title: 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.
authors.affiliation: Analytical Chemistry - Biointerfaces, Ruhr-University Bochum, NC4, Universitätsstr, 150, 44780 Bochum, Germany.
authors.forename: A R
authors.initials: AR
authors.lastname: von Gundlach
authors.affiliation: Helmholtz-Zentrum Geesthacht, Zentrum für Material- und Küstenforschung GmbH, Max-Planck-Straße 1, 21502 Geesthacht, Germany.
authors.forename: V M
authors.initials: VM
authors.lastname: Garamus
authors.affiliation: Analytical Chemistry - Biointerfaces, Ruhr-University Bochum, NC4, Universitätsstr, 150, 44780 Bochum, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Gorniak
authors.affiliation: Life Health and Chemical Sciences, Open University, Walton Hall, Milton Keynes MK7 6AA, United Kingdom.
authors.forename: H A
authors.initials: HA
authors.lastname: Davies
authors.affiliation: Institute for Applied Computer Science (IAI), Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Reischl
authors.affiliation: Institute for Applied Computer Science (IAI), Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Mikut
authors.affiliation: Institute of Infection and Immunology, St. George's University of London (SGUL), Cranmer Terrace, London SW17 0RE, United Kingdom.
authors.forename: K
authors.initials: K
authors.lastname: Hilpert
authors.affiliation: Analytical Chemistry - Biointerfaces, Ruhr-University Bochum, NC4, Universitätsstr, 150, 44780 Bochum, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Rosenhahn
mesh_terms: D000617:Aminoglycosides / Q000737:chemistry / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000494:pharmacology*; D002701:Chloramphenicol / Q000737:chemistry / Q000494:pharmacology; D002939:Ciprofloxacin / Q000737:chemistry / Q000494:pharmacology; D055808:Drug Discovery; D048168:Escherichia coli K12 / Q000187:drug effects* / Q000254:growth & development / Q000648:ultrastructure; D057166:High-Throughput Screening Assays; D046529:Microscopy, Electron, Transmission; D011112:Polymyxin B / Q000737:chemistry / Q000494:pharmacology; D025341:Principal Component Analysis; D012293:Rifampin / Q000737:chemistry / Q000494:pharmacology; D053838:Scattering, Small Angle; D013752:Tetracycline / Q000737:chemistry / Q000494:pharmacology; D014961:X-Ray Diffraction; D047090:beta-Lactams / Q000737:chemistry / Q000494:pharmacology
chemical_list: D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D047090:beta-Lactams; D002939:Ciprofloxacin; D002701:Chloramphenicol; D013752:Tetracycline; D011112:Polymyxin B; D012293:Rifampin
keywords: Antimicrobial peptides; BioSAXS; E.coli; Mode of action; TEM