Analysis of the oxidative stress regulon identifies soxS as a genetic target for resistance reversal in multi-drug resistant Klebsiella pneumoniae

AbstractIn bacteria, the defense system deployd to counter oxidative stress is orchestrated by three transcriptional factors – SoxS, SoxR, and OxyR. Although the regulon that these factors control is known in many bacteria, similar data is not available for Klebsiella pneumoniae. To address this data gap, oxidative stress was artificially induced in K. pneumoniae MGH 78578 using paraquat and the corresponding oxidative stress regulon recorded using RNA-seq. The soxS gene was significantly induced during oxidative stress and a knock-out mutant was constructed, to explore its functionality. The wild-type and mutant were grown in the presence of paraquat and subjected to RNA-seq to elucidate the soxS regulon in K. pneumoniae MGH78578. Genes that are commonly regulated both in the oxidative stress regulon and soxS regulon were identified and denoted as the ‘oxidative SoxS regulon’ – these included a stringent group of genes specifically regulated by SoxS. Efflux pump encoding genes such as acrAB-tolC, acrE, and global regulators such as marRAB were identified as part of this regulon. Consequently, the isogenic soxS mutant was found to exhibit a reduction in the minimum bactericidal concentration against tetracycline compared to that of the wild type. Impaired efflux activity, allowing tetracycline to be accumulated in the cytoplasm to bactericidal levels, was further evaluated using a tetraphenylphosphonium (TPP+) accumulation assay. The soxS mutant was also susceptible to tetracycline in vivo, in a zebrafish embryo model. We conclude that the soxS gene could be considered as a genetic target against which an inhibitor could be developed in the future and used in combinatorial therapy with tetracycline to combat infections associated with multi-drug resistant K. pneumoniae.

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MYB134-RNAi poplar plants show reduced tannin synthesis in leaves but not roots, and increased susceptibility to oxidative stress

Journal of Experimental Botany ◽

10.1093/jxb/eraa371 ◽

2020 ◽

Vol 71 (20) ◽

pp. 6601-6611

Author(s):

Geraldine Gourlay ◽

Dawei Ma ◽

Axel Schmidt ◽

C Peter Constabel

Keyword(s):

Oxidative Stress ◽

Reactive Oxygen Species ◽

Condensed Tannin ◽

Reactive Oxygen ◽

Oxygen Species ◽

Rna Seq ◽

Wild Type ◽

And Control ◽

Increased Susceptibility

Abstract The importance of the poplar MYB134 gene in controlling condensed tannin (CT) biosynthesis was tested by suppressing its expression using RNA interference (RNAi). MYB134-RNAi plants grew normally but showed reduced accumulation of stress-induced CTs in leaves. RNA-seq analysis indicated that flavonoid- and CT-related genes, as well as additional CT regulators, were strongly and specifically down-regulated by MYB134 suppression. This confirmed that the primary MYB134 target is the leaf flavonoid and CT pathway. Root CT accumulation was not impacted by MYB suppression, suggesting that additional CT regulators are active in roots and emphasizing the complexity of the regulation of CTs in poplar. To test the effect of CT down-regulation on oxidative stress resistance, leaves of MYB134-RNAi and control plants were exposed to the reactive oxygen species generator methyl viologen. MYB134-RNAi leaves sustained significantly more photosystem II damage, as seen in reduced chlorophyll fluorescence, compared with wild-type leaves. MYB134-RNAi leaves also contained more hydrogen peroxide, a reactive oxygen species, compared with the wild type. Our data thus corroborate the hypothesis that CT can act as an antioxidant in vivo and protect against oxidative stress. Overall, MYB134 was shown to be a central player in the regulation of CT synthesis in leaves.

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Klebsiella virus UPM2146 lyses multiple drug-resistant Klebsiella pneumoniae in vitro and in vivo

PLoS ONE ◽

10.1371/journal.pone.0245354 ◽

2021 ◽

Vol 16 (1) ◽

pp. e0245354

Author(s):

Omar Assafiri ◽

Adelene Ai-Lian Song ◽

Geok Hun Tan ◽

Irwan Hanish ◽

Amalia Mohd Hashim ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Multiple Drug ◽

