Active maintenance of proton motive force mediates starvation-induced bacterial antibiotic tolerance in Escherichia coli.
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ABSTRACT:
SUBMITTER: Wang M
PROVIDER: S-EPMC8440630 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:It is well known that loss of aerobic respiration in Gram-negative bacteria can diminish the efficacy of a variety of bactericidal antibiotics, which has lead to subsequent demonstrations that the formation of reactive oxygen species (ROS) and the proton motive force (PMF) can both play a role in antibiotic toxicity. The susceptibility of Gram-negative bacteria to aminoglycoside antibiotics, particularly gentamicin, has previously been linked to both the production of ROS and the rate of antibiotic uptake that is mediated by the PMF, although the relative contributions of ROS and PMF to aminoglycoside toxicity has remained poorly understood. Herein, gentamicin was shown to elicit a very modest increase in ROS levels in an aerobically grown Escherichia coli clinical isolate. The well-characterised uncoupler 2,4-dinitrophenol (DNP) was used to disrupt the PMF, which resulted in a significant decrease in gentamicin lethality towards E. coli. DNP did not significantly alter respiratory oxygen consumption, supporting the hypothesis that this uncoupler does not increase ROS production via elevated respiratory oxidase activity. These observations support the hypothesis that maintenance of PMF rather than induction of ROS production underpins the mechanism for how the respiratory chain potentiates the toxicity of aminoglycosides. This was further supported by the demonstration that the uncoupler DNP elicits a dramatic decrease in gentamicin lethality under anaerobic conditions. Together, these data strongly suggest that maintenance of the PMF is the dominant mechanism for the respiratory chain in potentiating the toxic effects of aminoglycosides.
| S-EPMC8739286 | biostudies-literature
Project description:There is a growing need for new antibiotics. Compounds that target the proton motive force (PMF), uncouplers, represent one possible class of compounds that might be developed because they are already used to treat parasitic infections, and there is interest in their use for the treatment of other diseases, such as diabetes. Here, we tested a series of compounds, most with known antiinfective activity, for uncoupler activity. Many cationic amphiphiles tested positive, and some targeted isoprenoid biosynthesis or affected lipid bilayer structure. As an example, we found that clomiphene, a recently discovered undecaprenyl diphosphate synthase inhibitor active against Staphylococcus aureus, is an uncoupler. Using in silico screening, we then found that the anti-glioblastoma multiforme drug lead vacquinol is an inhibitor of Mycobacterium tuberculosis tuberculosinyl adenosine synthase, as well as being an uncoupler. Because vacquinol is also an inhibitor of M. tuberculosis cell growth, we used similarity searches based on the vacquinol structure, finding analogs with potent (?0.5-2 ?g/mL) activity against M. tuberculosis and S. aureus. Our results give a logical explanation of the observation that most new tuberculosis drug leads discovered by phenotypic screens and genome sequencing are highly lipophilic (logP ?5.7) bases with membrane targets because such species are expected to partition into hydrophobic membranes, inhibiting membrane proteins, in addition to collapsing the PMF. This multiple targeting is expected to be of importance in overcoming the development of drug resistance because targeting membrane physical properties is expected to be less susceptible to the development of resistance.
| S-EPMC4697371 | biostudies-literature
Project description:There is considerable interest in improving plant productivity by altering the dynamic responses of photosynthesis in tune with natural conditions. This is exemplified by the 'energy-dependent' form of non-photochemical quenching (qE), the formation and decay of which can be considerably slower than natural light fluctuations, limiting photochemical yield. In addition, we recently reported that rapidly fluctuating light can produce field recombination-induced photodamage (FRIP), where large spikes in electric field across the thylakoid membrane (Δψ) induce photosystem II recombination reactions that produce damaging singlet oxygen (1O2). Both qE and FRIP are directly linked to the thylakoid proton motive force (pmf), and in particular, the slow kinetics of partitioning pmf into its ΔpH and Δψ components. Using a series of computational simulations, we explored the possibility of 'hacking' pmf partitioning as a target for improving photosynthesis. Under a range of illumination conditions, increasing the rate of counter-ion fluxes across the thylakoid membrane should lead to more rapid dissipation of Δψ and formation of ΔpH. This would result in increased rates for the formation and decay of qE while resulting in a more rapid decline in the amplitudes of Δψ-spikes and decreasing 1O2 production. These results suggest that ion fluxes may be a viable target for plant breeding or engineering. However, these changes also induce transient, but substantial mismatches in the ATP : NADPH output ratio as well as in the osmotic balance between the lumen and stroma, either of which may explain why evolution has not already accelerated thylakoid ion fluxes. Overall, though the model is simplified, it recapitulates many of the responses seen in vivo, while spotlighting critical aspects of the complex interactions between pmf components and photosynthetic processes. By making the programme available, we hope to enable the community of photosynthesis researchers to further explore and test specific hypotheses.This article is part of the themed issue 'Enhancing photosynthesis in crop plants: targets for improvement'.
