Bacterial Cytological Profiling Research Articles

Imaging‐based profiling for elucidation of antibacterial mechanisms of action

AbstractIn this review, we aim to summarize experimental data and approaches to identifying cellular targets or mechanisms of action of antibacterials based on imaging techniques. Imaging‐based profiling methods, such as bacterial cytological profiling, dynamic bacterial morphology imaging, and others, have become a useful research tool for mechanistic studies of new antibiotics as well as combinations with conventional ones and other therapeutic options. The main methodological and experimental details and obtained results are summarized and discussed. The review covers the literature up to February 2024.

An arylsulfonamide that targets cell wall biosynthesis in Mycobacterium tuberculosis.

We investigated the mechanism of action of an arylsulfonamide with whole-cell activity against Mycobacterium tuberculosis. We newly synthesized the molecule and confirmed it had activity against both extracellular and intracellular bacilli. The molecule had some activity against HepG2 cells but maintained some selectivity. Bacterial cytological profiling suggested that the mechanism of action was via disruption of cell wall synthesis, with similarities to an inhibitor of the mycolic acid exporter MmpL3. The compound induced expression from the IniB promoter and caused a boost in ATP production but did not induce reactive oxygen species. A mutation in MmpL3 (S591I) led to low-level resistance. Taken together, these data confirm the molecule targets cell wall biosynthesis with MmpL3 as the most probable target.

Identification and preclinical evaluation of MMV676558 as a promising therapeutic candidate against Clostridioides difficile

Clostridioides difficile, a gram-positive, toxin-producing, spore-forming anaerobe, is a major cause of antibiotic-associated diarrhoea. The bacterium's intrinsic drug resistance limits current treatment options to fidaxomicin and vancomycin for initial episodes, with anti-toxin B monoclonal antibody or faecal microbiota transplantation recommended for complicated or recurrent cases. This underscores the urgent need for novel therapeutics. In this study, we screened the MMV Pathogen Box at a 10 µM concentration against C. difficile R20291. Primary hits were evaluated for minimum inhibitory concentrations (MIC), killing kinetics, and biofilm inhibition. Bacterial cytological profiling (BCP) and transmission electron microscopy (TEM) were employed to study the mode of action. MMV676558 was further tested in a mouse model to assess survival, histopathology, and gut microbiota effects. We identified nineteen hits that inhibited over 50 % of C. difficile growth. MIC assays revealed three hits with MICs below 16 µg/mL: MMV676558, MMV688755, and MMV690027. These hits were effective against various C. difficile ribotypes. Killing kinetics were comparable or superior to vancomycin and fidaxomicin, and biofilm assays showed inhibitory effects. BCP and TEM analyses suggested membrane function disruption as the mode of action. Furthermore, MMV676558 demonstrated a protective effect in mice, with favourable histopathology and gut microbiota profiles. Given the urgent threat posed by C. difficile antibiotic resistance, discovering new treatments is a top priority. Our study identified three promising hits from the MMV Pathogen Box, with MMV676558 showing significant in vivo potential for further evaluation.

Recent Advances and Techniques for Identifying Novel Antibacterial Targets.

With the emergence of drug-resistant bacteria, the development of new antibiotics is urgently required. Target-based drug discovery is the most frequently employed approach for the drug development process. However, traditional drug target identification techniques are costly and time-consuming. As research continues, innovative approaches for antibacterial target identification have been developed which enabled us to discover drug targets more easily and quickly. In this review, methods for finding drug targets from omics databases have been discussed in detail including principles, procedures, advantages, and potential limitations. The role of phage-driven and bacterial cytological profiling approaches is also discussed. Moreover, current article demonstrates the advancements being made in the establishment of computational tools, machine learning algorithms, and databases for antibacterial target identification. Bacterial drug targets successfully identified by employing these aforementioned techniques are described as well. The goal of this review is to attract the interest of synthetic chemists, biologists, and computational researchers to discuss and improve these methods for easier and quicker development of new drugs.

Dissecting antibiotic effects on the cell envelope using bacterial cytological profiling: a phenotypic analysis starter kit.

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.

Design, Synthesis, Molecular Modeling, Biological Activity, and Mechanism of Action of Novel Amino Acid Derivatives of Norfloxacin.