Drug Resistant ◽

Zebrafish Model ◽

Whole Genome Analysis ◽

Narrow Host Range ◽

Opportunistic Bacteria ◽

Multiple Drug Resistant

Klebsiella pneumoniae are opportunistic bacteria found in the gut. In recent years they have been associated with nosocomial infections. The increased incidence of multiple drug-resistant K. pneumoniae makes it necessary to find new alternatives to treat the disease. In this study, phage UPM2146 was isolated from a polluted lake which can lyse its host K. pneumoniae ATCC BAA-2146. Observation from TEM shows that UPM2146 belongs to Caudoviriales (Order) based on morphological appearance. Whole genome analysis of UPM2146 showed that its genome comprises 160,795 bp encoding for 214 putative open reading frames (ORFs). Phylogenetic analysis revealed that the phage belongs to Ackermannviridae (Family) under the Caudoviriales. UPM2146 produces clear plaques with high titers of 1010 PFU/ml. The phage has an adsorption period of 4 min, latent period of 20 min, rise period of 5 min, and releases approximately 20 PFU/ bacteria at Multiplicity of Infection (MOI) of 0.001. UPM2146 has a narrow host-range and can lyse 5 out of 22 K. pneumoniae isolates (22.72%) based on spot test and efficiency of plating (EOP). The zebrafish larvae model was used to test the efficacy of UPM2146 in lysing its host. Based on colony forming unit counts, UPM2146 was able to completely lyse its host at 10 hours onwards. Moreover, we show that the phage is safe to be used in the treatment against K. pneumoniae infections in the zebrafish model.

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The role of the NMD factor UPF3B in olfactory sensory neurons

eLife ◽

10.7554/elife.57525 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 1

Author(s):

Kun Tan ◽

Samantha H Jones ◽

Blue B Lake ◽

Jennifer N Dumdie ◽

Eleen Y Shum ◽

...

Keyword(s):

Human Cognition ◽

Cell Populations ◽

Rna Seq ◽

Wild Type ◽

Single Cell Rna Sequencing ◽

Nonsense Mediated Rna Decay ◽

Different Levels ◽

Selection Of

The UPF3B-dependent branch of the nonsense-mediated RNA decay (NMD) pathway is critical for human cognition. Here, we examined the role of UPF3B in the olfactory system. Single-cell RNA-sequencing (scRNA-seq) analysis demonstrated considerable heterogeneity of olfactory sensory neuron (OSN) cell populations in wild-type (WT) mice, and revealed that UPF3B loss influences specific subsets of these cell populations. UPF3B also regulates the expression of a large cadre of antimicrobial genes in OSNs, and promotes the selection of specific olfactory receptor (Olfr) genes for expression in mature OSNs (mOSNs). RNA-seq and Ribotag analyses identified classes of mRNAs expressed and translated at different levels in WT and Upf3b-null mOSNs. Integrating multiple computational approaches, UPF3B-dependent NMD target transcripts that are candidates to mediate the functions of NMD in mOSNs were identified in vivo. Together, our data provides a valuable resource for the olfactory field and insights into the roles of NMD in vivo.

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Loss of Sirt3 accelerates arterial thrombosis by increasing formation of neutrophil extracellular traps and plasma tissue factor activity

Cardiovascular Research ◽

10.1093/cvr/cvy036 ◽

2018 ◽

Vol 114 (8) ◽

pp. 1178-1188 ◽

Cited By ~ 14

Author(s):

Daniel S Gaul ◽

Julien Weber ◽

Lambertus J van Tits ◽

Susanna Sluka ◽

Lisa Pasterk ◽

...

Keyword(s):

Oxidative Stress ◽

Myocardial Infarction ◽

Bone Marrow ◽

Arterial Thrombosis ◽

Ex Vivo ◽

Neutrophil Extracellular Traps ◽

Wild Type ◽

Rotational Thromboelastometry ◽

Extracellular Traps

AbstractAimsSirtuin 3 (Sirt3) is a mitochondrial, nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase that reduces oxidative stress by activation of superoxide dismutase 2 (SOD2). Oxidative stress enhances arterial thrombosis. This study investigated the effects of genetic Sirt3 deletion on arterial thrombosis in mice in an inflammatory setting and assessed the clinical relevance of these findings in patients with ST-elevation myocardial infarction (STEMI).Methods and resultsUsing a laser-induced carotid thrombosis model with lipopolysaccharide (LPS) challenge, in vivo time to thrombotic occlusion in Sirt3−/− mice (n = 6) was reduced by half compared to Sirt3+/+ wild-type (n = 8, P < 0.01) controls. Ex vivo analyses of whole blood using rotational thromboelastometry revealed accelerated clot formation and increased clot stability in Sirt3−/− compared to wild-type blood. rotational thromboelastometry of cell-depleted plasma showed accelerated clotting initiation in Sirt3−/− mice, whereas overall clot formation and firmness remained unaffected. Ex vivo LPS-induced neutrophil extracellular trap formation was increased in Sirt3−/− bone marrow-derived neutrophils. Plasma tissue factor (TF) levels and activity were elevated in Sirt3−/− mice, whereas plasma levels of other coagulation factors and TF expression in arterial walls remained unchanged. SOD2 expression in bone marrow -derived Sirt3−/− neutrophils was reduced. In STEMI patients, transcriptional levels of Sirt3 and its target SOD2 were lower in CD14+ leukocytes compared with healthy donors (n = 10 each, P < 0.01).ConclusionsSirt3 loss-of-function enhances experimental thrombosis in vivo via an increase of neutrophil extracellular traps and elevation of TF suggesting thrombo-protective effects of endogenous Sirt3. Acute coronary thrombosis in STEMI patients is associated with lower expression levels of SIRT3 and SOD2 in CD14+ leukocytes. Therefore, enhancing SIRT3 activity by pan-sirtuin activating NAD+-boosters may provide a novel therapeutic target to prevent or treat thrombotic arterial occlusion in myocardial infarction or stroke.

