The Dual Mode of Antibacterial Action of the Synthetic Small Molecule DCAP Involves Lipid II Binding.

The synthetic small molecule DCAP is a chemically well-characterized compound with antibiotic activity against Gram-positive and Gram-negative bacteria, including drug-resistant pathogens. Until now, its mechanism of action was proposed to rely exclusively on targeting the bacterial membrane, thereby causing membrane depolarization, and increasing membrane permeability (Eun et al. 2012, J. Am. Chem. Soc. 134 (28), 11322-11325; Hurley et al. 2015, ACS Med. Chem. Lett. 6, 466-471). Here, we show that the antibiotic activity of DCAP results from a dual mode of action that is more targeted and multifaceted than previously anticipated. Using microbiological and biochemical assays in combination with fluorescence microscopy, we provide evidence that DCAP interacts with undecaprenyl pyrophosphate-coupled cell envelope precursors, thereby blocking peptidoglycan biosynthesis and impairing cell division site organization. Our work discloses a concise model for the mode of action of DCAP which involves the binding to a specific target molecule to exert pleiotropic effects on cell wall biosynthetic and divisome machineries.

Microscopy-Based Multiwell Assay to Characterize Disturbed Bacterial Morphogenesis Upon Antibiotic Action.

The urgent need of new antimicrobial agents to combat life-threatening bacterial infections demands the identification and characterization of novel compounds that interfere with new and unprecedented target pathways or structures in multiresistant bacteria. Here, bacterial cell division has emerged as a new and promising target pathway for antibiotic intervention. Compounds, which inhibit division, commonly induce a characteristic filamentation phenotype of rod-shaped bacteria, such as Bacillus subtilis. Hence, this filamentation phenotype can be used to identify and characterize novel compounds that primarily target bacterial cell division. Since novel compounds of both synthetic and natural product origin are often available in small amounts only, thereby limiting the number of assays during mode of action studies, we here describe a semiautomated, microscopy-based approach that requires only small volumes of compounds to allow for the real-time observation of their effects on living bacteria, such as filamentation or cell lysis, in high-throughput 96-well-based formats. We provide a detailed workflow for the initial characterization of multiple compounds at once and further tools for the initial, microscopy-based characterization of their antibacterial mode of action.

Quantitative Analysis of Microscopy Data to Evaluate Bacterial Responses to Antibiotic Treatment.

Microscopy is a powerful method to evaluate the direct effects of antibiotic action on the single cell level. As with other methodologies, microscopy data is obtained through sufficient biological and technical replicate experiments, where evaluation of the sample is generally followed over time. Even if a single antibiotic is tested for a defined time, the most certain outcome is large amounts of raw data that requires systematic analysis. Although microscopy is a helpful qualitative method, the recorded information is stored as defined quantifiable units, the pixels. When this information is transferred to diverse bioinformatic tools, it is possible to analyze the microscopy data while avoiding the inherent bias associated to manual quantification. Here, we briefly describe methods for the analysis of microscopy images using open-source programs, with a special focus on bacteria exposed to antibiotics.

Progression of the late-stage divisome is unaffected by the depletion of the cytoplasmic FtsZ pool.

Cell division is a central and essential process in most bacteria, and also due to its complexity and highly coordinated nature, it has emerged as a promising new antibiotic target pathway in recent years. We have previously shown that ADEP antibiotics preferably induce the degradation of the major cell division protein FtsZ, thereby primarily leading to a depletion of the cytoplasmic FtsZ pool that is needed for treadmilling FtsZ rings. To further investigate the physiological consequences of ADEP treatment, we here studied the effect of ADEP on the different stages of the FtsZ ring in rod-shaped bacteria. Our data reveal the disintegration of early FtsZ rings during ADEP treatment in Bacillus subtilis, indicating an essential role of the cytoplasmic FtsZ pool and thus FtsZ ring dynamics during initiation and maturation of the divisome. However, progressed FtsZ rings finalized cytokinesis once the septal peptidoglycan synthase PBP2b, a late-stage cell division protein, colocalized at the division site, thus implying that the concentration of the cytoplasmic FtsZ pool and FtsZ ring dynamics are less critical during the late stages of divisome assembly and progression.

Cell division protein FtsZ: from structure and mechanism to antibiotic target.

Antimicrobial resistance to virtually all clinically applied antibiotic classes severely limits the available options to treat bacterial infections. Hence, there is an urgent need to develop and evaluate new antibiotics and targets with resistance-breaking properties. Bacterial cell division has emerged as a new antibiotic target pathway to counteract multidrug-resistant pathogens. New approaches in antibiotic discovery and bacterial cell biology helped to identify compounds that either directly interact with the major cell division protein FtsZ, thereby perturbing the function and dynamics of the cell division machinery, or affect the structural integrity of FtsZ by inducing its degradation. The impressive antimicrobial activities and resistance-breaking properties of certain compounds validate the inhibition of bacterial cell division as a promising strategy for antibiotic intervention.