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.

A marine-derived fatty acid targets the cell membrane of Gram-positive bacteria.

With the lack of new antibiotics in the drug discovery pipeline, coupled with accelerated evolution of antibiotic resistance, new sources of antibiotics that target pathogens of clinical importance are paramount. Here, we use bacterial cytological profiling to identify the mechanism of action of the monounsaturated fatty acid (Z)-13-methyltetra-4-decenoic acid isolated from the marine bacterium Olleya marilimosa with antibacterial effects against Gram-positive bacteria. The fatty acid antibiotic was found to rapidly destabilize the cell membrane by pore formation and membrane aggregation in Bacillus subtilis, suggesting that this fatty acid may be a promising adjuvant used in combination to enhance antibiotic sensitivity.

High-Resolution Bacterial Cytological Profiling Reveals Intrapopulation Morphological Variations upon Antibiotic Exposure.

Phenotypic heterogeneity is crucial to bacterial survival and could provide insights into the mechanism of action (MOA) of antibiotics, especially those with polypharmacological actions. Although phenotypic changes among individual cells could be detected by existing profiling methods, due to the data complexity, only population average data were commonly used, thereby overlooking the heterogeneity. In this study, we developed a high-resolution bacterial cytological profiling method that can capture morphological variations of bacteria upon antibiotic treatment. With an unprecedented single-cell resolution, this method classifies morphological changes of individual cells into known MOAs with an overall accuracy above 90%. We next showed that combinations of two antibiotics induce altered cell morphologies that are either unique or similar to that of an antibiotic in the combinations. With these combinatorial profiles, this method successfully revealed multiple cytological changes caused by a natural product-derived compound that, by itself, is inactive against Acinetobacter baumannii but synergistically exerts its multiple antibacterial activities in the presence of colistin. The findings have paved the way for future single-cell profiling in bacteria and have highlighted previously underappreciated intrapopulation variations caused by antibiotic perturbation.

Mansonone G and its derivatives exhibit membrane permeabilizing activities against bacteria.

In an era where the rate of bacteria evolving to be resistant to clinically-used antibiotics far exceeds that of antibiotic discovery, the search for new sources of antibacterial agents has expanded tremendously. In recent years, interest in plant-based natural products as promising sources of antibacterial agents has taken an upward trend. Mansonones, botanically-derived naphthoqionones, having many uses in Asian traditional medicine-including anti-infective roles-have sparked interest as a possible source of antibacterial agents. Here, we show that mansonone G, extracted from Mansonia gagei Drumm. heartwoods, possessed antibacterial activities towards Bacillus subtilis, Staphylococcus aureus and Escherichia coli lptD4213, inhibiting the growth of the bacteria at 15.6 μM, 62.5 μM and 125 μM, respectively. Fourteen derivatives of mansonone G were synthesized successfully and were found to have a similar antibacterial spectrum to that of the parent compound, with some derivatives possessing improved antibacterial activities. Bacterial cytological profiling analysis showed that mansonone G harbors membrane permeabilizing activities against B. subtilis and E. coli lptD4213. Temporal analysis of SYTOX Green staining among individual cells showed that mansonone G rapidly permeabilized bacterial membrane within 10 min, with SYTOX Green intensity reaching 13-fold above that of the control. Collectively, these findings highlight the importance of mansonone G and its derivatives as potential antibacterial agents, paving the way for further modifications in order to improve their antibacterial spectrum.

In vitro Antimicrobial Activity and the Mechanism of Berberine Against Methicillin-Resistant Staphylococcus aureus Isolated from Bloodstream Infection Patients.

