Elevated Membrane Potential as a Tetracycline Resistance Mechanism in Escherichia coli.

The metabolic environment is responsible for antibiotic resistance, which highlights the way in which the antibiotic resistance mechanism works. Here, GC-MS-based metabolomics with iTRAQ-based proteomics was used to characterize a metabolic state in tetracycline-resistant Escherichia coli K12 (E. coli-RTET) compared with tetracycline-sensitive E. coli K12. The repressed pyruvate cycle against the elevation of the proton motive force (PMF) and ATP constructed the most characteristic feature as a consequence of tetracycline resistance. To understand the role of the elevated PMF in tetracycline resistance, PMF inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and the pH gradient were used to investigate how the elevation influences bacterial viability and intracellular antibiotic concentration. A strong synergy was detected between CCCP and tetracycline to the viability, which was consistent with increasing intracellular drug and decreasing external pH. Furthermore, E. coli-RTET and E. coli-RGEN with high and low PMF concentrations were susceptible to gentamicin and tetracycline, respectively. The elevated PMF in E. coli-RTET was attributed to the activation of other metabolic pathways, except for the pyruvate cycle, including a malate-oxaloacetate-phosphoenolpyruvate-pyruvate-malate cycle. These results not only revealed a PMF-dependent mechanism for tetracycline resistance but also provided a solution to tetracycline-resistant pathogens by aminoglycosides and aminoglycoside-resistant bacteria by tetracyclines.

Development of novel indole-quinoline hybrid molecules targeting bacterial proton motive force.

AIMS: This study aimed to develop an editable structural scaffold for improving drug development, including pharmacokinetics and pharmacodynamics of antibiotics by using synthetic compounds derived from a (hetero)aryl-quinoline hybrid scaffold. METHODS AND RESULTS: In this study, 18 CF3-substituted (hetero)aryl-quinoline hybrid molecules were examined for their potential antibacterial activity against Staphylococcus aureus by determining minimal inhibitory concentrations. These 18 synthetic compounds represent modifications to key regions of the quinoline N-oxide scaffold, enabling us to conduct a structure-activity relationship analysis for antibacterial potency. Among the compounds, 3 m exhibited potency against with both methicillin resistant S. aureus strains, as well as other Gram-positive bacteria, including Enterococcus faecalis and Bacillus subtilis. We demonstrated that 3 m disrupted the bacterial proton motive force (PMF) through monitoring the PMF and conducting the molecular dynamics simulations. Furthermore, we show that this mechanism of action, disrupting PMF, is challenging for S. aureus to overcome. We also validated this PMF inhibition mechanism of 3 m in an Acinetobacter baumannii strain with weaken lipopolysaccharides. Additionally, in Gram-negative bacteria, we demonstrated that 3 m exhibited a synergistic effect with colistin that disrupts the outer membrane of Gram-negative bacteria. CONCLUSIONS: Our approach to developing editable synthetic novel antibacterials underscores the utility of CF3-substituted (hetero)aryl-quinoline scaffold for designing compounds targeting the bacterial proton motive force, and for further drug development, including pharmacokinetics and pharmacodynamics.

Melatonin prevents conjugative transfer of plasmid-mediated antibiotic resistance genes by disrupting proton motive force.

The widespread dissemination of antibiotic resistance genes (ARGs) is a serious problem and constitutes a threat for public health. Plasmid-mediated conjugative transfer of ARGs is recognized as one of the most important pathways accounting for this global crisis. Inhibiting the conjugative transfer of resistant gene-bearing plasmids provides a feasible strategy to prevent the spread of antibiotic resistance. Here we found that melatonin, a neurohormone secreted from pineal gland, substantially inhibited the horizontal transfer of RP4-7 plasmid in a dose-dependent manner. Furthermore, melatonin could also suppress the conjugal frequency of different types of clinical plasmids that carrying colistin resistance gene mcr-1 rather than blaNDM or tet(X) genes. Next, we investigated the mechanisms underlying the inhibitory effect of melatonin on conjugation. As a result, we showed that the addition of melatonin markedly reduced bacterial membrane permeability and inhibited the oxidative stress. In line with these observations, the conjugative transfer-related genes were regulated accordingly. Most importantly, we uncovered that melatonin disrupted bacterial proton motive force (PMF), which is an essential bacterial energy metabolism substance and is important for conjugative process. Collectively, these results provide implications that some non-antibiotics such as melatonin are effective inhibitors of transmission of ARGs and raise a promising strategy to confront the increasing resistant infections.

