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Membrane fluidity and thickness have emerged as crucial factors for the activity of and resistance to several antimicrobials. However, the lack of tools to study membrane fluidity and, in particular, thickness in living bacteria limits our understanding of this interplay. The Bacillus subtilis histidine kinase/phosphatase DesK is a molecular sensor that directly detects membrane thickness. It controls activity of DesR, which regulates expression of the lipid desaturase Des, known for its role in cold adaptation and daptomycin susceptibility. We hypothesized that this property could be exploited to develop biosensors and reporters for antibiotic-induced changes in membrane fluidity and thickness. To test this, we designed three assays based on the des system: activation of the Pdes promoter as reporter for membrane thickening, localization of DesK-GFP(green-fluorescent protein) as proxy for rigidified membrane domains, and antibiotic sensitivity of des, desK, and desR deletion mutants as readout for the importance of membrane rigidification/thickening under the tested condition. While we could not confirm the suitability of the des system as reporter for antibiotic-induced changes in membrane thickness, we did observe that des expression is only activated by mild temperature shocks, likely due to partitioning of the sensor DesK into fluid membrane domains upon phase separation, precluding effective thickness sensing under harsh cold shock and antibiotic stress conditions. Similarly, we did not observe any sensitivity of the deletion mutants to either temperature or antibiotic stress, raising the question to what extent the des system contributes to fluidity adaptation under these conditions. IMPORTANCE: The B. subtilis des system is a prime model for direct molecular membrane thickness sensor and, as such, has been well studied in vitro. Our study shows that our understanding of its function in vivo and its importance under temperature and antibiotic stress is still very limited. Specifically, our results suggest that (i) the des system senses very subtle membrane fluidity changes that escape detection by established fluidity reporters like laurdan; (ii) membrane thickness sensing by DesK is impaired by phase separation due to partitioning of the protein into the fluid phase; and (iii) fluidity adaptations by Des are too subtle to elicit growth defects under rigidifying conditions, raising the question of how much the des system contributes to adaptation of overall membrane fluidity.
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Bacillus subtilis , Proteínas de Bactérias , Membrana Celular , Fluidez de Membrana , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Bacillus subtilis/enzimologia , Fluidez de Membrana/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Membrana Celular/metabolismo , Membrana Celular/efeitos dos fármacos , Antibacterianos/farmacologia , Histidina Quinase/metabolismo , Histidina Quinase/genética , Regulação Bacteriana da Expressão Gênica , Separação de FasesRESUMO
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.IMPORTANCEPhenotypic analysis assays using specialized fluorescence fusions and dyes have become increasingly popular in antibiotic mode of action analysis. However, it can be difficult to implement these methods due to the need for specialized equipment and/or the complexity of bacterial cell biology and physiology, making the interpretation of results difficult for non-experts. This is especially problematic for compounds that have multiple or pleiotropic effects, such as inhibitors of the bacterial cell envelope. In order to make phenotypic analysis assays accessible to labs, whose primary expertise is not bacterial cell biology, or with limited equipment and resources, a set of simple and broadly accessible assays is needed that is easy to implement, execute, and interpret. Here, we have curated a set of assays and strains that does not need highly specialized equipment, can be performed in most labs, and is straightforward to interpret without knowing the intricacies of bacterial cell biology.
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Microorganisms within the marine environment have been shown to be very effective sources of naturally produced antimicrobial peptides (AMPs). Several nonribosomal peptides were identified based on genome mining predictions of Streptomyces sp. H-KF8, a marine Actinomycetota isolated from a remote Northern Chilean Patagonian fjord. Based on these predictions, a series of eight peptides, including cyclic peptides, were designed and chemically synthesized. Six of these peptides showed antimicrobial activity. Mode of action studies suggest that two of these peptides potentially act on the cell membrane via a novel mechanism allowing the passage of small ions, resulting in the dissipation of the membrane potential. This study shows that though structurally similar peptides, determined by NMR spectroscopy, the incorporation of small sequence mutations results in a dramatic influence on their bioactivity including mode of action. The qualified hit sequence can serve as a basis for more potent AMPs in future studies.
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Actinobacteria , Streptomyces , Peptídeos Antimicrobianos , Streptomyces/genética , Streptomyces/química , Peptídeos/farmacologia , Peptídeos/metabolismo , Peptídeos Cíclicos/químicaRESUMO
Membrane fluidity is a critical parameter of cellular membranes, which cells continuously strive to maintain within a viable range. Interference with the correct membrane fluidity state can strongly inhibit cell function. Triggered changes in membrane fluidity and associated impacts on lipid domains have been postulated to contribute to the mechanism of action of membrane targeting antimicrobials, but the corresponding analyses have been hampered by the absence of readily available analytical tools. Here, we expand upon the protocols outlined in the first edition of this book, providing further and alternative protocols that can be used to measure changes in membrane fluidity. We provide detailed protocols, which allow straightforward in vivo and in vitro measurement of antibiotic compound-triggered changes in membrane fluidity and fluid membrane microdomains. Furthermore, we summarize useful strains constructed by us and others to characterize and confirm lipid specificity of membrane antimicrobials directly in vivo.