| S-EPMC5566881 | biostudies-literature
Project description:Bacteria become highly tolerant to antibiotics when nutrients are limited. The inactivity of antibiotic targets caused by starvation-induced growth arrest is thought to be a key mechanism producing tolerance. Here we show that the antibiotic tolerance of nutrient-limited and biofilm Pseudomonas aeruginosa is mediated by active responses to starvation, rather than by the passive effects of growth arrest. The protective mechanism is controlled by the starvation-signaling stringent response (SR), and our experiments link SR-mediated tolerance to reduced levels of oxidant stress in bacterial cells. Furthermore, inactivating this protective mechanism sensitized biofilms by several orders of magnitude to four different classes of antibiotics and markedly enhanced the efficacy of antibiotic treatment in experimental infections.
| S-EPMC4046891 | biostudies-literature
Project description:Myxococcus xanthus is a Gram-negative bacterium that glides over surfaces without the aid of flagella. Two motility systems are used for locomotion: social-motility, powered by the retraction of type IV pili, and adventurous (A)-motility, powered by unknown mechanism(s). We have shown that AgmU, an A-motility protein, is part of a multiprotein complex that spans the inner membrane and periplasm of M. xanthus. In this paper, we present evidence that periplasmic AgmU decorates a looped continuous helix that rotates clockwise as cells glide forward, reversing its rotation when cells reverse polarity. Inhibitor studies showed that the AgmU helix rotation is driven by proton motive force (PMF) and depends on actin-like MreB cytoskeletal filaments. The AgmU motility complex was found to interact with MotAB homologs. Our data are consistent with a mechanochemical model in which PMF-driven motors, similar to bacterial flagella stator complexes, run along an endless looped helical track, driving rotation of the track; deformation of the cell surface by the AgmU-associated proteins creates pressure waves in the slime, pushing cells forward.
| S-EPMC3038734 | biostudies-literature
Project description:The thylakoid proton motive force (pmf) generated during photosynthesis is the essential driving force for ATP production; it is also a central regulator of light capture and electron transfer. We investigated the effects of elevated pmf on photosynthesis in a library of Arabidopsis thaliana mutants with altered rates of thylakoid lumen proton efflux, leading to a range of steady-state pmf extents. We observed the expected pmf-dependent alterations in photosynthetic regulation, but also strong effects on the rate of photosystem II (PSII) photodamage. Detailed analyses indicate this effect is related to an elevated electric field (Δψ) component of the pmf, rather than lumen acidification, which in vivo increased PSII charge recombination rates, producing singlet oxygen and subsequent photodamage. The effects are seen even in wild type plants, especially under fluctuating illumination, suggesting that Δψ-induced photodamage represents a previously unrecognized limiting factor for plant productivity under dynamic environmental conditions seen in the field.