Nitric oxide disrupts bacterial cytokinesis by poisoning purine metabolism.

Cytostasis is the most salient manifestation of the potent antimicrobial activity of nitric oxide (NO), yet the mechanism by which NO disrupts bacterial cell division is unknown. Here, we show that in respiring Escherichia coli, Salmonella, and Bacillus subtilis, NO arrests the first step in division, namely, the GTP-dependent assembly of the bacterial tubulin homolog FtsZ into a cytokinetic ring. FtsZ assembly fails in respiring cells because NO inactivates inosine 5'-monophosphate dehydrogenase in de novo purine nucleotide biosynthesis and quinol oxidases in the electron transport chain, leading to drastic depletion of nucleoside triphosphates, including the GTP needed for the polymerization of FtsZ. Despite inhibiting respiration and dissipating proton motive force, NO does not destroy Z ring formation and only modestly decreases nucleoside triphosphates in glycolytic cells, which obtain much of their ATP by substrate-level phosphorylation and overexpress inosine 5'-monophosphate dehydrogenase. Purine metabolism dictates the susceptibility of early morphogenic steps in cytokinesis to NO toxicity.

L-lysine potentiates aminoglycosides against Acinetobacter baumannii via regulation of proton motive force and antibiotics uptake.

Acinetobacter baumannii, a Gram-negative opportunistic pathogen, is a leading cause of hospital- and community-acquired infections. Acinetobacter baumannii can rapidly acquire diverse resistance mechanisms and undergo genetic modifications that confer resistance and persistence to all currently used clinical antibiotics. In this study, we found exogenous L-lysine sensitizes Acinetobacter baumannii, other Gram-negative bacteria (Escherichia coli and Klebsiella pneumoniae) and a Gram-positive bacterium (Mycobacterium smegmatis) to aminoglycosides. Importantly, the combination of L-lysine with aminoglycosides killed clinically isolated multidrug-resistant Acinetobacter baumannii and persister cells. The exogenous L-lysine can increase proton motive force via transmembrane chemical gradient, resulting in aminoglycoside acumination that further accounts for reactive oxygen species production. The combination of L-lysine and antibiotics highlights a promising strategy against bacterial infection.

Ammonia uptake by transmembrane pH gradient poly(isoprene)-block-poly(ethylene glycol) polymersomes.

Transmembrane pH gradient poly(isoprene)-block-poly(ethylene glycol) (PI-b-PEG) polymersomes were investigated for their potential use in the detoxification of ammonia, a metabolite that is excessively present in patients suffering from urea cycle disorders and advanced liver diseases, and which causes neurotoxic effects (e.g., hepatic encephalopathy). Polymers varying in PI and PEG block length were synthesized via nitroxide-mediated polymerization and screened for their ability to self-assemble into polymersomes in aqueous media. Ammonia sequestration by the polymersomes was investigated in vitro. While most vesicular systems were able to capture ammonia in simulated intestinal fluids, uptake was lost in partially dehydrated medium mimicking conditions in the colon. Polymeric crosslinking of residual olefinic bonds in the PI block increased polymersome stability, partially preserving the ammonia capture capacity in the simulated colon environment. These more stable vesicular systems hold promise for the chronic oral treatment of hyperammonemia.

Natural Flavones from Morus alba against Methicillin-Resistant Staphylococcus aureus via Targeting the Proton Motive Force and Membrane Permeability.

