Project description:Deeper understanding of antibiotic-induced physiological responses is critical to identifying means for enhancing our current antibiotic arsenal. Bactericidal antibiotics with diverse targets have been hypothesized to kill bacteria, in part, by inducing production of damaging reactive species. This notion has been supported by many groups, but recently challenged. Here we robustly test the hypothesis using biochemical, enzymatic and biophysical assays along with genetic and phenotypic experiments. We first used a novel intracellular hydrogen peroxide (H2O2) sensor, together with a chemically diverse panel of fluorescent dyes sensitive to an array of reactive species, to demonstrate that antibiotics broadly induce redox stress. Subsequent gene expression analyses reveal that complex antibiotic-induced oxidative stress responses are distinct from canonical responses generated by supra-physiological levels of H2O2. We next developed a method to dynamically quantify cellular respiration and found that bactericidal antibiotics elevate oxygen consumption, indicating significant alterations to bacterial redox physiology. We further show that catalase or DNA mismatch repair enzyme overexpression, as well as antioxidant pre-treatment limit antibiotic lethality, indicating that reactive oxygen species causatively contribute to antibiotic killing. Critically, the killing efficacy of antibiotics was diminished under strict anaerobic conditions, but could be enhanced by exposure to molecular oxygen or addition of alternative electron acceptors, suggesting that environmental factors play a role in killing cells physiologically primed for death. This work provides direct evidence that bactericidal antibiotics, downstream of their target-specific interactions, induce complex redox alterations that contribute to cellular damage and death, thus supporting an evolving, expanded model of antibiotic lethality. Here, we used microarrays to analyze oxidative stress responses to bactericidal antibiotic treatment in wildtype and mutant E coli WT or mutant E coli cells were grown to OD ~0.3. Untreated cells were harvested at time 0 as controls. Treated cells given the appropriate chemical perturbation and harvested 1 hour post-treatment. All experiments were performed in technical triplicate.

Project description:Deeper understanding of antibiotic-induced physiological responses is critical to identifying means for enhancing our current antibiotic arsenal. Bactericidal antibiotics with diverse targets have been hypothesized to kill bacteria, in part, by inducing production of damaging reactive species. This notion has been supported by many groups, but recently challenged. Here we robustly test the hypothesis using biochemical, enzymatic and biophysical assays along with genetic and phenotypic experiments. We first used a novel intracellular hydrogen peroxide (H2O2) sensor, together with a chemically diverse panel of fluorescent dyes sensitive to an array of reactive species, to demonstrate that antibiotics broadly induce redox stress. Subsequent gene expression analyses reveal that complex antibiotic-induced oxidative stress responses are distinct from canonical responses generated by supra-physiological levels of H2O2. We next developed a method to dynamically quantify cellular respiration and found that bactericidal antibiotics elevate oxygen consumption, indicating significant alterations to bacterial redox physiology. We further show that catalase or DNA mismatch repair enzyme overexpression, as well as antioxidant pre-treatment limit antibiotic lethality, indicating that reactive oxygen species causatively contribute to antibiotic killing. Critically, the killing efficacy of antibiotics was diminished under strict anaerobic conditions, but could be enhanced by exposure to molecular oxygen or addition of alternative electron acceptors, suggesting that environmental factors play a role in killing cells physiologically primed for death. This work provides direct evidence that bactericidal antibiotics, downstream of their target-specific interactions, induce complex redox alterations that contribute to cellular damage and death, thus supporting an evolving, expanded model of antibiotic lethality. Here, we used microarrays to analyze oxidative stress responses to bactericidal antibiotic treatment in wildtype and mutant E coli

Project description:The overuse of antibiotics has made public health and safety face a serious crisis. It is urgent to develop new clinical treatment methods to combat drug-resistant bacteria to alleviate the health crisis. The efficiency of antibiotics is closely related to the metabolic state of bacteria. However, studies on fluoroquinolone-resistant Salmonella are relatively rare. In this study, non-targeted metabolomics was used to analyze the metabolic difference between sarafloxacin-susceptible Salmonella Typhimurium (SAR-S) and sarafloxacin-resistant Salmonella Typhimurium (SAR-R), and found that the central carbon metabolism was weakened due to drug resistance. We also confirmed that exogenous L-leucine increased the killing effect of sarafloxacin on SAR-R and other clinically resistant Salmonella serotypes. Exogenous L-leucine stimulated the metabolic state of bacteria, especially the TCA cycle, which increased the working efficiency of the electron transfer chain and increased the intracellular NADH, ATP concentrationa nd reactive oxygen species level. Furthermore, we used real-time quantitative PCR to confirm the changes in various metabolic characteristics. Our results suggest that when the metabolism of drug-resistant bacteria is reprogrammed, the bactericidal effect of antibiotics improves. This study further enhances research in the anti-drug resistance field at the metabolic level and provides theoretical support for solving the current problem of sarafloxacin drug resistance, a unique fluoroquinolone drug for animals. To sum up, our research results showed that exogenous L-leucine promotes the bactericidal effect of antibiotics through metabolic modification of drug-resistant bacteria, which has the potential as a new adjuvant of antibiotics. LC-MS positive mode is reported in the current study <a href='https://www.ebi.ac.uk/metabolights/MTBLS7711' rel='noopener noreferrer' target='_blank'>MTBLS7711</a>.LC-MS negative mode is reported in <a href='https://www.ebi.ac.uk/metabolights/MTBLS7713' rel='noopener noreferrer' target='_blank'>MTBLS7713</a>.

