impact. Hence, the World Health Organization emphasized the urgent need for the development of antibiotics with novel mechanisms of action to counteract the steady decline of approved antibiotics since the early 1980s. One highly promising and alternative strategy to conventional antibiotics is based on antimicrobial peptides (AMPs), effector molecules of the innate immune system that provide a first line of defense against invaders. The peculiarity of AMPs in comparison to conventional antibiotics is that they interfere physically with the barrier function of the cell envelope of bacteria and do not interact with specific target molecules. Furthermore, owing to their killing of bacteria within minutes, evolvement of resistance is less likely. However despite the insight on molecular interactions gained from studies on model systems, there is no comprehensive picture of how AMPs affect bacterial cells and actually kill bacteria. In particular, the kinetic and spatial evolution of processes at the different structural levels of the cell envelope (e.g. cell wall and cell membrane) is currently unknown. In order to fully understand their mechanism of action it is essential to bridge this gap. To address this issue we suggest performing time-resolved X-ray scattering experiments on live Escherichia coli and monitor their response to AMPs. This highly innovative approach will enable us for the first time to probe changes at the molecular and microscopic level in real time. Preliminary experiments at the synchrotron in Grenoble demonstrated for the first time that structural changes induced by AMPs occur on the sub-second time scale, which is much faster than anticipated. Our studies will initially focus on AMPs derived from human lactoferrin, a protein enriched in breast milk, developed within a European project coordinated by the main applicant. The primary target of these peptides is known to be the bacterial cell envelope. The aim of the proposal is to dissect structural changes in bacteria especially cell envelope upon AMP attacks and to follow their progression as killing proceeds. The gained insight will be transferred to future studies on other AMPs and other clinically relevant bacteria such as Staphylococcus aureus, paving the way towards a comprehensive understanding of the molecular killing mechanism of AMPs. This knowledge will facilitate their development into novel and effective compounds for therapeutic applications in infectious diseases caused by antibiotic-resistant pathogens.

Infectious diseases caused by multi-resistant pathogenic bacteria gain rapidly grounds world-wide, especially in health-care units. Owing to this growing threat the WHO ranked antibiotic resistance as a priority disease and emphasized the development of novel antibiotics to counteract the steady decline of approved antibiotics since the early 80s. This calls in particular for alternative antimicrobial agents having completely different mechanisms of action. One highly promising strategy is based on antimicrobial peptides (AMPs), effector molecules of innate immunity that provide a first line of defense against a substantial array of pathogenic microorganisms. The molecular mode of action of membrane-active AMPs has been extensively studied in various model systems using a plethora of biophysical techniques. Despite this impressive insight on molecular interactions in systems of reduced complexity, there is no comprehensive picture of how AMPs affect bacterial cells in vivo and how they actually kill bacteria. Thus, the aim of the project was to explore the mode of action of selected AMPs on live bacteria using "state-of-the-art" X-ray and neutron scattering techniques combined with advanced modelling established in our group. This approach represents a novel and powerful technique to study the effects of antimicrobial peptides in real time and obtaining information at multiple spatial and time scales without the need of any invasive staining or labelling technique. This unique strategy allowed us to unravel the kinetics of AMP interactions at different hierarchical levels as well as to delineate the sequence of processes leading to killing of bacteria. We demonstrated that AMPs saturate the surface of bacteria in less than three seconds concomitantly leading to major structural changes on the cell envelope such as vesicle formation and membrane detachment diminishing its barrier function. Within few more seconds AMPs translocate through the cell envelope, where they subsequently accumulate in the cytosolic region of the bacteria and lead to metabolic shutdown within minutes due to binding to negatively charged essential cell constituents such as DNA, RNA, ribosomes and proteins. Hence, our results support the hypothesis that AMPs act very fast (within seconds to minutes) and target various cell structures and components, which makes it unlikely that bacteria develop resistance. In other words, our results suggest that the combination of translocation speed and efficient shut down of bacterial metabolism by interfering with several cell constituents are generic factors to be considered in designing future AMPs to combat infectious diseases. Thus, the outcome will be highly relevant for antibiotic drug development.