| S-EPMC5050024 | biostudies-literature
Project description:The Escherichia coli TonB system consists of the cytoplasmic membrane proteins TonB, ExbB, and ExbD and multiple outer membrane active transporters for diverse iron siderophores and vitamin B12. The cytoplasmic membrane proteins harvest and transmit the proton motive force (PMF) to outer membrane transporters. This system, which spans the cell envelope, has only one component with a significant cytoplasmic presence, ExbB. Characterization of sequential 10-residue deletions in the ExbB cytoplasmic loop (residues 40 to 129; referred to as ?10 proteins) revealed that it was required for all TonB-dependent activities, including interaction between the periplasmic domains of TonB and ExbD. Expression of eight out of nine of the ?10 proteins at chromosomal levels led to immediate, but reversible, growth arrest. Arrest was not due to collapse of the PMF and did not require the presence of ExbD or TonB. All ?10 proteins that caused growth arrest were dominant for that phenotype. However, several were not dominant for iron transport, indicating that growth arrest was an intrinsic property of the ?10 variants, whether or not they could associate with wild-type ExbB proteins. The lack of dominance in iron transport also ruled out trivial explanations for growth arrest, such as high-level induction. Taken together, the data suggest that growth arrest reflected a changed interaction between the ExbB cytoplasmic loop and one or more unknown growth-regulatory proteins. Consistent with that, a large proportion of the ExbB cytoplasmic loop between transmembrane domain 1 (TMD1) and TMD2 is predicted to be disordered, suggesting the need for interaction with one or more cytoplasmic proteins to induce a final structure.
| S-EPMC3807443 | biostudies-literature
Project description:To identify novel inhibitors of Mycobacterium tuberculosis cell envelope biosynthesis, we employed a two-step approach. First, we screened the diverse synthetic small molecule 71,544-compound Enamine library for growth inhibitors using the non-pathogenic surrogate Mycobacterium bovis BCG as screening strain and turbidity as readout. Second, 16 confirmed hits were tested for their ability to induce the cell envelope stress responsive promoter piniBAC controlling expression of red fluorescent protein in an M. bovis BCG reporter strain. Using a fluorescence readout, the acetamide E11 was identified. Resistant mutant selection and whole genome sequencing revealed the mycolic acid transporter Mmpl3 as a candidate target of E11. Biochemical analysis using mycobacterial spheroplasts and various membrane assays suggest that E11 indirectly inhibits MmpL3-facilitated translocation of trehalose monomycolates by proton motive force disruption. E11 showed potent bactericidal activity against growing and non-growing M. tuberculosis, low cytotoxic, and hemolytic activity and a dynamic structure activity relationship. In addition to activity against M. tuberculosis, E11 was active against the non-tuberculous mycobacterium M. abscessus, an emerging opportunistic pathogen. In conclusion, we identified a novel bactericidal anti-mycobacterial lead compound targeting MmpL3 providing an attractive starting point for optimization.
| S-EPMC6289072 | biostudies-literature
Project description:Extraintestinal pathogenic Escherichia coli (ExPEC) causes a variety of acute infections in its hosts, and multidrug-resistant strains present significant challenges to public health and animal husbandry. Therefore, it is necessary to explore new drug targets to control E. coli epidemics. Previous studies have reported that ppk mutants of Burkholderia pseudomallei and Mycobacterium tuberculosis are more susceptible than the wild types (WTs) to stress. Therefore, we investigated the stress response to antibiotics mediated by polyphosphate kinase (PPK) in ExPEC strain PCN033. We observed that planktonic cells of a ppk knockout strain (Δppk) were more susceptible to antibiotics than was WT. However, biofilm-grown Δppk cells showed similar susceptibility to that of the WT and were more tolerant than the planktonic cells. During the planktonic lifestyle, the expression of genes involved in antibiotic tolerance (including resistance-conferring genes, and antibiotic influx, and efflux genes) did not change in the Δppk mutant without antibiotic treatment. However, the resistance-conferring gene bla and efflux genes were upregulated more in the WT than in the Δppk mutant by treatment with tazobactam. After treatment with gentamycin, the efflux genes and influx genes were upregulated and downregulated, respectively, more in the WT than in the Δppk mutant. The expression of genes involved in biofilm regulation also changed after treatment with tazobactam or gentamycin, and which is consistent with the results of the biofilm formation. Together, these observations indicate that PPK is important for the antibiotic stress response during the planktonic growth of ExPEC and might be a potential drug target in bacteria.
| S-EPMC4871857 | biostudies-literature
Project description: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 ?-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.
| S-EPMC6365497 | biostudies-literature