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Abstract 123: Age-Related Endothelial Senescence is Driven by Thrombospondin-1-Activated NADPH Oxidase Nox1

Hypertension ◽

10.1161/hyp.66.suppl_1.123 ◽

2015 ◽

Vol 66 (suppl_1) ◽

Author(s):

Daniel N Meijles ◽

Imad Al Ghouleh ◽

Sanghamitra Sahoo ◽

Jefferson H Amaral ◽

Heather Knupp ◽

...

Keyword(s):

Oxidative Stress ◽

Nadph Oxidase ◽

Vascular Diseases ◽

Dependent Manner ◽

Wild Type ◽

Molecular Control ◽

Redox Biology ◽

Age Related ◽

P53 Activation

Organismal aging represents an independent risk factor underlying many vascular diseases, including systemic and pulmonary hypertension, and atherosclerosis. While the mechanisms driving aging are largely elusive, a steady persistent increase in tissue oxidative stress has been associated with senescence. Previously we showed TSP1 elicits NADPH oxidase (Nox)-dependent vascular smooth muscle cell oxidative stress. However mechanisms by which TSP1 affects endothelial redox biology are unknown. Here, we tested the hypothesis that TSP1 induces endothelial oxidative stress-linked senescence in aging. Using rapid autopsy disease-free human pulmonary (PA) artery, we identified a significant positive correlation between age, protein levels of TSP1, Nox1 and the cell-cycle repressor p21cip (p<0.05). Age also positively associated with increased Amplex Red-detected PA hydrogen peroxide levels (p<0.05). Moreover, treatment of human PA endothelial cells (HPAEC) with TSP1 (2.2nM; 24h) increased expression (~1.9 fold; p<0.05) and activation of Nox1 (~1.7 fold; p<0.05) compared to control, as assessed by Western blot and SOD-inhibitable cytochrome c reduction. Western blotting and immunofluorescence showed a TSP1-mediated increase in p53 activation, indicative of the DNA damage response. Moreover, TSP1 significantly increased HPAEC senescence in a p53/p21cip/Rb-dependent manner, as assessed by immunofluorescent detection of subcellular localization and senescence-associated β-galactosidase staining. To explore this pathway in vivo, middle-aged (8-10 month) wild-type and TSP1-null mice were utilized. In the TSP1-null, reduced lung senescence, oxidative stress, Nox1 levels and p21cip expression were observed compared to wild-type supporting findings in human samples and cell experiments. Finally, prophylactic treatment with specific Nox1 inhibitor NoxA1ds (10μM) attenuated TSP1-induced HPAEC ROS, p53 activation, p21cip expression and senescence. Taken together, our results provide molecular insight into the functional interplay between TSP1 and Nox1 in the regulation of endothelial senescence, with implications for molecular control of the aging process.

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Knock out of sHSP genes determines some modifications in the probiotic attitude of Lactiplantibacillus plantarum

Biotechnology Letters ◽

10.1007/s10529-020-03041-6 ◽

2020 ◽

Author(s):

Angela Longo ◽

Pasquale Russo ◽

Vittorio Capozzi ◽

Giuseppe Spano ◽

Daniela Fiocco

Keyword(s):

