Characterization of the in vivo mechanisms of action of cell envelope-targeting antibiotics

Sammanfattning: Multidrug-resistant bacteria pose a major threat to society. Once easily treatable diseases are now challenging to cure. With the golden age of antibiotics being over, the discovery of new antibiotics has been stagnant, and no new antibiotic class has been introduced into the clinic since 2003. Therefore, it is crucial to discover new antibiotics and to optimize existing antimicrobial compounds. This thesis aims to provide more information on the mechanisms of antibiotics on living bacteria and can be divided into two parts. One part provides detailed information on techniques that can be utilized to study the mode of action of new antibiotic compounds. Here, methods are discussed in detail and curated to a method set that can be easily utilized by other researchers. The curated assays were then applied, and a phenotypic reference set was created. In the second part, these methods were used to investigate the mechanisms of new antimicrobial compounds and lead structures to provide important information for their further development. The fluorescence dyes laurdan, Nile red, and DiIC12 that are able to track fluidity changes in the bacterial membranes in response to antibiotic treatment were described. In addition to the detailed protocols and their evaluation, the methods were applied to investigate the effects of daptomycin. These methods were able to detect that daptomycin changes the overall membrane fluidity differently in B. subtilis and S. aureus . Furthermore, I have successfully applied the curated methods, consisting of BCP, membrane potential (DiSC3(5), and cell wall synthesis assays (MreB mobility and PliaI ) to study several new compounds, e.g., antimicrobial peptides from marine streptomycetes. Two of these new antimicrobial peptides led to the consecutive permeabilization of the outer and inner membrane in E. coli . Interestingly, they also affected DNA packing. Further, an in-depth study of a new promising drug candidate, ES24, was conducted, showing its unique target of the Sec-secretion pathway and an additional DNA-damaging effect. Overall, this thesis provides important insights into the techniques used in mode of action studies, describes an easily accessible method set for other researchers, and provides important information on the modes of action of new antimicrobial compounds. Mode of action, cell-envelope targeting, antibiotics, antimicrobial peptides