PurposeTo investigate the antimicrobial activity of berberine and the mechanism by which it combats methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from patients with bloodstream infections.MethodsFifteen clinical MRSA isolates were collected, and their Multi-locus Sequence Types (MLST) were examined. The minimum inhibitory concentration (MIC) and combined antibacterial activity of berberine alone, and when combined with clindamycin and rifampicin separately, were determined. Additionally, two MRSA strains (ST239 and ST5) were selected to perform the time-killing assay and biofilm formation test. Cell wall alterations and cell membrane integrity were measured by confocal laser scanning microscopy (CLSM) and electron microscopy to assess the influence on cell morphology.ResultsOur data showed berberine was effective against MRSA at MIC values varying from 256 to 64 mg*L−1 for different MLST types. Berberine alone, and when combined with clindamycin and rifampicin separately, displayed excellent antibacterial activity which reduced the bacterial counts by 2lgCFU*mL within 24h and significantly weakened biofilm formation compared with control strain. Additionally, bacterial cytological profiling indicates that berberine destroyed the structure of the cell walls, membrane integrity and further changed the cell morphology with concentration increased.ConclusionIn our study, berberine has excellent anti-MRSA activities and has synergistic antibacterial property when combined with clindamycin and rifamycin separately, and the mechanism of activities involves the destruction of cell wall and membrane.

Bis-Indole Alkaloids Isolated from the Sponge Spongosorites calcicola Disrupt Cell Membranes of MRSA.

Antimicrobial resistance (AMR) is a global health challenge with methicillin resistant Staphylococcus aureus (MRSA), a leading cause of nosocomial infection. In the search for novel antibiotics, marine sponges have become model organisms as they produce diverse bioactive compounds. We investigated and compared the antibacterial potential of 3 bis-indole alkaloids—bromodeoxytopsentin, bromotopsentin and spongotine A—isolated from the Northeastern Atlantic sponge Spongosorites calcicola. Antimicrobial activity was determined by MIC and time-kill assays. The mechanism of action of bis-indoles was assessed using bacterial cytological profiling via fluorescence microscopy. Finally, we investigated the ability of bis-indole alkaloids to decrease the cytotoxicity of pathogens upon co-incubation with HeLa cells through the measurement of mammalian cell lysis. The bis-indoles were bactericidal to clinically relevant Gram-positive pathogens including MRSA and to the Gram-negative gastroenteric pathogen Vibrio parahaemolyticus. Furthermore, the alkaloids were synergistic in combination with conventional antibiotics. Antimicrobial activity of the bis-indole alkaloids was due to rapid disruption and permeabilization of the bacterial cell membrane. Significantly, the bis-indoles reduced pathogen cytotoxicity toward mammalian cells, indicating their ability to prevent bacterial virulence. In conclusion, sponge bis-indole alkaloids are membrane-permeabilizing agents that represent good antibiotic candidates because of their potency against Gram-positive and Gram-negative bacterial pathogens.

Isolation and characterization of Streptomyces bacteriophages and Streptomyces strains encoding biosynthetic arsenals.

The threat to public health posed by drug-resistant bacteria is rapidly increasing, as some of healthcare's most potent antibiotics are becoming obsolete. Approximately two-thirds of the world's antibiotics are derived from natural products produced by Streptomyces encoded biosynthetic gene clusters. Thus, to identify novel gene clusters, we sequenced the genomes of four bioactive Streptomyces strains isolated from the soil in San Diego County and used Bacterial Cytological Profiling adapted for agar plate culturing in order to examine the mechanisms of bacterial inhibition exhibited by these strains. In the four strains, we identified 104 biosynthetic gene clusters. Some of these clusters were predicted to produce previously studied antibiotics; however, the known mechanisms of these molecules could not fully account for the antibacterial activity exhibited by the strains, suggesting that novel clusters might encode antibiotics. When assessed for their ability to inhibit the growth of clinically isolated pathogens, three Streptomyces strains demonstrated activity against methicillin-resistant Staphylococcus aureus. Additionally, due to the utility of bacteriophages for genetically manipulating bacterial strains via transduction, we also isolated four new phages (BartholomewSD, IceWarrior, Shawty, and TrvxScott) against S. platensis. A genomic analysis of our phages revealed nearly 200 uncharacterized proteins, including a new site-specific serine integrase that could prove to be a useful genetic tool. Sequence analysis of the Streptomyces strains identified CRISPR-Cas systems and specific spacer sequences that allowed us to predict phage host ranges. Ultimately, this study identified Streptomyces strains with the potential to produce novel chemical matter as well as integrase-encoding phages that could potentially be used to manipulate these strains.