Small Heat Shock Protein ◽

Cellular Adhesion ◽

Probiotic Properties ◽

Wild Type ◽

Knock Out ◽

Human Enterocyte ◽

Transcriptional Induction ◽

Pro Inflammatory Cytokines

Abstract Objective We investigated whether the knock out of small heat shock protein (sHSP) genes (hsp1, hsp2 and hsp3) impact on probiotic features of Lactiplantibacillus plantarum WCFS1, aiming to find specific microbial effectors involved in microbe-host interplay. Results The probiotic properties of L. plantarum WCFS1 wild type, hsp1, hsp2 and hsp3 mutant clones were evaluated and compared through in vitro trials. Oro-gastro-intestinal assays pointed to significantly lower survival for hsp1 and hsp2 mutants under stomach-like conditions, and for hsp3 mutant under intestinal stress. Adhesion to human enterocyte-like cells was similar for all clones, though the hsp2 mutant exhibited higher adhesiveness. L. plantarum cells attenuated the transcriptional induction of pro-inflammatory cytokines on lipopolysaccharide-treated human macrophages, with some exception for the hsp1 mutant. Intriguingly, this clone also induced a higher IL10/IL12 ratio, which is assumed to indicate the anti-inflammatory potential of probiotics. Conclusions sHSP genes deletion determined some differences in gut stress resistance, cellular adhesion and immuno-modulation, also implying effects on in vivo interaction with the host. HSP1 might contribute to immunomodulatory mechanisms, though additional experiments are necessary to test this feature.

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Pejvakin-mediated pexophagy protects auditory hair cells against noise-induced damage

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1821844116 ◽

2019 ◽

Vol 116 (16) ◽

pp. 8010-8017 ◽

Cited By ~ 12

Author(s):

Jean Defourny ◽

Alain Aghaie ◽

Isabelle Perfettini ◽

Paul Avan ◽

Sedigheh Delmaghani ◽

...

Keyword(s):

Oxidative Stress ◽

Hair Cells ◽

Cell Damage ◽

Peroxisome Proliferation ◽

Wild Type ◽

Auditory Hair Cells ◽

C Terminus ◽

Cysteine Mutagenesis ◽

Autophagic Degradation

Noise overexposure causes oxidative stress, leading to auditory hair cell damage. Adaptive peroxisome proliferation involving pejvakin, a peroxisome-associated protein from the gasdermin family, has been shown to protect against this harmful oxidative stress. However, the role of pejvakin in peroxisome dynamics and homeostasis remains unclear. Here we show that sound overstimulation induces an early and rapid selective autophagic degradation of peroxisomes (pexophagy) in auditory hair cells from wild-type, but not pejvakin-deficient (Pjvk−/−), mice. Noise overexposure triggers recruitment of the autophagosome-associated protein MAP1LC3B (LC3B; microtubule-associated protein 1 light chain 3β) to peroxisomes in wild-type, but not Pjvk−/−, mice. We also show that pejvakin–LC3B binding involves an LC3-interacting region within the predicted chaperone domain of pejvakin. In transfected cells and in vivo transduced auditory hair cells, cysteine mutagenesis experiments demonstrated the requirement for both C328 and C343, the two cysteine residues closest to the C terminus of pejvakin, for reactive oxygen species-induced pejvakin–LC3B interaction and pexophagy. The viral transduction of auditory hair cells from Pjvk−/− mice in vivo with both Pjvk and Lc3b cDNAs completely restored sound-induced pexophagy, fully prevented the development of oxidative stress, and resulted in normal levels of peroxisome proliferation, whereas Pjvk cDNA alone yielded only a partial correction of the defects. Overall, our results demonstrate that pexophagy plays a key role in noise-induced peroxisome proliferation and identify defective pexophagy as a cause of noise-induced hearing loss. They suggest that pejvakin acts as a redox-activated pexophagy receptor/adaptor, thereby identifying a previously unknown function of gasdermin family proteins.

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In Vivo Titration of Mitomycin C Action by Four Escherichia coli Genomic Regions on Multicopy Plasmids

Journal of Bacteriology ◽

10.1128/jb.183.7.2259-2264.2001 ◽

2001 ◽

Vol 183 (7) ◽

pp. 2259-2264 ◽

Cited By ~ 26

Author(s):

Yan Wei ◽

Amy C. Vollmer ◽

Robert A. LaRossa

Keyword(s):

Escherichia Coli ◽

Mitomycin C ◽

Transcriptional Activation ◽

Efflux Pump ◽

Wild Type ◽

Potent Inducer ◽

E Coli ◽

Genomic Regions

ABSTRACT Mitomycin C (MMC), a DNA-damaging agent, is a potent inducer of the bacterial SOS response; surprisingly, it has not been used to select resistant mutants from wild-type Escherichia coli. MMC resistance is caused by the presence of any of four distinctE. coli genes (mdfA, gyrl, rob, andsdiA) on high-copy-number vectors. mdfAencodes a membrane efflux pump whose overexpression results in broad-spectrum chemical resistance. The gyrI (also called sbmC) gene product inhibits DNA gyrase activity in vitro, while the rob protein appears to function in transcriptional activation of efflux pumps. SdiA is a transcriptional activator of ftsQAZ genes involved in cell division.