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Fluidez de Membrana , Microscopia , Espectrometria de Fluorescência , Membrana Celular , LipídeosRESUMO
Eeyarestatin 24 (ES24) is a promising new antibiotic with broad-spectrum activity. It shares structural similarity with nitrofurantoin (NFT), yet appears to have a distinct and novel mechanism: ES24 was found to inhibit SecYEG-mediated protein transport and membrane insertion in Gram-negative bacteria. However, possible additional targets have not yet been explored. Moreover, its activity was notably better against Gram-positive bacteria, for which its mechanism of action had not yet been investigated. We have used transcriptomic stress response profiling, phenotypic assays, and protein secretion analyses to investigate the mode of action of ES24 in comparison with NFT using the Gram-positive model bacterium Bacillus subtilis and have compared our findings to Gram-negative Escherichia coli. Here, we show the inhibition of Sec-dependent protein secretion in B. subtilis and additionally provide evidence for DNA damage, probably caused by the generation of reactive derivatives of ES24. Interestingly, ES24 caused a gradual dissipation of the membrane potential, which led to delocalization of cytokinetic proteins and subsequent cell elongation in E. coli. However, none of those effects were observed in B. subtilis, thereby suggesting that ES24 displays distinct mechanistic differences with respect to Gram-positive and Gram-negative bacteria. Despite its structural similarity to NFT, ES24 profoundly differed in our phenotypic analysis, which implies that it does not share the NFT mechanism of generalized macromolecule and structural damage. Importantly, ES24 outperformed NFT in vivo in a zebrafish embryo pneumococcal infection model. Our results suggest that ES24 not only inhibits the Sec translocon, but also targets bacterial DNA and, in Gram-negative bacteria, the cell membrane.
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Antibacterianos , Escherichia coli , Animais , Escherichia coli/genética , Escherichia coli/metabolismo , DNA Bacteriano , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Peixe-Zebra , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas , Transporte ProteicoRESUMO
Two series of N4-substituted piperazinyl amino acid derivatives of norfloxacin (24 new compounds) were designed and synthesized to attain structural surrogates with additional binding sites and enhanced antibacterial activity. Synthesized derivatives showed increased antibacterial and antimycobacterial activity compared to their lead structure, norfloxacin. Molecular modeling studies supported the notion that the derivatives can establish additional bonds with the target enzymes gyrase and topoisomerase IV. In vitro enzyme inhibition assays confirmed that the tested compounds were significant inhibitors of these enzymes. Inhibition of gyrase and topoisomerase IV was then confirmed in living bacterial cells using bacterial cytological profiling of both Gram-negative Escherichia coli and Gram-positive Bacillus subtilis, revealing a typical topoisomerase inhibition phenotype characterized by severe nucleoid packing defects. Several derivatives exhibited additional effects on the Gram-positive cell wall synthesis machinery and/or the cytoplasmic membrane, which likely contributed to their increased antibacterial activity. While we could not identify specific cell wall or membrane targets, membrane depolarization was not observed. Our experiments further suggest that cell wall synthesis inhibition most likely occurs outside the membrane-bound lipid II cycle.
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Fluoroquinolones are broad-spectrum antibiotics that target gyrase and topoisomerase IV, involved in DNA compaction and segregation. We synthesized 28 novel norfloxacin hydroxamic acid derivatives with additional metal-chelating and hydrophobic pharmacophores, designed to enable interactions with additional drug targets. Several compounds showed equal or better activity than norfloxacin against Gram-positive, Gram-negative, and mycobacteria, with MICs as low as 0.18 µM. The most interesting derivatives were selected for in silico, in vitro, and in vivo mode of action studies. Molecular docking, enzyme inhibition, and bacterial cytological profiling confirmed inhibition of gyrase and topoisomerase IV for all except two tested derivatives (10f and 11f). Further phenotypic analysis revealed polypharmacological effects on peptidoglycan synthesis for four derivatives (16a, 17a, 17b, 20b). Interestingly, compounds 17a, 17b, and 20b, showed never seen before effects on cell wall synthetic enzymes, including MreB, MurG, and PonA, suggesting a novel mechanism of action, possibly impairing the lipid II cycle.
