Small angle X-ray scattering as a high-throughput method to classify antimicrobial modes of action

Abstract

Multi-drug resistant bacteria are currently undermining our health care system worldwide. While novel antimicrobial drugs, such as antimicrobial peptides, are urgently needed, identification of new modes of action is money and time consuming, and in addition current approaches are not available in a high throughput manner. Here we explore how small angle X-ray scattering (SAXS) as high throughput method can contribute to classify the mode of action for novel antimicrobials and therefore supports fast decision making in drug development. Using data bases for natural occurring antimicrobial peptides or predicting novel artificial peptides, many candidates can be discovered that will kill a selected target bacterium. However, in order to narrow down the selection it is important to know if these peptides follow all the same mode of action. In addition, the mode of action should be different from conventional antibiotics, in consequence peptide candidates can be developed further into drugs against multi-drug resistant bacteria. Here we used one short antimicrobial peptide with unknown mode of action and compared the ultrastructural changes of Escherichia coli cells after treatment with the peptide to cells treated with classic antibiotics. The key finding is that SAXS as a structure sensitive tool provides a rapid feedback on drug induced ultrastructural alterations in whole E. coli cells. We could demonstrate that ultrastructural changes depend on the used antibiotics and their specific mode of action. This is demonstrated using several well characterized antimicrobial compounds and the analysis of resulting SAXS curves by principal component analysis. To understand the result of the PCA analysis, the data is correlated with TEM images. In contrast to real space imaging techniques, SAXS allows to obtain nanoscale information averaged over approximately one million cells. The measurement takes only seconds, while conventional tests to identify a mode of action require days or weeks per single substance. The antimicrobial peptide showed a different mode of action as all tested antibiotics including polymyxin B and is therefore a good candidate for further drug development. We envision SAXS to become a useful tool within the high-throughput screening pipeline of modern drug discovery. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

BioSAXS measurements reveal that two antimicrobial peptides induce similar molecular changes in Gram-negative and Gram-positive bacteria

“…In addition, the cytoplasm becomes much more homogenous as compared to the control. Interestingly, the peptides in this study result in a very different response compared to a peptide (RLKRWWKFL) described in our previous studies, indicating different modes of action (Von Gundlach et al, 2016a). In addition, we could not detect any similarities, for example damages to the cell wall or membrane, dramatic changes in the inside of the cells, typically seen with polymyxin B, a cyclic lipopeptide with detergentlike mode of action (Von Gundlach et al, 2016a).…”

“…The observed effect is consequencently a mixture of ultrastructural changes on the bacterial level caused by direct and indirect effects of the antimicrobial compound as well as direct and indirect effects of the stress response of the bacteria. For each compound these effects will be concentration and time dependent (Von Gundlach et al, 2016a).…”

“…It is possible to use peptide libraries to screen and optimize antimicrobial peptides (Ashby et al, 2017). With the already large number of antimicrobial peptides and there various modes of actions a method to classify such peptides according to their mode of action would be of high value and BioSAXS was recently used to develop such a classification method (Von Gundlach et al, 2016a) Small angle X-ray scattering of biological samples (BioSAXS), for example proteins, is a powerful method for the characterization of both ordered and disordered structures in biological samples that provides information about the sizes and shapes ranging from a few kDa to GDa (Chen et al, 2018;Kikhney and Svergun, 2015). In the last decades, X-ray technology has matured to allow the study of protein crystals and proteins in solution down to atomic resolution.…”

“…The novelty of this work is the application of SAXS to reveal structural changes on the length scale between 3 nm -120 nm within bacteria as consequence of treatment with antimicrobial substances. Subtle structural intracellular rearrangements in the bacteria can accurately be probed across large bacterial populations (hundreds of thousands of bacteria) within seconds (Von Gundlach et al, 2016a). We recently demonstrated that antimicrobial compounds with different modes of action lead to different ultrastructural changes, detectable by BioSAXS (Von Gundlach et al, 2016b).…”

Structure-based screening of binding affinities via small-angle X-ray scattering

“…Notably, no assumption is made of the substrate material. It is possible that V R can quantify structural alterations in other existing applications of SAXS, for instance cellular ultrastructure,(44) whole-cell morphology, (45) and beyond.…”