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Skeletal muscle weakness due to deficiency of CuZn-superoxide dismutase is associated with loss of functional innervation

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00093.2011 ◽

2011 ◽

Vol 301 (5) ◽

pp. R1400-R1407 ◽

Cited By ~ 49

Author(s):

Lisa M. Larkin ◽

Carol S. Davis ◽

Catrina Sims-Robinson ◽

Tatiana Y. Kostrominova ◽

Holly Van Remmen ◽

...

Keyword(s):

Oxidative Stress ◽

Superoxide Dismutase ◽

Muscle Weakness ◽

Muscle Atrophy ◽

Muscle Stimulation ◽

Wild Type ◽

Functional Changes ◽

Fiber Number ◽

Cuzn Superoxide Dismutase

An association between oxidative stress and muscle atrophy and weakness in vivo is supported by elevated oxidative damage and accelerated loss of muscle mass and force with aging in CuZn-superoxide dismutase-deficient ( Sod1−/−) mice. The purpose was to determine the basis for low specific force (N/cm2) of gastrocnemius muscles in Sod1−/− mice and establish the extent to which structural and functional changes in muscles of Sod1−/− mice resemble those associated with normal aging. We tested the hypothesis that muscle weakness in Sod1−/− mice is due to functionally denervated fibers by comparing forces during nerve and direct muscle stimulation. No differences were observed for wild-type mice at any age in the forces generated in response to nerve and muscle stimulation. Nerve- and muscle-stimulated forces were also not different for 4-wk-old Sod1−/− mice, whereas, for 8- and 20-mo-old mice, forces during muscle stimulation were 16 and 30% greater, respectively, than those obtained using nerve stimulation. In addition to functional evidence of denervation with aging, fiber number was not different for Sod1−/− and wild-type mice at 4 wk, but 50% lower for Sod1−/− mice by 20 mo, and denervated motor end plates were prevalent in Sod1−/− mice at both 8 and 20 mo and in WT mice by 28 mo. The data suggest ongoing denervation in muscles of Sod1−/− mice that results in fiber loss and muscle atrophy. Moreover, the findings support using Sod1−/− mice to explore mechanistic links between oxidative stress and the progression of deficits in muscle structure and function.

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Potential Role of Dual NF-κB and Oxidative Stress Pathway Inhibitor, OT-304 as a Novel Drug Resistance-Reversing Agent.

Blood ◽

10.1182/blood.v110.11.4211.4211 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4211-4211

Author(s):

Shaker A. Mousa ◽

Ghanshyam Patil ◽

Abdelhadi Rebbaa

Keyword(s):

Oxidative Stress ◽

Drug Resistance ◽

Tumor Cells ◽

Genetic Alterations ◽

Potential Candidate ◽

Drug Resistant ◽

P Glycoprotein ◽

And Oxidative Stress

Abstract The development of resistance to chemotherapy represents an adaptive biological response by tumor cells that leads to treatment failure and patient relapse. During the course of their evolution (intrinsic resistance) or in response to chemotherapy (acquired resistance), tumor cells may undergo genetic alterations to possess a drug resistant phenotype. Dysregulation of membrane transport proteins and cellular enzymes, as well as altered susceptibility to commit to apoptosis are among the mechanisms that contribute to the genesis of acquired drug resistance. Recently, the development of approaches to prevent and/or to reverse this phenomenon has attracted special interest and a number of drug candidates have been identified. Despite strong effects observed for these candidates in vitro, however, most of them fail in vivo. In the present study, we have identified a novel small molecule inhibitor of dual NF-κB and oxidative stress pathways, OT-304, as a potential candidate to reverse drug resistance. Initial investigations indicate that this compound effectively inhibits proliferation of doxorubicin-sensitive and doxorubicin-resistant cells to the same extent, suggesting that it is capable of bypassing the development of drug resistance. Additional experiments reveal that OT-304 enhances cancer cell sensitivity to doxorubicin and to etoposide, particularly in cells characterized by the over-expression of the drug transporter P-glycoprotein. These findings suggest that either the expression/and or the function of P-glycoprotein could be affected by OT-304. In vivo studies using tumor xenografts in nude mice showed that OT-304 is also capable of preventing the growth of drug resistant cancer cells. This later finding further confirms the role of OT-304 as a drug resistance-reversing agent and warrants further pre-clinical and clinical investigation to determine its efficacy in treating aggressive tumors.

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