Iron Triggers λSo Prophage Induction and Release of Extracellular DNA in Shewanella oneidensis MR-1 Biofilms

ABSTRACTProphages are ubiquitous elements within bacterial chromosomes and affect host physiology and ecology in multiple ways. We have previously demonstrated that phage-induced lysis is required for extracellular DNA (eDNA) release and normal biofilm formation inShewanella oneidensisMR-1. Here, we investigated the regulatory mechanisms of prophage λSo spatiotemporal induction in biofilms. To this end, we used a functional fluorescence fusion to monitor λSo activation in various mutant backgrounds and in response to different physiological conditions. λSo induction occurred mainly in a subpopulation of filamentous cells in a strictly RecA-dependent manner, implicating oxidative stress-induced DNA damage as the major trigger. Accordingly, mutants affected in the oxidative stress response (ΔoxyR) or iron homeostasis (Δfur) displayed drastically increased levels of phage induction and abnormal biofilm formation, while planktonic cells were not or only marginally affected. To further investigate the role of oxidative stress, we performed a mutant screen and identified two independent amino acid substitutions in OxyR (T104N and L197P) that suppress induction of λSo by hydrogen peroxide (H2O2). However, λSo induction was not suppressed in biofilms formed by both mutants, suggesting a minor role of intracellular H2O2in this process. In contrast, addition of iron to biofilms strongly enhanced λSo induction and eDNA release, while both processes were significantly suppressed at low iron levels, strongly indicating that iron is the limiting factor. We conclude that uptake of iron during biofilm formation triggers λSo-mediated lysis of a subpopulation of cells, likely by an increase in iron-mediated DNA damage sensed by RecA.

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Genetic and Biochemical Analysis of CodY-Mediated Cell Aggregation in Staphylococcus aureus Reveals an Interaction between Extracellular DNA and Polysaccharide in the Extracellular Matrix

Journal of Bacteriology ◽

10.1128/jb.00593-19 ◽

2020 ◽

Vol 202 (8) ◽

Cited By ~ 2

Author(s):

Kevin D. Mlynek ◽

Logan L. Bulock ◽

Carl J. Stone ◽

Luke J. Curran ◽

Marat R. Sadykov ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Surface ◽

Biofilm Formation ◽

Biochemical Analysis ◽

Cell Aggregation ◽

Extracellular Dna ◽

Dependent Manner ◽

Content Type ◽

Mutant Cells

ABSTRACT The global regulator CodY links nutrient availability to the regulation of virulence factor gene expression in Staphylococcus aureus, including many genes whose products affect biofilm formation. Antithetical phenotypes of both biofilm deficiency and accumulation have been reported for codY-null mutants; thus, the role of CodY in biofilm development remains unclear. codY mutant cells of a strain producing a robust biofilm elaborate proaggregation surface-associated features not present on codY mutant cells that do not produce a robust biofilm. Biochemical analysis of the clinical isolate SA564, which aggregates when deficient for CodY, revealed that these features are sensitive to nuclease treatment and are resistant to protease exposure. Genetic analyses revealed that disrupting lgt (the diacylglycerol transferase gene) in codY mutant cells severely weakened aggregation, indicating a role for lipoproteins in the attachment of the biofilm matrix to the cell surface. An additional and critical role of IcaB in producing functional poly-N-acetylglucosamine (PIA) polysaccharide in extracellular DNA (eDNA)-dependent biofilm formation was shown. Moreover, overproducing PIA is sufficient to promote aggregation in a DNA-dependent manner regardless of source of nucleic acids. Taken together, our results point to PIA synthesis as the primary determinant of biofilm formation when CodY activity is reduced and suggest a modified electrostatic net model for matrix attachment whereby PIA associates with eDNA, which interacts with the cell surface via covalently attached membrane lipoproteins. This work counters the prevailing view that polysaccharide- and eDNA/protein-based biofilms are mutually exclusive. Rather, we demonstrate that eDNA and PIA can work synergistically to form a biofilm. IMPORTANCE Staphylococcus aureus remains a global health concern and exemplifies the ability of an opportunistic pathogen to adapt and persist within multiple environments, including host tissue. Not only does biofilm contribute to persistence and immune evasion in the host environment, it also may aid in the transition to invasive disease. Thus, understanding how biofilms form is critical for developing strategies for dispersing biofilms and improving biofilm disease-related outcomes. Using biochemical, genetic, and cell biology approaches, we reveal a synergistic interaction between PIA and eDNA that promotes cell aggregation and biofilm formation in a CodY-dependent manner in S. aureus. We also reveal that envelope-associated lipoproteins mediate attachment of the biofilm matrix to the cell surface.