Bacterial cytological profiling identifies transmembrane anion transport as the mechanism of action for a urea-based antibiotic

ABSTRACT There is a growing fear that bacteria are emerging that are resistant to most known antibiotics, which could lead to an increasing number of fatalities due to bacterial infections. There is therefore an urgent need for the development of novel antibacterial products, preferably with novel mode of actions for which there are no known resistance pathways. We have previously found serendipitously that a simple urea-based compound functions as a potent antibacterial agent. In this manuscript, we use bacterial cytological profiling (BCP) to determine its mechanism of action. BCP reveals that the urea is acting through a membrane-based pathway that is comparable to, but distinct from, the mechanism of cationophore calcimycin. Additional liposome-based assays suggest that the likely mechanism of action of this mono-urea is transmembrane anion transport. This finding highlights that synthetic anionophores could be used as potential antibiotics.

Bacterial Cytological Profiling Identifies Rhodanine-Containing PAINS Analogs as Specific Inhibitors of Escherichia coli Thymidylate Kinase In Vivo.

In this study, we sought to determine whether an in vivo assay for studying antibiotic mechanisms of action could provide insight into the activity of compounds that may inhibit multiple targets. Thus, we conducted an activity screen of 31 structural analogs of rhodanine-containing pan-assay interference compounds (PAINS). We identified nine active molecules against Escherichia coli and classified them according to their in vivo mechanisms of action. The mechanisms of action of PAINS are generally difficult to identify due to their promiscuity. However, we leveraged bacterial cytological profiling, a fluorescence microscopy technique, to study these complex mechanisms. Ultimately, we found that although some of our molecules promiscuously inhibit multiple cellular pathways, a few molecules specifically inhibit DNA replication despite structural similarity to related PAINS. A genetic analysis of resistant mutants revealed thymidylate kinase (essential for DNA synthesis) as an intracellular target of some of these rhodanine-containing antibiotics. This finding was supported by in vitro activity assays, as well as experiments utilizing a thymidylate kinase overexpression system. The analog that demonstrated the half-maximal inhibitory concentration in vitro and MIC in vivo displayed the greatest specificity for inhibition of the DNA replication pathway, despite containing a rhodamine moiety. Although it is thought that PAINS cannot be developed as antibiotics, this work showcases novel inhibitors of E. coli thymidylate kinase. Moreover, perhaps more importantly, this work highlights the utility of bacterial cytological profiling for studying the in vivo specificity of antibiotics and demonstrates that bacterial cytological profiling can identify multiple pathways that are inhibited by an individual molecule. IMPORTANCE We demonstrate that bacterial cytological profiling is a powerful tool for directing antibiotic discovery efforts because it can be used to determine the specificity of an antibiotic's in vivo mechanism of action. By assaying analogs of PAINS, molecules that are notoriously intractable and nonspecific, we (surprisingly) identify molecules with specific activity against E. coli thymidylate kinase. This suggests that structural modifications to PAINS can confer stronger inhibition by targeting a specific cellular pathway. While in vitro inhibition assays are susceptible to false-positive results (especially from PAINS), bacterial cytological profiling provides the resolution to identify molecules with specific in vivo activity.

Peptides Affecting the Outer Membrane Lipid Asymmetry System (MlaA-OmpC/F) Reduce Avian Pathogenic Escherichia coli (APEC) Colonization in Chickens.

