Dissecting antibiotic effects on the cell envelope using bacterial cytological profiling: a phenotypic analysis starter kit
Journal article, 2024

Phenotypic analysis assays such as bacterial cytological profiling (BCP) have become increasingly popular for antibiotic mode of action analysis. A plethora of dyes, protein fusions, and reporter strains are available and have been used for this purpose, enabling both rapid mode of action categorization and in-depth analysis of antibiotic mechanisms. However, non-expert researchers may struggle choosing suitable assays and interpreting results. This is a particular problem for antibiotics that have multiple or complex targets, such as the bacterial cell envelope. Here, we set out to curate a minimal set of accessible and affordable phenotypic assays that allow distinction between membrane and cell wall targets, can identify dual-action inhibitors, and can be implemented in most research environments. To this end, we employed BCP, membrane potential, fluidity, and cell wall synthesis assays. To assess specificity and ease of interpretation, we tested three well-characterized and commercially available reference antibiotics: the potassium ionophore valinomycin, the lipid II-binding glycopeptide vancomycin, and the dual-action lantibiotic nisin, which binds lipid II and forms a membrane pore. Based on our experiments, we suggest a minimal set of BCP, a membrane-potentiometric probe, and fluorescent protein fusions to MinD and MreB as basic assay set and recommend complementing these assays with Laurdan-based fluidity measurements and a PliaI reporter fusion, where indicated. We believe that our results can provide guidance for researchers who wish to use phenotypic analysis for mode of action studies but do not possess the specialized equipment or expert knowledge to employ the full breadth of possible techniques.

vancomycin

mechanism of action

valinomycin

antimicrobial agents

bacterial cytological profiling

fluorescence assays

nisin

Author

Ann-Britt Schafer

University of Gothenburg

Margareth Sidarta

University of Gothenburg

Ireny Abdelmesseh Nekhala Abdelmesseh

Chalmers, Life Sciences, Chemical Biology

Gabriela Marinho Righetto

University of Gothenburg

Aysha Arshad

Chalmers, Life Sciences, Chemical Biology

Michaela Wenzel

University of Gothenburg

Microbiology spectrum

2165-0497 (eISSN)

Vol. 12 3

Subject Categories

Microbiology

Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)

Microbiology in the medical area

DOI

10.1128/spectrum.03275-23

PubMed

38289933

More information

Latest update

3/23/2024