The emergence and rapid spread of methicillin-resistant Staphylococcus aureus (MRSA) critically requires alternative therapeutic options. New antibacterial drugs and strategies are urgently needed to combat MRSA-associated infections. Here, we investigated the antibacterial activity of flavones from Morus alba and the potential mode of action against MRSA. Kuwanon G, kuwanon H, mulberrin, and morusin displayed high efficiency in killing diverse MRSA isolates. On the basis of structure-activity analysis, the cyclohexene-phenyl ketones and isopentenyl groups were critical to increase the membrane permeability and to dissipate the proton motive force. Meanwhile, mechanistic studies further showed that kuwanon G displayed rapid bactericidal activity in vitrowith difficulty in developing drug resistance. Kuwanon G targeted phosphatidylglycerol and cardiolipin in the cytoplasmic membrane through the formation of hydrogen bonds and electrostatic interactions. Additionally, kuwanon G promoted wound healing in a mouse model of MRSA skin infection. In summary, these results indicate that flavones are promising lead compounds to treat MRSA-associated infections through disrupting the proton motive force and membrane permeability.

Host metabolites stimulate the bacterial proton motive force to enhance the activity of aminoglycoside antibiotics.

Antibiotic susceptibility of bacterial pathogens is typically evaluated using in vitro assays that do not consider the complex host microenvironment. This may help explaining a significant discrepancy between antibiotic efficacy in vitro and in vivo, with some antibiotics being effective in vitro but not in vivo or vice versa. Nevertheless, it is well-known that antibiotic susceptibility of bacteria is driven by environmental factors. Lung epithelial cells enhance the activity of aminoglycoside antibiotics against the opportunistic pathogen Pseudomonas aeruginosa, yet the mechanism behind is unknown. The present study addresses this gap and provides mechanistic understanding on how lung epithelial cells stimulate aminoglycoside activity. To investigate the influence of the local host microenvironment on antibiotic activity, an in vivo-like three-dimensional (3-D) lung epithelial cell model was used. We report that conditioned medium of 3-D lung cells, containing secreted but not cellular components, potentiated the bactericidal activity of aminoglycosides against P. aeruginosa, including resistant clinical isolates, and several other pathogens. In contrast, conditioned medium obtained from the same cell type, but grown as conventional (2-D) monolayers did not influence antibiotic efficacy. We found that 3-D lung cells secreted endogenous metabolites (including succinate and glutamate) that enhanced aminoglycoside activity, and provide evidence that bacterial pyruvate metabolism is linked to the observed potentiation of antimicrobial activity. Biochemical and phenotypic assays indicated that 3-D cell conditioned medium stimulated the proton motive force (PMF), resulting in increased bacterial intracellular pH. The latter stimulated antibiotic uptake, as determined using fluorescently labelled tobramycin in combination with flow cytometry analysis. Our findings reveal a cross-talk between host and bacterial metabolic pathways, that influence downstream activity of antibiotics. Understanding the underlying basis of the discrepancy between the activity of antibiotics in vitro and in vivo may lead to improved diagnostic approaches and pave the way towards novel means to stimulate antibiotic activity.

Gboxin is an oxidative phosphorylation inhibitor that targets glioblastoma.

Cancer-specific inhibitors that reflect the unique metabolic needs of cancer cells are rare. Here we describe Gboxin, a small molecule that specifically inhibits the growth of primary mouse and human glioblastoma cells but not that of mouse embryonic fibroblasts or neonatal astrocytes. Gboxin rapidly and irreversibly compromises oxygen consumption in glioblastoma cells. Gboxin relies on its positive charge to associate with mitochondrial oxidative phosphorylation complexes in a manner that is dependent on the proton gradient of the inner mitochondrial membrane, and it inhibits the activity of F0F1 ATP synthase. Gboxin-resistant cells require a functional mitochondrial permeability transition pore that regulates pH and thus impedes the accumulation of Gboxin in the mitochondrial matrix. Administration of a metabolically stable Gboxin analogue inhibits glioblastoma allografts and patient-derived xenografts. Gboxin toxicity extends to established human cancer cell lines of diverse organ origin, and shows that the increased proton gradient and pH in cancer cell mitochondria is a mode of action that can be targeted in the development of antitumour reagents.

2-aminoimidazoles collapse mycobacterial proton motive force and block the electron transport chain.