Project description:The overuse of antibiotics has made public health and safety face a serious crisis. It is urgent to develop new clinical treatment methods to combat drug-resistant bacteria to alleviate the health crisis. The efficiency of antibiotics is closely related to the metabolic state of bacteria. However, studies on fluoroquinolone-resistant Salmonella are relatively rare. In this study, non-targeted metabolomics was used to analyze the metabolic difference between sarafloxacin-susceptible Salmonella Typhimurium (SAR-S) and sarafloxacin-resistant Salmonella Typhimurium (SAR-R), and found that the central carbon metabolism was weakened due to drug resistance. We also confirmed that exogenous L-leucine increased the killing effect of sarafloxacin on SAR-R and other clinically resistant Salmonella serotypes. Exogenous L-leucine stimulated the metabolic state of bacteria, especially the TCA cycle, which increased the working efficiency of the electron transfer chain and increased the intracellular NADH, ATP concentration and reactive oxygen species level. Furthermore, we used real-time quantitative PCR to confirm the changes in various metabolic characteristics. Our results suggest that when the metabolism of drug-resistant bacteria is reprogrammed, the bactericidal effect of antibiotics improves. This study further enhances research in the anti-drug resistance field at the metabolic level and provides theoretical support for solving the current problem of sarafloxacin drug resistance, a unique fluoroquinolone drug for animals. To sum up, our research results showed that exogenous L-leucine promotes the bactericidal effect of antibiotics through metabolic modification of drug-resistant bacteria, which has the potential as a new adjuvant of antibiotics. LC-MS negative mode is reported in the current study <a href='https://www.ebi.ac.uk/metabolights/MTBLS7713' rel='noopener noreferrer' target='_blank'>MTBLS7713</a>.LC-MS positive mode is reported in <a href='https://www.ebi.ac.uk/metabolights/MTBLS7711' rel='noopener noreferrer' target='_blank'>MTBLS7711</a>.

Project description:This model is from the article: Modelling the Role of the Hsp70/Hsp90 System in the Maintenance of Protein Homeostasis Proctor CJ, Lorimer IAJ PLoS ONE2011; 6(7): e22038. doi:10.1371/journal.pone.0022038, Abstract: Neurodegeneration is an age-related disorder which is characterised by the accumulation of aggregated protein and neuronal cell death. There are many different neurodegenerative diseases which are classified according to the specific proteins involved and the regions of the brain which are affected. Despite individual differences, there are common mechanisms at the sub-cellular level leading to loss of protein homeostasis. The two central systems in protein homeostasis are the chaperone system, which promotes correct protein folding, and the cellular proteolytic system, which degrades misfolded or damaged proteins. Since these systems and their interactions are very complex, we use mathematical modelling to aid understanding of the processes involved. The model developed in this study focuses on the role of Hsp70 (IPR00103) and Hsp90 (IPR001404) chaperones in preventing both protein aggregation and cell death. Simulations were performed under three different conditions: no stress; transient stress due to an increase in reactive oxygen species; and high stress due to sustained increases in reactive oxygen species. The model predicts that protein homeostasis can be maintained during short periods of stress. However, under long periods of stress, the chaperone system becomes overwhelmed and the probability of cell death pathways being activated increases. Simulations were also run in which cell death mediated by the JNK (P45983) and p38 (Q16539) pathways was inhibited. The model predicts that inhibiting either or both of these pathways may delay cell death but does not stop the aggregation process and that eventually cells die due to aggregated protein inhibiting proteasomal function. This problem can be overcome if the sequestration of aggregated protein into inclusion bodies is enhanced. This model predicts responses to reactive oxygen species-mediated stress that are consistent with currently available experimental data. The model can be used to assess specific interventions to reduce cell death due to impaired protein homeostasis. Note: Simulations were performed under three different conditions: 1) normal condition (no stress), 2) moderate stress due to an increase in reactive oxygen species (ROS) i.e. ROS levels were increased by a factor of 4 at time=4hours for a period of 1 hour (not 2 hours as mentioned in the figure 5 legend of the reference publication. This is a typo in the paper and is clarified by the author) and 3) high stress due to sustained increase in reactive oxygen species (ROS) (here ROS increases with time). The model that corresponds to the normal condition is submitted as a main model in the BioModels Database. The other two models, that corresponds to the moderate stress conditions and high stress conditions are available in SBML format as supporting files [go to Curation tab]. Supplementary figures S3 (normal condition), S4 (moderate stress condition) and S6 (high stress condition) are reproduced here.

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Streptococcus pneumoniae R6

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