The Expression of Superoxide Dismutase (SOD) and a Putative ABC Transporter Permease Is Inversely Correlated during Biofilm Formation in Listeria monocytogenes 4b G

Little is known about the molecular basis of biofilm formation in Listeria monocytogenes. The superoxide dismutase (SOD) of the deletion mutant of lm.G_1771 gene, which encodes for a putative ABC transporter permease, is highly expressed in biofilm. In this study, the sod gene deletion mutant Δsod, and double deletion mutant of the sod and lm. G_1771 genes Δ1771Δsod were used to investigate the role of SOD and its relationship to the expression of the putative ABC transporter permease in biofilm formation. Our results showed that the ability to form a biofilm was significantly reduced in the Δsod mutant and the Δ1771Δsod double mutant. Both Δsod and Δ1771Δsod mutants exhibited slow growth phenotypes and produced more reactive oxygen species (ROS). The growth was inhibited in the mutants by methyl viologen (MV, internal oxygen radical generator) treatment. In addition, the expression of one oxidation resistance gene (kat), two stress regulators encoding genes (perR and sigB), and one DNA repair gene (recA) were analyzed in both the wild-type L. monocytogenes 4b G and the deletion mutants by RT-qPCR. The expression levels of the four genes were increased in the deletion mutants when biofilms were formed. Taken together, our data indicated that SOD played an important role in biofilm formation through coping with the oxidant burden in deficient antioxidant defenses.     

Deletion of the Mitochondrial Superoxide Dismutase sod-2 Extends Lifespan in Caenorhabditis elegans

The oxidative stress theory of aging postulates that aging results from the accumulation of molecular damage caused by reactive oxygen species (ROS) generated during normal metabolism. Superoxide dismutases (SODs) counteract this process by detoxifying superoxide. It has previously been shown that elimination of either cytoplasmic or mitochondrial SOD in yeast, flies, and mice results in decreased lifespan. In this experiment, we examine the effect of eliminating each of the five individual sod genes present in Caenorhabditis elegans. In contrast to what is observed in other model organisms, none of the sod deletion mutants shows decreased lifespan compared to wild-type worms, despite a clear increase in sensitivity to paraquat- and juglone-induced oxidative stress. In fact, even mutants lacking combinations of two or three sod genes survive at least as long as wild-type worms. Examination of gene expression in these mutants reveals mild compensatory up-regulation of other sod genes. Interestingly, we find that sod-2 mutants are long-lived despite a significant increase in oxidatively damaged proteins. Testing the effect of sod-2 deletion on known pathways of lifespan extension reveals a clear interaction with genes that affect mitochondrial function: sod-2 deletion markedly increases lifespan in clk-1 worms while clearly decreasing the lifespan of isp-1 worms. Combined with the mitochondrial localization of SOD-2 and the fact that sod-2 mutant worms exhibit phenotypes that are characteristic of long-lived mitochondrial mutants—including slow development, low brood size, and slow defecation—this suggests that deletion of sod-2 extends lifespan through a similar mechanism. This conclusion is supported by our demonstration of decreased oxygen consumption in sod-2 mutant worms. Overall, we show that increased oxidative stress caused by deletion of sod genes does not result in decreased lifespan in C. elegans and that deletion of sod-2 extends worm lifespan by altering mitochondrial function.     

A mitochondrial superoxide signal triggers increased longevity in Caenorhabditis elegans

The nuo-6 and isp-1 genes of C. elegans encode, respectively, subunits of complex I and III of the mitochondrial respiratory chain. Partial loss-of-function mutations in these genes decrease electron transport and greatly increase the longevity of C. elegans by a mechanism that is distinct from that induced by reducing their level of expression by RNAi. Electron transport is a major source of the superoxide anion (O^{⋅ –}), which in turn generates several types of toxic reactive oxygen species (ROS), and aging is accompanied by increased oxidative stress, which is an imbalance between the generation and detoxification of ROS. These observations have suggested that the longevity of such mitochondrial mutants might result from a reduction in ROS generation, which would be consistent with the mitochondrial oxidative stress theory of aging. It is difficult to measure ROS directly in living animals, and this has held back progress in determining their function in aging. Here we have adapted a technique of flow cytometry to directly measure ROS levels in isolated mitochondria to show that the generation of superoxide is elevated in the nuo-6 and isp-1 mitochondrial mutants, although overall ROS levels are not, and oxidative stress is low. Furthermore, we show that this elevation is necessary and sufficient to increase longevity, as it is abolished by the antioxidants NAC and vitamin C, and phenocopied by mild treatment with the prooxidant paraquat. Furthermore, the absence of effect of NAC and the additivity of the effect of paraquat on a variety of long- and short-lived mutants suggest that the pathway triggered by mitochondrial superoxide is distinct from previously studied mechanisms, including insulin signaling, dietary restriction, ubiquinone deficiency, the hypoxic response, and hormesis. These findings are not consistent with the mitochondrial oxidative stress theory of aging. Instead they show that increased superoxide generation acts as a signal in young mutant animals to trigger changes of gene expression that prevent or attenuate the effects of subsequent aging. We propose that superoxide is generated as a protective signal in response to molecular damage sustained during wild-type aging as well. This model provides a new explanation for the well-documented correlation between ROS and the aged phenotype as a gradual increase of molecular damage during aging would trigger a gradually stronger ROS response.     