There is an urgent need to develop new drugs against tuberculosis. In particular, it is critical to target drug tolerant Mycobacterium tuberculosis (M. tuberculosis), responsible, in part, for the lengthy antibiotic regimen required for treatment. We previously postulated that the presence of in vivo biofilm-like communities of M. tuberculosis could contribute to this drug tolerance. Consistent with this hypothesis, certain 2-aminoimidazole (2-AIs) molecules with anti-biofilm activity were shown to revert mycobacterial drug tolerance in an in vitro M. tuberculosis biofilm model. While exploring their mechanism of action, it was serendipitously observed that these 2-AI molecules also potentiated ß-lactam antibiotics by affecting mycobacterial protein secretion and lipid export. As these two bacterial processes are energy-dependent, herein it was evaluated if 2-AI compounds affect mycobacterial bioenergetics. At low concentrations, 2B8, the lead 2-AI compound, collapsed both components of the proton motive force, similar to other cationic amphiphiles. Interestingly, however, the minimum inhibitory concentration of 2B8 against M. tuberculosis correlated with a higher drug concentration determined to interfere with the mycobacterial electron transport chain. Collectively, this study elucidates the mechanism of action of 2-AIs against M. tuberculosis, providing a tool to better understand mycobacterial bioenergetics and develop compounds with improved anti-mycobacterial activity.

Gene disruption of ribosomal protein L5 (RPL5) decreased the sensitivity of CHO-K1 cells to uncoupler carbonylcyanide-3-chlorophenylhydrazone.

Protonophoric uncoupler carbonylcyanide-3-chlorophenylhydrazone (CCCP) decreases the proton motive force (ΔP) of the mitochondrial inner membrane and results in inhibition of oxidative phosphorylation. In this study, a CCCP-resistant clone was isolated from a random gene trap insertional mutant library of Chinese hamster ovary (CHO)-K1 cells which was constructed by infecting a retrovirus vector, ROSAßgeo. Although we expected the isolation of the mutants defective in nuclear genes responsible for mitochondrial functions, the disrupted gene of the isolated mutant that we named R1 cells was identified as one of the alleles for ribosomal protein 5 of large subunit (RPL5). The R1 cells express as much as 80% RPL5 protein compared with the parental CHO-K1 cells, possibly due to enhanced transcription from a remaining wild-type RPL5 allele in R1 cells. Furthermore, the protein amount is not decreased by CCCP in R1 cells, in contrast to its clear reduction by CCCP in parental cells. Since mutations of RPL5 and other ribosomal proteins are responsible for the ribosomopathies and cancer, the present mutant may be a useful cellular model of such human diseases from a viewpoint of energy metabolism as well as a tool for the study of ribosome biogenesis and extra-ribosomal function of the RPL5 protein.

SmpB down-regulates proton-motive force for the persister tolerance to aminoglycosides in Aeromonas veronii.

Bacterial persisters comprise a small fraction of phenotypically heterogeneous variants with transient capability for survival when exposed to high concentrations of antibiotic. In aquatic pathogenic bacteria Aeromonas veronii, Small Protein B (SmpB), the core factor of trans-translation system, was identified as a new persistence-related gene. The SmpB deletion exhibited a higher susceptibility and lower persister cell formation under aminoglycosides antibiotics pressure compared with wild type. The transcriptional and translational activities of smpB gene were significantly enhanced by the gentamicin challenge in exponential phase, but not changed in stationary phase. The transcriptomic analysis revealed that the smpB deletion stimulated the production of proton-motive force (PMF). The cell survival induced by carbonyl cyanide m-chlorophenyl hydrazone (CCCP) further verified that SmpB variation affected the quantities of PMF. Taken together, these results uncovered a novel mechanism of persister formation mediated by SmpB under aminoglycosides treatments.

Using pH Gradient Dissolution with In-Situ Flux Measurement to Evaluate Bioavailability and DDI for Formulated Poorly Soluble Drug Products.

