Transposon-Directed Insertion-Site Sequencing Reveals Glycolysis Gene gpmA as Part of the H2O2 Defense Mechanisms in Escherichia coli.

Hydrogen peroxide (H2O2) is a common effector of defense mechanisms against pathogenic infections. However, bacterial factors involved in H2O2 tolerance remain unclear. Here we used transposon-directed insertion-site sequencing (TraDIS), a technique allowing the screening of the whole genome, to identify genes implicated in H2O2 tolerance in Escherichia coli. Our TraDIS analysis identified 10 mutants with fitness defect upon H2O2 exposure, among which previously H2O2-associated genes (oxyR, dps, dksA, rpoS, hfq and polA) and other genes with no known association with H2O2 tolerance in E. coli (corA, rbsR, nhaA and gpmA). This is the first description of the impact of gpmA, a gene involved in glycolysis, on the susceptibility of E. coli to H2O2. Indeed, confirmatory experiments showed that the deletion of gpmA led to a specific hypersensitivity to H2O2 comparable to the deletion of the major H2O2 scavenger gene katG. This hypersensitivity was not due to an alteration of catalase function and was independent of the carbon source or the presence of oxygen. Transcription of gpmA was upregulated under H2O2 exposure, highlighting its role under oxidative stress. In summary, our TraDIS approach identified gpmA as a member of the oxidative stress defense mechanism in E. coli.

Transcriptomic Analysis of E. coli after Exposure to a Sublethal Concentration of Hydrogen Peroxide Revealed a Coordinated Up-Regulation of the Cysteine Biosynthesis Pathway.

Hydrogen peroxide (H2O2) is a key defense component of host-microbe interaction. However, H2O2 concentrations generated by immune cells or epithelia are usually insufficient for bacterial killing and rather modulate bacterial responses. Here, we investigated the impact of sublethal H2O2 concentration on gene expression of E. coli BW25113 after 10 and 60 min of exposure. RNA-seq analysis revealed that approximately 12% of bacterial genes were strongly dysregulated 10 min following exposure to 2.5 mM H2O2. H2O2 exposure led to the activation of a specific antioxidant response and a general stress response. The latter was characterized by a transient down-regulation of genes involved in general metabolism, such as nucleic acid biosynthesis and translation, with a striking and coordinated down-regulation of genes involved in ribosome formation, and a sustained up-regulation of the SOS response. We confirmed the rapid transient and specific response mediated by the transcription factor OxyR leading to up-regulation of antioxidant systems, including the catalase-encoding gene (katG), that rapidly degrade extracellular H2O2 and promote bacterial survival. We documented a strong and transient up-regulation of genes involved in sulfur metabolism and cysteine biosynthesis, which are under the control of the transcription factor CysB. This strong specific transcriptional response to H2O2 exposure had no apparent impact on bacterial survival, but possibly replenishes the stores of oxidized cysteine and glutathione. In summary, our results demonstrate that different stress response mechanisms are activated by H2O2 exposure and highlight the cysteine synthesis as an antioxidant response in E. coli.

Hydrogen Peroxide Affects Growth of S. aureus Through Downregulation of Genes Involved in Pyrimidine Biosynthesis.

Reactive oxygen species (ROS) play a crucial role in the cellular defense against S. aureus, as evidenced by the importance of this pathogen in patients lacking the ROS-generating phagocyte NADPH oxidase NOX2. ROS concentrations required to kill S. aureus in vitro are much higher than those found in the phagosome. We therefore hypothesized that sublethal ROS concentrations may play a role in S. aureus gene dysregulation and investigated the in vitro transcriptomic response of S. aureus to sublethal concentrations of hydrogen peroxide (H2O2). A striking observation of these experiments was a coordinated and massive downregulation of genes involved in pyrimidine metabolism. Using transposon insertion mutants, we demonstrated that deletion of carA, a gene involved in pyrimidine synthesis, led to a significant growth defect and to an increased sensitivity of S. aureus to added H2O2. The phenotype of the carA mutant could be reversed through supplementation with the pyrimidine precursor uracil, or with a multicopy vector encoding carA. As opposed to the impact of ROS on extracellular survival, carA deletion did not affect the intracellular survival in neutrophils. Our results raise the possibility that ROS-dependent downregulation of pyrimidine metabolism might be a survival strategy of S. aureus, allowing colonization through intracellular survival, while decreasing the risk of killing the host through dampened extracellular growth.

Ultrasound-driven cardiac MRI.

PURPOSE: One of the challenges of cardiac MR imaging is the compensation of respiratory motion, which causes the heart and the surrounding tissues to move. Commonly-used methods to overcome this effect, breath-holding and MR navigation, present shortcomings in terms of available acquisition time or need to periodically interrupt the acquisition, respectively. In this work, an implementation of respiratory motion compensation that obtains information from abdominal ultrasound and continuously adapts the imaged slice position in real time is presented. METHODS: A custom workflow was developed, comprising an MR-compatible ultrasound acquisition system, a feature-motion-tracking system with polynomial predictive capability, and a custom MR sequence that continuously adapts the position of the acquired slice according to the tracked position. The system was evaluated on a moving phantom by comparing sharpness and image blurring between static and moving conditions, and in vivo by tracking the motion of the blood vessels of the liver to estimate the cardiac motion. Cine images of the heart were acquired during free breathing. RESULTS: In vitro, the predictive motion correction yielded significantly better results than non-predictive or non-corrected acquisitions (p â‰ª 0.01). In vivo, the predictive correction resulted in an image quality very similar to the breath-hold acquisition, whereas the uncorrected images show noticeable blurring artifacts. CONCLUSION: In this work, the possibility of using ultrasound navigation with tracking for the real-time adaptation of MR imaging slices was demonstrated. The implemented technique enabled efficient imaging of the heart with resolutions that would not be feasible in a single breath-hold.