Transcriptional response of Acinetobacter baumannii to hydrogen peroxide and role of mumR and oxyR in regulating this response
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ABSTRACT: Acinetobacter baumannii is a Gram-negative opportunistic pathogen that causes multiple infections, including pneumonia, bacteremia, and wound infections. Due to multiple intrinsic and acquired drug-resistance mechanisms, A. baumannii isolates are commonly multi-drug resistant and infections are notoriously difficult to treat. Therefore, it is important to identify mechanisms used by A. baumannii to survive stresses encountered during infection as a means of identifying new drug targets. In this study, we determined the transcriptional response of A. baumannii to hydrogen peroxide stress using RNASequencing. Upon exposure to hydrogen peroxide, A. baumannii differentially transcribes several hundred genes. In this study, we also determined the transcriptional profile of A. baumannii strains with the transcriptional regulators mumR or oxyR genetically inactivated and identified transcriptional differences between these strains and wild-type A. baumannii in response to hydrogen peroxide stress. In doing this, the function of A. baumannii OxyR in hydrogen peroxide stress resistance and regulation of genes required for hydrogen peroxide detoxification was defined. Moreover, the contribution of the uncharacterized regulator MumR to hydrogen peroxide stress resistance was also explored. This work reveals the transcriptome of an important human pathogen in the presence of hydrogen peroxide stress.
Project description:The idea was to examine the role of OxyR in control of the oxidative stress response of B. fragilis when exposed to either atmospheric oxygen, 5% oxygen atmosphere, or hydrogen peroxide Keywords: stress response
Project description:The intracellular pathogen Legionella pneumophila (Lp) is a strict aerobe, surviving and replicating in environments where it frequently encounters reactive oxygen species, such as the nutrient-poor water environment and inside host cells. In many proteobacteria, the oxidative stress response is regulated by the LysR-type regulator OxyR; however, the role played by the OxyR homologue in Lp is still unclear. Therefore, we undertook the characterisation of the phenotypes associated with the deletion of OxyR in Lp. OxyR is dispensable for growth in rich broth, in amoeba and in human cultured macrophages, and for the survival of Lp in water. Nevertheless, the mutant was found to be more sensitive to hydrogen peroxide than the wild-type when grown to post-exponential phase, but not when grown to exponential phase. Moreover, the mutant is defective in forming isolated colonies on charcoal yeast extract (CYE) agar plates, but supplementation with anti-ROS molecules, such as pyruvate, α-ketoglutarate and catalase, rescued this defect. Further characterisation of this phenotype using a transcriptional reporter fusion and microarray analysis revealed that the deletion mutant is not defective for the expression of known anti-ROS genes which suggests that the growth defect on agar plates and the higher susceptibility to hydrogen peroxide are due to a broad change in the transcriptional response. Furthermore, the growth defect is suppressed when the mutant is grown on CYE plates made with agarose, suggesting that a compound present in typical agar is toxic for the oxyR mutant.
Project description:Series of DNA microarrays (4) comparing the M. catarrhalis strain 035E and M. catarrhalis oxyR mutant response to 50 mM hydrogen peroxide. Wild-type cells and oxyR mutant cells were exposed to 50 mM hydrogen peroxide for 15 minutes. RNA was extracted and DNA microarray analysis performed. 4 biologic replicates were studied. One dye swap was included in this series analysis. Control oxyR mutant cells exposed to water
Project description:Series of DNA microarrays (4) comparing the M. catarrhalis strain 035E and M. catarrhalis oxyR mutant response to 50 mM hydrogen peroxide.
Project description:Salmonella enterica serovar Typhimurium is a Gram-negative bacterium, facultative anaerobe and intracellular pathogen that causes enteric fever in mice. Once orally ingested, Salmonella invades and traverse the mucosal intestinal epithelia, where it is phagocytized by specialized cells including macrophages, dendritic cells and neutrophils. Within these cells, the bacterium is kept in a compartment termed Salmonella containing vacuole where it is exposed to different adverse conditions including nutrient deprivation, acid pH, reactive oxygen (ROS) as well as nitrogen (RNS) species and low oxygen levels. Among the signals encountered by the bacteria, oxidative stress is one of the main challenges that it has to overcome in order to survive. In this context, the OxyR and SoxRS proteins are the most studied regulators involved in response to ROS. However, in the past years growing evidence suggests that the ArcAB two-component system might play a key role in modulating gene expression in response to ROS. Furthermore, the global regulator ArcA is required for the resistance of Escherichia coli, S. Enteritidis and Typhimurium to hydrogen peroxide (H2O2), however, the ArcA regulon under oxidative stress conditions remains elusive. Therefore, the aim of this work was to demonstrate that ArcAB regulates the expression of genes in response to hydrogen peroxide and determine the ArcA regulon under this condition. To achieve this, we evaluated transcriptomic changes in strain 14028s, ∆arcA in response to H2O2. Total RNA was harvested from three biological replicates of wt and arcA mutant cultures exposed or unexposed to 1.5 mM hydrogen peroxide for 20 min in LB medium.
