Project description:Pneumococcal bacteriocins (pneumocins) are antibacterial toxins that mediate intra- and interspecies competition within the human host. What triggers pneumocin expression is poorly understood. Using RNA-sequencing, we mapped the regulon of the pneumocin cluster (blp) of Streptococcus pneumoniae D39 and show that several antibiotics activate the blp-genes. Real-time gene expression measurements showed that while the promoter driving expression of the two-component regulatory system blpS/H was constitutive, the remaining blp-promoters (e.g. controlling pneumocin expression, immunity and the inducer peptide BlpC) was pH-dependent, induced in the late exponential phase and occurred in all cells. Intriguingly, competence for genetic transformation, mediated by the ComD/E two-component quorum system, was induced by the same stimuli. To test for regulatory interplay, we utilized synthetic BlpC and competence-stimulating peptide (CSP). Strikingly, immediately upon addition of CSP, the blp-promoters were activated in a comD/E dependent manner. After a delay, blp-expression was highly induced but strictly dependent on the presence of blpRH and blpC. This raised the question how BlpC is exported since phylogenetic analysis showed that the putative exporter for BlpC, blpAB, is not intact in strain D39 and most other strains. However, all sequenced pneumococcal strains contain intact comAB genes, encoding the transport system for CSP. In fact, we show that high expression of the blp-genes requires comAB. Together, we demonstrate that regulation of pneumocin expression is intertwined with competence, explaining why certain antibiotics induce blp-expression. Antibiotic-induced pneumocin expression might promote survival under antibiotic stress by killing and liberating DNA from neighboring (antibiotic-resistant) bacteria, which can then be used for transformation or repair. Pairwise comparison of untreated and antibiotic-treated cells (either DNA-seq or RNA-seq). It was performed with deep sequencing, using an Illumina HiSeq 2000 machine with 100 nt paired-end reads. Control and HPUra-treated samples were analysed from duplicate samples, while other conditions were from single samples.

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: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.

Dataset Information

Postgraduate Scientific Research Innovation Plan of Bengbu Medical College

Publications

Proteomic analysis of meropenem-induced outer membrane vesicles released by carbapenem-resistant <i>Klebsiella pneumoniae</i>.

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