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The Role of Oxidative Stress in Koenimbine-Induced DNA Damage and Heat Shock Protein Modulation in HepG2 Cells

Integrative Cancer Therapies ◽

10.1177/1534735416678982 ◽

2016 ◽

Vol 16 (4) ◽

pp. 563-571 ◽

Cited By ~ 4

Author(s):

Yahya Hasan Hobani

Keyword(s):

Oxidative Stress ◽

Dna Damage ◽

Ascorbic Acid ◽

Heat Shock ◽

Hepg2 Cells ◽

Time Dependent ◽

Dependent Manner ◽

Hsp 70 ◽

Hsp 90

Background. Murraya koenigii (L.) Spreng, is a significant herb of traditional Ayurvedic system of medicine. Koenimbine, a carbazole alkaloid isolated from this plant holds antiproliferative and apoptotic effects. The aim of this study was to assess koenimbine-induced DNA damage and to clarify the role of free radicals in cell death mechanisms, using HepG2 cells. Methods. The level of cytotoxicity was assayed by MTT assay. To elucidate the role of glutathione (GSH), the intracellular GSH level was analyzed. The effect of koenimbine in the cell mitochondria was evaluated using mitochondrial membrane potential (MMP) changes. Single cell gel electrophoresis assay was used to examine the level of DNA damage. Heat shock proteins, Hsp 70 and Hsp 90 expressions were checked at mRNA and protein level. Ascorbic acid and catalase were used as control antioxidants. Results. It was observed that koenimbine considerably increased DNA damage in HepG2 cells at subcytotoxic concentrations. Koenimbine induced the increased levels of reactive oxygen species (ROS) and reduction of GSH level in HepG2 cells, together with time-dependent loss of MMP. In addition, results clearly showed that koenimbine encouraged cells to express Hsp 70 and Hsp 90 in a concentration-dependent manner up to a concentration of 100 µM and a time-dependent manner at 24-hour incubation both at transcriptional and translational levels. The antioxidant capacity of ascorbic acid was found to be not as prominent as to catalase throughout the study. Conclusion. Based on these data it can be concluded that koenimbine causes DNA strand breaks in HepG2 cells, probably through oxidative stress. Moreover, the oxidative stress induced was closely associated with MMP reduction and GSH depletion associated with HSP modulation at subcytotoxic concentration.

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Roles of Two Shewanella oneidensis MR-1 Extracellular Endonucleases

Applied and Environmental Microbiology ◽

10.1128/aem.00643-11 ◽

2011 ◽

Vol 77 (15) ◽

pp. 5342-5351 ◽

Cited By ~ 37

Author(s):

Julia Gödeke ◽

Magnus Heun ◽

Sebastian Bubendorfer ◽

Kristina Paul ◽

Kai M. Thormann

Keyword(s):

Biofilm Formation ◽

Cell Envelope ◽

Shewanella Oneidensis ◽

Thick Layer ◽

Sole Source ◽

Extracellular Dna ◽

Matrix Component ◽

Content Type ◽

Static Conditions

ABSTRACTThe dissimilatory iron-reducing bacteriumShewanella oneidensisMR-1 is capable of using extracellular DNA (eDNA) as the sole source of carbon, phosphorus, and nitrogen. In addition, we recently demonstrated thatS. oneidensisMR-1 requires eDNA as a structural component during all stages of biofilm formation. In this study, we characterize the roles of twoShewanellaextracellular endonucleases, ExeS and ExeM. While ExeS is likely secreted into the medium, ExeM is predicted to remain associated with the cell envelope. BothexeMandexeSare highly expressed under phosphate-limited conditions. Mutants lackingexeSand/orexeMexhibit decreased eDNA degradation; however, the capability ofS. oneidensisMR-1 to use DNA as the sole source of phosphorus is only affected in mutants lackingexeM. Neither of the two endonucleases alleviates toxic effects of increased eDNA concentrations. The deletion ofexeMand/orexeSsignificantly affects biofilm formation ofS. oneidensisMR-1 under static conditions, and expression ofexeMandexeSdrastically increases during static biofilm formation. Under hydrodynamic conditions, a deletion ofexeMleads to altered biofilms that consist of densely packed structures which are covered by a thick layer of eDNA. Based on these results, we hypothesize that a major role of ExeS and, in particular, ExeM ofS. oneidensisMR-1, is to degrade eDNA as a matrix component during biofilm formation to improve nutrient supply and to enable detachment.