Project description:Using Nanopore sequencing, our study has revealed a close correlation between genomic methylation levels and antibiotic resistance rates in Acinetobacter Baumannii. Specifically, the combined genome-wide DNA methylome and transcriptome analysis revealed the first epigenetic-based antibiotic-resistance mechanism in A. baumannii. Our findings suggest that the precise location of methylation sites along the chromosome could provide new diagnostic markers and drug targets to improve the management of multidrug-resistant A. baumannii infections.
Project description:Salmonella enterica serovar Typhimurium is a Gram-negative bacterium, facultative anaerobe and intracellular pathogen that causes enteric fever in mice. Once orally ingested, Salmonella invades and traverse the mucosal intestinal epithelia, where it is phagocytized by specialized cells including macrophages, dendritic cells and neutrophils. Within these cells, the bacterium is kept in a compartment termed Salmonella containing vacuole where it is exposed to different adverse conditions including nutrient deprivation, acid pH, reactive oxygen (ROS) as well as nitrogen (RNS) species and low oxygen levels. Among the signals encountered by the bacteria, oxidative stress is one of the main challenges that it has to overcome in order to survive. In this context, the OxyR and SoxRS proteins are the most studied regulators involved in response to ROS. However, in the past years growing evidence suggests that the ArcAB two-component system might play a key role in modulating gene expression in response to ROS. Furthermore, the global regulator ArcA is required for the resistance of Escherichia coli, S. Enteritidis and Typhimurium to hydrogen peroxide (H2O2), however, the ArcA regulon under oxidative stress conditions remains elusive. Therefore, the aim of this work was to demonstrate that ArcAB regulates the expression of genes in response to hydrogen peroxide and determine the ArcA regulon under this condition. To achieve this, we evaluated transcriptomic changes in strain 14028s, ∆arcA in response to H2O2.
Project description:Candida albicans is a fungal opportunistic pathogen responsible for cause severe infections in immunocompromised patients. Despite nowadays an effective antifungal armoury is available, the emergence of antifungal resistance is promoting the urge to develop alternative treatments to combat this fungal pathogen. For this purpose it is essential to go deepen in the knowledge of C. albicans response to several stressors. With this aim we performed a proteomic study of C. albicans exposed to 5 and 10 mM of hydrogen peroxide and 40 and 60 mM of acetic acid taking advantage of data independent acquisition mass spectrometry. Four biological replicates of each condition were analysed. For protein identification we used a C. albicans spectral library previously constructed by DDA analysis allowing the DIA quantitation of nearby 2000 proteins. Changes in protein abundance after the treatments reflected highly different proteomic patterns after each treatment. The exposure of C. albicans to hydrogen peroxide led to an increase in many proteins related to oxidative stress response, proteasome and protein folding while few proteins from mitochondria decreased their abundance. In contrast, acetic acid treatment triggered the decrease in the abundance of proteins related to aminoacid biosynthesis, actin polymerization, oxidative stress response and proteasome. This extensive proteomic study provided valuable information for the development of new drug targets against decisive proteins for cell survival.
Project description:The bacterial pathogen, Acinetobacter baumannii, is a leading cause of drug-resistant infections. Here, we investigated the potential of developing nanobodies that specifically recognize A. baumannii over other Gram-negative bacteria. Through generation and panning of a synthetic nanobody library, we identified several potential lead candidates. We demonstrate how incorporation of next generation sequencing analysis can aid in selection of lead candidates for further characterization. Using monoclonal phage display, we validated the binding of several lead nanobodies to A. baumannii. Subsequent purification and biochemical characterization revealed one particularly robust nanobody that broadly and specifically bound A. baumannii compared to other common drug resistant pathogens. These findings support the potentially for nanobodies to selectively target A. baumannii and the identification of lead candidates for possible future diagnostic and therapeutic development.