This study described a pH-gradient dissolution method combined with flux measurements as an in vitro tool for assessing the risk of bioavailability reduction due to drug-drug interactions (DDI) caused by acid reducing agents (ARAs). The device incorporates absorption chambers into USP II dissolution vessels, with fiber optic UV-probes monitoring concentration in situ. Dosage forms of Genentech BCS class II drugs, GDC-0810, GDC-0941, and compound A, were tested by starting the dissolution in either pH 1.6 or pH 4.0 media then converting to FaSSIF after 30 min. GDC-0810 showed no significant difference in flux between the two conversion experiments. A supersaturation phase was observed for GDC-0941 in the pH 1.6 experiments after media conversion to FaSSIF; however, it did not appear to occur in the pH 4.0 experiment due to low drug solubility at pH 4.0, resulting in a 95% decrease in flux compared to pH 1.6 experiment. The extent of flux reduction and the total accumulated API mass in the absorption chamber agreed well with the 89% reduction in mean Cmax and the 82% reduction in mean AUC from dog PK study between animals treated with pentagastrin and famotidine. Testing of the compound A optimized formulation tablets showed a 25% reduction in flux and in vitro absorbed amount by changing pH 1.6 to 4.0, correlating well with the AUC decrease in clinical studies. Good correlation between in vitro data and in vivo PK data demonstrated the applicability of the method for formulators to develop drug products mitigating DDI from ARAs.

Bicarbonate Alters Bacterial Susceptibility to Antibiotics by Targeting the Proton Motive Force.

The antibacterial properties of sodium bicarbonate have been known for years, yet the molecular understanding of its mechanism of action is still lacking. Utilizing chemical-chemical combinations, we first explored the effect of bicarbonate on the activity of conventional antibiotics to infer on the mechanism. Remarkably, the activity of 8 classes of antibiotics differed in the presence of this ubiquitous buffer. These interactions and a study of mechanism of action revealed that, at physiological concentrations, bicarbonate is a selective dissipater of the pH gradient of the proton motive force across the cytoplasmic membrane of both Gram-negative and Gram-positive bacteria. Further, while components that make up innate immunity have been extensively studied, a link to bicarbonate, the dominant buffer in the extracellular fluid, has never been made. Here, we also explored the effects of bicarbonate on components of innate immunity. Although the immune response and the buffering system have distinct functions in the body, we posit there is interplay between these, as the antimicrobial properties of several components of innate immunity were enhanced by a physiological concentration of bicarbonate. Our findings implicate bicarbonate as an overlooked potentiator of host immunity in the defense against pathogens. Overall, the unique mechanism of action of bicarbonate has far-reaching and predictable effects on the activity of innate immune components and antibiotics. We conclude that bicarbonate has remarkable power as an antibiotic adjuvant and suggest that there is great potential to exploit this activity in the discovery and development of new antibacterial drugs by leveraging testing paradigms that better reflect the physiological concentration of bicarbonate.

Insight into synergetic mechanisms of tetracycline and the selective serotonin reuptake inhibitor, sertraline, in a tetracycline-resistant strain of Escherichia coli.

Sertraline, an antidepressive drug, has been reported to inhibit general bacterial efflux pumps. In the present study, we report for the first time a synergistic effect of sertraline and tetracycline in a TetA-encoded tetracycline-resistant strain of Escherichia coli. Synergy between sertraline and tetracycline in an E. coli strain with TetA-mediated tetracycline resistance (E. coli APEC_O2) was assessed by the MIC and checkerboard assays. The global transcriptome of E. coli APEC_O2 exposed to ½ MIC concentrations of sertraline and/or tetracycline was analyzed to elucidate the interaction mechanism between sertraline and tetracycline. The fractional inhibitory concentration index for tetracycline and sertraline in E. coli APEC_O2 was 0.5. In addition, in the presence of ½ MIC of sertraline, the sensitivity of E. coli APEC_O2 to tetracycline could be restored according to clinical standards (from 64 to 4 mg l-1). RNA data suggest changes in respiration that is likely to decrease intracellular pH and thereby the proton-motive force, which provides the energy for the tetracycline efflux pump. Furthermore, sertraline and tetracycline may induce a change from oxidation to fermentation in the E.coli, which further decreases pH, resulting in cell death. This study shows that sertraline interacts with tetracycline in a synergistic and AcrAB-TolC pump-independent manner. The combinational treatment was further shown to induce many changes in the global transcriptome, including altered tetA and tetR expression. The results indicate that sertraline may be used as a helper compound with the aim to reverse tetracycline resistance encoded by tetA.