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Protective Role of Perillic Acid Against Radiation−Induced Oxidative Stress, Cytokine Profile, DNA Damage, and Intestinal Toxicity in Mice

Journal of Environmental Pathology Toxicology and Oncology ◽

10.1615/jenvironpatholtoxicoloncol.v29.i3.40 ◽

2010 ◽

Vol 29 (3) ◽

pp. 199-212 ◽

Cited By ~ 11

Author(s):

P. Pratheeshkumar ◽

T.J. Raphael ◽

Girija Kuttan

Keyword(s):

Oxidative Stress ◽

Dna Damage ◽

Protective Role ◽

Cytokine Profile ◽

Intestinal Toxicity ◽

Radiation Induced

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Non-canonical Functions of Telomerase Reverse Transcriptase: Emerging Roles and Biological Relevance

Current Topics in Medicinal Chemistry ◽

10.2174/1568026620666200131125110 ◽

2020 ◽

Vol 20 (6) ◽

pp. 498-507 ◽

Cited By ~ 4

Author(s):

Connor A.H. Thompson ◽

Judy M.Y. Wong

Keyword(s):

Oxidative Stress ◽

Dna Damage ◽

Reverse Transcriptase ◽

Cancer Cells ◽

Telomere Length ◽

Telomere Maintenance ◽

Telomerase Reverse Transcriptase ◽

Alternative Mechanism ◽

Dependent Manner ◽

Mitochondrial Integrity

Increasing evidence from research on telomerase suggests that in addition to its catalytic telomere repeat synthesis activity, telomerase may have other biologically important functions. The canonical roles of telomerase are at the telomere ends where they elongate telomeres and maintain genomic stability and cellular lifespan. The catalytic protein component Telomerase Reverse Transcriptase (TERT) is preferentially expressed at high levels in cancer cells despite the existence of an alternative mechanism for telomere maintenance (alternative lengthening of telomeres or ALT). TERT is also expressed at higher levels than necessary for maintaining functional telomere length, suggesting other possible adaptive functions. Emerging non-canonical roles of TERT include regulation of non-telomeric DNA damage responses, promotion of cell growth and proliferation, acceleration of cell cycle kinetics, and control of mitochondrial integrity following oxidative stress. Non-canonical activities of TERT primarily show cellular protective effects, and nuclear TERT has been shown to protect against cell death following double-stranded DNA damage, independent of its role in telomere length maintenance. TERT has been suggested to act as a chromatin modulator and participate in the transcriptional regulation of gene expression. TERT has also been reported to regulate transcript levels through an RNA-dependent RNA Polymerase (RdRP) activity and produce siRNAs in a Dicer-dependent manner. At the mitochondria, TERT is suggested to protect against oxidative stress-induced mtDNA damage and promote mitochondrial integrity. These extra-telomeric functions of TERT may be advantageous in the context of increased proliferation and metabolic stress often found in rapidly-dividing cancer cells. Understanding the spectrum of non-canonical functions of telomerase may have important implications for the rational design of anti-cancer chemotherapeutic drugs.

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RND Efflux Systems Contribute to Resistance and Virulence of Aliarcobacter butzleri

Antibiotics ◽

10.3390/antibiotics10070823 ◽

2021 ◽

Vol 10 (7) ◽

pp. 823

Author(s):

Cristiana Mateus ◽

Ana Rita Nunes ◽

Mónica Oleastro ◽

Fernanda Domingues ◽

Susana Ferreira

Keyword(s):