Avian pathogenic Escherichia coli (APEC), an extraintestinal pathogenic E. coli (ExPEC), causes colibacillosis in chickens and is reportedly associated with urinary tract infections and meningitis in humans. Development of resistance is a major limitation of current ExPEC antibiotic therapy. New antibacterials that can circumvent resistance problem such as antimicrobial peptides (AMPs) are critically needed. Here, we evaluated the efficacy of Lactobacillus rhamnosus GG (LGG)-derived peptides against APEC and uncovered their potential antibacterial targets. Three peptides (NPSRQERR [P1], PDENK [P2], and VHTAPK [P3]) displayed inhibitory activity against APEC. These peptides were effective against APEC in biofilm and chicken macrophage HD11 cells. Treatment with these peptides reduced the cecum colonization (0.5 to 1.3 log) of APEC in chickens. Microbiota analysis revealed two peptides (P1 and P2) decreased Enterobacteriaceae abundance with minimal impact on overall cecal microbiota of chickens. Bacterial cytological profiling showed peptides disrupt APEC membranes either by causing membrane shedding, rupturing, or flaccidity. Furthermore, gene expression analysis revealed that peptides downregulated the expression of ompC (>13.0-fold), ompF (>11.3-fold), and mlaA (>4.9-fold), genes responsible for the maintenance of outer membrane (OM) lipid asymmetry. Consistently, immunoblot analysis also showed decreased levels of OmpC and MlaA proteins in APEC treated with peptides. Alanine scanning studies revealed residues crucial (P1, N, E, R and P; P2, D and E; P3, T, P, and K) for their activity. Overall, our study identified peptides with a new antibacterial target that can be developed to control APEC infections in chickens, thereby curtailing poultry-originated human ExPEC infections. IMPORTANCE Avian pathogenic Escherichia coli (APEC) is a subgroup of extraintestinal pathogenic E. coli (ExPEC) and considered a foodborne zoonotic pathogen transmitted through consumption of contaminated poultry products. APEC shares genetic similarities with human ExPECs, including uropathogenic E. coli (UPEC) and neonatal meningitis E. coli (NMEC). Our study identified Lactobacillus rhamnosus GG (LGG)-derived peptides (P1 [NPSRQERR], P2 [PDENK], and P3 [VHTAPK]) effective in reducing APEC infection in chickens. Antimicrobial peptides (AMPs) are regarded as ideal candidates for antibacterial development because of their low propensity for resistance development and ability to kill resistant bacteria. Mechanistic studies showed peptides disrupt the APEC membrane by affecting the MlaA-OmpC/F system responsible for the maintenance of outer membrane (OM) lipid asymmetry, a promising new druggable target to overcome resistance problems in Gram-negative bacteria. Altogether, these peptides can provide a valuable approach for development of novel anti-ExPEC therapies, including APEC, human ExPECs, and other related Gram-negative pathogens. Furthermore, effective control of APEC infections in chickens can curb poultry-originated ExPEC infections in humans.

Technologies for High-Throughput Identification of Antibiotic Mechanism of Action.

There are two main strategies for antibiotic discovery: target-based and phenotypic screening. The latter has been much more successful in delivering first-in-class antibiotics, despite the major bottleneck of delayed Mechanism-of-Action (MOA) identification. Although finding new antimicrobial compounds is a very challenging task, identifying their MOA has proven equally challenging. MOA identification is important because it is a great facilitator of lead optimization and improves the chances of commercialization. Moreover, the ability to rapidly detect MOA could enable a shift from an activity-based discovery paradigm towards a mechanism-based approach. This would allow to probe the grey chemical matter, an underexplored source of structural novelty. In this study we review techniques with throughput suitable to screen large libraries and sufficient sensitivity to distinguish MOA. In particular, the techniques used in chemical genetics (e.g., based on overexpression and knockout/knockdown collections), promoter-reporter libraries, transcriptomics (e.g., using microarrays and RNA sequencing), proteomics (e.g., either gel-based or gel-free techniques), metabolomics (e.g., resourcing to nuclear magnetic resonance or mass spectrometry techniques), bacterial cytological profiling, and vibrational spectroscopy (e.g., Fourier-transform infrared or Raman scattering spectroscopy) were discussed. Ultimately, new and reinvigorated phenotypic assays bring renewed hope in the discovery of a new generation of antibiotics.

Identifying the effect of vancomycin on health care-associated methicillin-resistant Staphylococcus aureus strains using bacteriological and physiological media.

BackgroundThe evolving antibiotic-resistant behavior of health care–associated methicillin-resistant Staphylococcus aureus (HA-MRSA) USA100 strains are of major concern. They are resistant to a broad class of antibiotics such as macrolides, aminoglycosides, fluoroquinolones, and many more.FindingsThe selection of appropriate antibiotic susceptibility examination media is very important. Thus, we use bacteriological (cation-adjusted Mueller-Hinton broth) as well as physiological (R10LB) media to determine the effect of vancomycin on USA100 strains. The study includes the profiling behavior of HA-MRSA USA100 D592 and D712 strains in the presence of vancomycin through various high-throughput assays. The US100 D592 and D712 strains were characterized at sub-inhibitory concentrations through growth curves, RNA sequencing, bacterial cytological profiling, and exo-metabolomics high throughput experiments.ConclusionsThe study reveals the vancomycin resistance behavior of HA-MRSA USA100 strains in dual media conditions using wide-ranging experiments.