Energy Requirements for Protein Secretion via the Flagellar Type III Secretion System.

Protein transport across the cytoplasmic membrane is coupled to energy derived from adenosine triphosphate hydrolysis or the protein motive force (pmf). A sophisticated, multi-component type III secretion system exports substrate proteins of both the bacterial flagellum and virulence-associated injectisome system of many Gram-negative pathogens. The type-III secretion system is primarily a pmf-driven protein exporter. Here, I describe methods to investigate the export of substrate proteins into the culture supernatant under conditions that manipulate the pmf.

Disruption of Autolysis in Bacillus subtilis using TiO2 Nanoparticles.

In contrast to many nanotoxicity studies where nanoparticles (NPs) are observed to be toxic or reduce viable cells in a population of bacteria, we observed that increasing concentration of TiO2 NPs increased the cell survival of Bacillus subtilis in autolysis-inducing buffer by 0.5 to 5 orders of magnitude over an 8 hour exposure. Molecular investigations revealed that TiO2 NPs prevent or delay cell autolysis, an important survival and growth-regulating process in bacterial populations. Overall, the results suggest two potential mechanisms for the disruption of autolysis by TiO2 NPs in a concentration dependent manner: (i) directly, through TiO2 NP deposition on the cell wall, delaying the collapse of the protonmotive-force and preventing the onset of autolysis; and (ii) indirectly, through adsorption of autolysins on TiO2 NP, limiting the activity of released autolysins and preventing further lytic activity. Enhanced darkfield microscopy coupled to hyperspectral analysis was used to map TiO2 deposition on B. subtilis cell walls and released enzymes, supporting both mechanisms of autolysis interference. The disruption of autolysis in B. subtilis cultures by TiO2 NPs suggests the mechanisms and kinetics of cell death may be influenced by nano-scale metal oxide materials, which are abundant in natural systems.

Two-peptide bacteriocin PlnEF causes cell membrane damage to Lactobacillus plantarum.

Biologically active, artificially synthesized two-peptide bacteriocin PlnEF was used to study its mode of action on sensitive bacteria Lactobacillus plantarum pl2. The data obtained showed that PlnEF induced membrane permeabilization, allowing for the efflux of electrolytes, which was evidenced by the increased extracellular conductivity, the dissipation of transmembrane electrical potential and pH gradient, and rapid intracellular ATP depletion after L. plantarum pl2 cells were treated with PlnEF for minutes. Laser confocal microscopy showed that PlnEF accumulated very quickly in L. plantarum pl2 cells and the accumulation of PlnEF caused damage to cell membrane. Scanning electron microscopy and transmission electron microscopy further showed that PlnEF induced morphological changes and structure disruption to L. plantarum pl2 cells, such as the formation of blebs, microspheres, membrane deformation and cell lysis. In summary, the data obtained show that PlnEF caused cell membrane damage to L. plantarum pl2 cells. Our study reveals the antimicrobial mechanism of two-peptide bacteriocin PlnEF against L. plantarum.

Evidence that bacteriophage λ lysogens may induce in response to the proton motive force uncoupler CCCP.

We describe a genetic ß-galactoside reporter system using a disk diffusion assay on MacConkey Lactose agar petri plates to monitor maintenance of the bacteriophage λ prophage state and viral induction in Escherichia coli K-12. Evidence is presented that the phage λ major lytic promoters, pL and pR, are activated when cells containing the reporters are exposed to the energy poison carbonyl cyanide m-chlorophenyl hydrazine, CCCP. This uncoupler of oxidative phosphorylation inhibits ATP synthesis by collapsing the proton motive force. Expression of the λ lytic promoters in response to CCCP requires host RecA function and an autocleavable CI repressor, as does SOS induction of the λ prophage that occurs by a DNA damage-dependent pathway. λ Cro function is required for CCCP-mediated activation of the λ lytic promoters. CCCP does not induce an sfi-lacZ SOS reporter.