Oxidative Stress ◽

Human Serum ◽

Ethidium Bromide ◽

Biofilm Formation ◽

Bacterial Resistance ◽

Efflux Pumps ◽

Bacterial Virulence ◽

Relative Fitness ◽

Mutant Strains

Aliarcobacter butzleri is an emergent enteropathogen that can be found in a range of environments. This bacterium presents a vast repertoire of efflux pumps, such as the ones belonging to the resistance nodulation cell division family, which may be associated with bacterial resistance, as well as virulence. Thus, this work aimed to evaluate the contribution of three RND efflux systems, AreABC, AreDEF and AreGHI, in the resistance and virulence of A. butzleri. Mutant strains were constructed by inactivation of the gene that encodes the inner membrane protein of these systems. The bacterial resistance profile of parental and mutant strains to several antimicrobials was assessed, as was the intracellular accumulation of the ethidium bromide dye. Regarding bacterial virulence, the role of these three efflux pumps on growth, strain fitness, motility, biofilm formation ability, survival in adverse conditions (oxidative stress and bile salts) and human serum and in vitro adhesion and invasion to Caco-2 cells was evaluated. We observed that the mutants from the three efflux pumps were more susceptible to several classes of antimicrobials than the parental strain and presented an increase in the accumulation of ethidium bromide, indicating a potential role of the efflux pumps in the extrusion of antimicrobials. The mutant strains had no bacterial growth defects; nonetheless, they presented a reduction in relative fitness. For the three mutants, an increase in the susceptibility to oxidative stress was observed, while only the mutant for AreGHI efflux pump showed a relevant role in bile stress survival. All the mutant strains showed an impairment in biofilm formation ability, were more susceptible to human serum and were less adherent to intestinal epithelial cells. Overall, the results support the contribution of the efflux pumps AreABC, AreDEF and AreGHI of A. butzleri to antimicrobial resistance, as well as to bacterial virulence.

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Role of hepcidin in oxidative stress and cell death of cultured mouse renal collecting duct cells: protection against iron and sensitization to cadmium

Archives of Toxicology ◽

10.1007/s00204-021-03106-z ◽

2021 ◽

Author(s):

Stephanie Probst ◽

Johannes Fels ◽

Bettina Scharner ◽

Natascha A. Wolff ◽

Eleni Roussa ◽

...

Keyword(s):

Oxidative Stress ◽

Cell Death ◽

Cell Fate ◽

Catalase Activity ◽

Metal Ion ◽

Iron Homeostasis ◽

Collecting Duct ◽

Duct Cell ◽

Plasmid Transfection

AbstractThe liver hormone hepcidin regulates systemic iron homeostasis. Hepcidin is also expressed by the kidney, but exclusively in distal nephron segments. Several studies suggest hepcidin protects against kidney damage involving Fe2+ overload. The nephrotoxic non-essential metal ion Cd2+ can displace Fe2+ from cellular biomolecules, causing oxidative stress and cell death. The role of hepcidin in Fe2+ and Cd2+ toxicity was assessed in mouse renal cortical [mCCD(cl.1)] and inner medullary [mIMCD3] collecting duct cell lines. Cells were exposed to equipotent Cd2+ (0.5–5 μmol/l) and/or Fe2+ (50–100 μmol/l) for 4–24 h. Hepcidin (Hamp1) was transiently silenced by RNAi or overexpressed by plasmid transfection. Hepcidin or catalase expression were evaluated by RT-PCR, qPCR, immunoblotting or immunofluorescence microscopy, and cell fate by MTT, apoptosis and necrosis assays. Reactive oxygen species (ROS) were detected using CellROX™ Green and catalase activity by fluorometry. Hepcidin upregulation protected against Fe2+-induced mIMCD3 cell death by increasing catalase activity and reducing ROS, but exacerbated Cd2+-induced catalase dysfunction, increasing ROS and cell death. Opposite effects were observed with Hamp1 siRNA. Similar to Hamp1 silencing, increased intracellular Fe2+ prevented Cd2+ damage, ROS formation and catalase disruption whereas chelation of intracellular Fe2+ with desferrioxamine augmented Cd2+ damage, corresponding to hepcidin upregulation. Comparable effects were observed in mCCD(cl.1) cells, indicating equivalent functions of renal hepcidin in different collecting duct segments. In conclusion, hepcidin likely binds Fe2+, but not Cd2+. Because Fe2+ and Cd2+ compete for functional binding sites in proteins, hepcidin affects their free metal ion pools and differentially impacts downstream processes and cell fate.