1289. Pravibismane is a Potent, Broad Spectrum Anti-Infective Small Molecule that Rapidly Disrupts Bacterial Bioenergetics and Halts Bacterial Growth

BackgroundThe rise in resistance to existing antimicrobials has prompted a need for the development of novel antibiotics. Microbion has identified a novel compound, pravibismane, with potent broad spectrum anti-infective and anti-biofilm activity.MethodsHere we used a variety of assays, including Bacterial Cytological Profiling (BCP), to analyze pravibismane in E.coli to gain insight into its likely mechanism of action (MOA). The BCP profile of pravibismane suggested it rapidly shut down cell growth, potentially by turning off cellular gene or protein expression. This was confirmed using a plasmid based GFP induction assay in E.coli tolC that showed pravibismane strongly reduced expression of GFP. The kinetics, reversibility and MOA of pravibismane was further characterized by using time-lapse microscopy, wash out experiments and measurements of both membrane potential and relative intracellular ATP levels.ResultsWe found that pravibismane acts rapidly (within 30 mins) to completely halt cell growth rather than causing immediate cell lysis such as that observed with non-specific cell damaging agents bleach or detergent. Inhibitor wash out experiments in which cells were exposed to pravibismane for 2 hours, washed to remove the compound, and then observed using time-lapse microscopy revealed that the effect of pravibismane is reversible and that cells recovered 8-12 hrs after removing the compound. Wash out experiments with an E.coli tolC strain carrying a plasmid with an IPTG inducible GFP demonstrated that transcription and translation ultimately resumed in most cells after washout. The bioenergetics of the membrane was measured using DiBAC 4(5), a membrane potential sensitive dye which can enter depolarized cells, which revealed that pravibismane caused depolarization of the membrane within 30 mins of exposure in a concentration dependent manner. Finally, a luciferase assay determined pravibismane reduced ATP levels (resulting in decreased luminescence) within 15 mins of exposure in a concentration dependent manner unlike antibiotic controls that had modest or no effect on luminescence.ConclusionOur results suggest that pravibismane acts rapidly to disrupt cellular bioenergetics, resulting in the immediate cessation of cell growth and protein expression.DisclosuresBrett Baker, M.Sc., D.C., Microbion Corporation (Board Member, Employee)

A How-To Guide for Mode of Action Analysis of Antimicrobial Peptides.

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.

A Potent Host Defense Peptide Triggers DNA Damage and Is Active against Multidrug-Resistant Gram-Negative Pathogens.

Gram-negative bacteria are some of the biggest threats to public health due to a large prevalence of antibiotic resistance. The difficulty in treating bacterial infections, stemming from their double membrane structure combined with efflux pumps in the outer membrane, has resulted in a much greater need for antimicrobials with activity against these pathogens. Tunicate host defense peptide (HDP), Clavanin A, is capable of not only inhibiting Gram-negative growth but also potentiating activity in the presence of Zn(II). Here, we provide evidence that the improvements of Clavanin A activity in the presence of Zn(II) are due to its novel mechanism of action. We employed E.coli TD172 (ΔrecA::kan) and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to show in cellulae that DNA damage occurs upon treatment with Clavanin A. In vitro assays demonstrated that Zn(II) ions are required for the nuclease activity of the peptide. The quantum mechanics/molecular mechanics (QM/MM) calculations were used to investigate the mechanism of DNA damage. In the rate-determining step of the proposed mechanism, due to its Lewis acidity, the Zn(II) ion activates the scissile P-O bond of DNA and creates a hydroxyl nucleophile from a water molecule. A subsequent attack by this group to the electrophilic phosphorus cleaves the scissile phosphoester bond. Additionally, we utilized bacterial cytological profiling (BCP), circular dichroism (CD) spectroscopy in the presence of lipid vesicles, and surface plasmon resonance combined with electrical impedance spectroscopy in order to address the apparent discrepancies between our results and the previous studies regarding the mechanism of action of Clavanin A. Finally, our approach may lead to the identification of additional Clavanin A like HDPs and promote the development of antimicrobial peptide based therapeutics.