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Fluoroquinolones are an important class of antibiotics with broad-spectrum antibacterial and antitubercular activity. Here, we describe the design and synthesis of a series of 38 N4-substituted piperazinyl norfloxacin derivatives. Their activity and mechanism of action were characterized using in silico, in vitro, and in vivo approaches. Several compounds displayed interesting activities against both Gram-negative and Gram-positive bacteria, and few displayed antimycobacterial activity, whereby some were as potent as norfloxacin and ciprofloxacin. Molecular docking experiments suggested that the new derivatives inhibit both DNA gyrase and DNA topoisomerase IV in a similar manner as norfloxacin. Selecting the most promising candidates for experimental mode of action analysis, we confirmed DNA gyrase and topoisomerase IV as targets of all tested compounds using enzymatic in vitro assays. Phenotypic analysis of both Escherichia coli and Bacillus subtilis confirmed a typical gyrase inhibition phenotype for all of the tested compounds. Assessment of possible additional targets revealed three compounds with unique effects on the B. subtilis cell wall synthesis machinery, suggesting that they may have an additional target in this pathway. Comparison with known cell wall synthesis inhibitors showed that the new compounds elicit a distinct and, so far, unique phenotype, suggesting that they act differently from known cell wall synthesis inhibitors. Interestingly, our phenotypic analysis revealed that both norfloxacin and ciprofloxacin displayed additional cellular effects as well, which may be indicative of the so far unknown additional mechanisms of fluoroquinolones.
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Antimicrobial peptides (AMPs) are a promising alternative to classical antibiotics in the fight against multi-resistant bacteria. They are produced by organisms from all domains of life and constitute a nearly universal defense mechanism against infectious agents. No drug can be approved without information about its mechanism of action. In order to use them in a clinical setting, it is pivotal to understand how AMPs work. While many pore-forming AMPs are well-characterized in model membrane systems, non-pore-forming peptides are often poorly understood. Moreover, there is evidence that pore formation may not happen or not play a role in vivo. It is therefore imperative to study how AMPs interact with their targets in vivo and consequently kill microorganisms. This has been difficult in the past, since established methods did not provide much mechanistic detail. Especially, methods to study membrane-active compounds have been scarce. Recent advances, in particular in microscopy technology and cell biological labeling techniques, now allow studying mechanisms of AMPs in unprecedented detail. This review gives an overview of available in vivo methods to investigate the antibacterial mechanisms of AMPs. In addition to classical mode of action classification assays, we discuss global profiling techniques, such as genomic and proteomic approaches, as well as bacterial cytological profiling and other cell biological assays. We cover approaches to determine the effects of AMPs on cell morphology, outer membrane, cell wall, and inner membrane properties, cellular macromolecules, and protein targets. We particularly expand on methods to examine cytoplasmic membrane parameters, such as composition, thickness, organization, fluidity, potential, and the functionality of membrane-associated processes. This review aims to provide a guide for researchers, who seek a broad overview of the available methodology to study the mechanisms of AMPs in living bacteria.
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Peptídeos Catiônicos Antimicrobianos , Proteômica , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Testes de Sensibilidade Microbiana , Proteínas Citotóxicas Formadoras de PorosRESUMO
Trypanosomatid parasites cause devastating African sleeping sickness, Chagas disease, and Leishmaniasis that affect about 18 million people worldwide. Recently, we showed that the biogenesis of glycosomes could be the "Achilles' heel" of trypanosomatids suitable for the development of new therapies against trypanosomiases. This was shown for inhibitors of the import machinery of matrix proteins, while the distinct machinery for the topogenesis of glycosomal membrane proteins evaded investigation due to the lack of a druggable interface. Here we report on the identification of the highly divergent trypanosomal PEX3, a central component of the transport machinery of peroxisomal membrane proteins and the master regulator of peroxisome biogenesis. The trypanosomatid PEX3 shows very low degree of conservation and its identification was made possible by a combinatory approach identifying of PEX19-interacting proteins and secondary structure homology screening. The trypanosomal PEX3 localizes to glycosomes and directly interacts with the membrane protein import receptor PEX19. RNAi-studies revealed that the PEX3 is essential and that its depletion results in mislocalization of glycosomal proteins to the cytosol and a severe growth defect. Comparison of the parasites and human PEX3-PEX19 interface disclosed differences that might be accessible for drug development. The absolute requirement for biogenesis of glycosomes and its structural distinction from its human counterpart make PEX3 a prime drug target for the development of novel therapies against trypanosomiases. The identification paves the way for future drug development targeting PEX3, and for the analysis of additional partners involved in this crucial step of glycosome biogenesis.