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PmtA Regulates Pyocyanin Expression and Biofilm Formation in Pseudomonas aeruginosa

Frontiers in Microbiology ◽

10.3389/fmicb.2021.789765 ◽

2021 ◽

Vol 12 ◽

Author(s):

Amy V. Thees ◽

Kathryn M. Pietrosimone ◽

Clare K. Melchiorre ◽

Jeremiah N. Marden ◽

Joerg Graf ◽

...

Keyword(s):

Oxidative Stress ◽

Pseudomonas Aeruginosa ◽

Metal Binding ◽

Biofilm Formation ◽

Binding Capacity ◽

Opportunistic Pathogen ◽

Small Molecular Weight ◽

Heavy Metal Binding ◽

The Impact

The opportunistic pathogen Pseudomonas aeruginosa expresses a small molecular weight, cysteine-rich protein (PmtA), identified as a metallothionein (MT) protein family member. The MT family proteins have been well-characterized in eukaryotes as essential for zinc and copper homeostasis, protection against oxidative stress, and the ability to modify a variety of immune activities. Bacterial MTs share sequence homology, antioxidant chemistry, and heavy metal-binding capacity with eukaryotic MTs, however, the impact of bacterial MTs on virulence and infection have not been well-studied. In the present study, we investigated the role of PmtA in P. aeruginosa PAO1 using a PmtA-deficient strain (ΔpmtA). Here we demonstrated the virulence factor, pyocyanin, relies on the expression of PmtA. We showed that PmtA may be protective against oxidative stress, as an alternative antioxidant, glutathione, can rescue pyocyanin expression. Furthermore, the expression of phzM, which encodes a pyocyanin precursor enzyme, was decreased in the ΔpmtA mutant during early stationary phase. Upregulated pmtA expression was previously detected in confluent biofilms, which are essential for chronic infection, and we observed that the ΔpmtA mutant was disrupted for biofilm formation. As biofilms also modulate antibiotic susceptibility, we examined the ΔpmtA mutant susceptibility to antibiotics and found that the ΔpmtA mutant is more susceptible to cefepime and ciprofloxacin than the wild-type strain. Finally, we observed that the deletion of pmtA results in decreased virulence in a waxworm model. Taken together, our results support the conclusion that PmtA is necessary for the full virulence of P. aeruginosa and may represent a potential target for therapeutic intervention.

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Abstract 40: Prothrombotic Role of Platelet Tlr2 in Hyperlipidemia

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.40 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Sudipta Biswas ◽

Liang Xin ◽

Soumya Panigrahi ◽

Alejandro Zimman ◽

Valentin Yakubenko ◽

...

Keyword(s):

Oxidative Stress ◽

Platelet Reactivity ◽

Biological Activities ◽

Biologically Active ◽

Dependent Manner ◽

Prothrombotic State ◽

Downstream Signaling ◽

Subsequent Activation ◽

Biologically Active Derivatives

A prothrombotic state and increased platelet reactivity are common in hyperlipidemia and oxidative stress. Lipid peroxidation, a major consequence of oxidative stress, generates highly reactive products including hydroxy-w-oxoalkenoic acids that modify autologous proteins generating biologically active derivatives. Phosphatidylethanolamine, the second most abundant eukaryotic phospholipid can also be modified by hydroxy-w-oxoalkenoic acids. However, the conditions leading to accumulation of such derivatives in circulation and their biological activities remain poorly understood. We now show that carboxyalkylpyrrole-phosphatidylethanolamine derivatives (CAP-PE) accumulate in plasma of hyperlipidemic ApoE -/- mice. CAP-PE directly bind to TLR2 and induce platelet integrin alpha 2b beta 3 activation and P-selectin expression in TLR2 dependent manner. Platelet activation by CAP-PE includes assembly of TLR2/TLR1 receptor complex, induction of downstream signaling via MyD88/TIRAP, phosphorylation of IRAK4, and subsequent activation of TRAF6. This in turn activates the Src family kinases, Syk and PLC gamma 2 and platelet integrins. By intravital thrombosis studies we have demonstrated that CAP-PE accelerate thrombosis in TLR2 dependent manner. Furthermore, we demonstrate that TLR2 deficient mice are protected from accelerated thrombosis induced by hyperlipidemia. Taken together, our studies demonstrate a cross-talk between innate immunity and integrin activation signaling pathways in platelets and reveal that TLR2 plays a key role in platelet hyperreactivity and prothrombotic state in hyperlipidemia.

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