Mitochondrial and Cytoplasmic ROS Have Opposing Effects on Lifespan

Reactive oxygen species (ROS) are highly reactive, oxygen-containing molecules that can cause molecular damage within the cell. While the accumulation of ROS-mediated damage is widely believed to be one of the main causes of aging, ROS also act in signaling pathways. Recent work has demonstrated that increasing levels of superoxide, one form of ROS, through treatment with paraquat, results in increased lifespan. Interestingly, treatment with paraquat robustly increases the already long lifespan of the clk-1 mitochondrial mutant, but not other long-lived mitochondrial mutants such as isp-1 or nuo-6. To genetically dissect the subcellular compartment in which elevated ROS act to increase lifespan, we deleted individual superoxide dismutase (sod) genes in clk-1 mutants, which are sensitized to ROS. We find that only deletion of the primary mitochondrial sod gene, sod-2 results in increased lifespan in clk-1 worms. In contrast, deletion of either of the two cytoplasmic sod genes, sod-1 or sod-5, significantly decreases the lifespan of clk-1 worms. Further, we show that increasing mitochondrial superoxide levels through deletion of sod-2 or treatment with paraquat can still increase lifespan in clk-1;sod-1 double mutants, which live shorter than clk-1 worms. The fact that mitochondrial superoxide can increase lifespan in worms with a detrimental level of cytoplasmic superoxide demonstrates that ROS have a compartment specific effect on lifespan – elevated ROS in the mitochondria acts to increase lifespan, while elevated ROS in the cytoplasm decreases lifespan. This work also suggests that both ROS-dependent and ROS-independent mechanisms contribute to the longevity of clk-1 worms.     

Succinate Dehydrogenase Upregulation Destabilize Complex I and Limits the Lifespan of gas-1 Mutant

Many Caenorhabditis elegans mutants with dysfunctional mitochondrial electron transport chain are surprisingly long lived. Both short-lived (gas-1(fc21)) and long-lived (nuo-6(qm200)) mutants of mitochondrial complex I have been identified. However, it is not clear what are the pathways determining the difference in longevity. We show that even in a short-lived gas-1(fc21) mutant, many longevity assurance pathways, shown to be important for lifespan prolongation in long-lived mutants, are active. Beside similar dependence on alternative metabolic pathways, short-lived gas-1(fc21) mutants and long-lived nuo-6(qm200) mutants also activate hypoxia-inducible factor –1α (HIF-1α) stress pathway and mitochondrial unfolded protein response (UPR^mt). The major difference that we detected between mutants of different longevity, is in the massive loss of complex I accompanied by upregulation of complex II levels, only in short-lived, gas-1(fc21) mutant. We show that high levels of complex II negatively regulate longevity in gas-1(fc21) mutant by decreasing the stability of complex I. Furthermore, our results demonstrate that increase in complex I stability, improves mitochondrial function and decreases mitochondrial stress, putting it inside a “window” of mitochondrial dysfunction that allows lifespan prolongation.     

Superoxide Dismutase Activity in Pseudomonas putida Affects Utilization of Sugars and Growth on Root Surfaces

To investigate the role of superoxide dismutases (SOD) in root colonization and oxidative stress, mutants of Pseudomonas putida lacking manganese-superoxide dismutase (MnSOD) (sodA), iron-superoxide dismutase (FeSOD) (sodB), or both were generated. The sodA sodB mutant did not grow on components washed from bean root surfaces or glucose in minimal medium. The sodB and sodA sodB mutants were more sensitive than wild type to oxidative stress generated within the cell by paraquat treatment. In single inoculation of SOD mutants on bean, only the sodA sodB double mutant was impaired in growth on root surfaces. In mixed inoculations with wild type, populations of the sodA mutant were equal to those of the wild type, but levels of the sodB mutant and, to a great extent, the sodA sodB mutant, were reduced. Confocal microscopy of young bean roots inoculated with green fluorescent protein-tagged cells showed that wild type and SOD single mutants colonized well predominantly at the root tip but that the sodA sodB double mutant grew poorly at the tip. Our results indicate that FeSOD in P. putida is more important than MnSOD in aerobic metabolism and oxidative stress. Inhibition of key metabolic enzymes by increased levels of superoxide anion may cause the impaired growth of SOD mutants in vitro and in planta.     

Longevity and resistance to stress correlate with DNA repair capacity in Caenorhabditis elegans

DNA repair is an important mechanism by which cells maintain genomic integrity. Decline in DNA repair capacity or defects in repair factors are thought to contribute to premature aging in mammals. The nematode Caenorhabditis elegans is a good model for studying longevity and DNA repair because of key advances in understanding the genetics of aging in this organism. Long-lived C. elegans mutants have been identified and shown to be resistant to oxidizing agents and UV irradiation, suggesting a genetically determined correlation between DNA repair capacity and life span. In this report, gene-specific DNA repair is compared in wild-type C. elegans and stress-resistant C. elegans mutants for the first time. DNA repair capacity is higher in long-lived C. elegans mutants than in wild-type animals. In addition, RNAi knockdown of the nucleotide excision repair gene xpa-1 increased sensitivity to UV and reduced the life span of long-lived C. elegans mutants. These findings support that DNA repair capacity correlates with longevity in C. elegans.     

Dietary resveratrol administration increases MnSOD expression and activity in mouse brain

trans-Resveratrol (3,4′,5-trihydroxystilbene; RES) is of interest for its reported protective effects in a variety of pathologies, including neurodegeneration. Many of these protective properties have been attributed to the ability of RES to reduce oxidative stress. In vitro studies have shown an increase in antioxidant enzyme activities following exposure to RES, including upregulation of mitochondrial superoxide dismutase, an enzyme that is capable of reducing both oxidative stress and cell death. We sought to determine if a similar increase in endogenous antioxidant enzymes is observed with RES treatment in vivo. Three separate modes of RES delivery were utilized; in a standard diet, a high fat diet and through a subcutaneous osmotic minipump. RES given in a high fat diet proved to be effective in elevating antioxidant capacity in brain resulting in an increase in both MnSOD protein level (140%) and activity (75%). The increase in MnSOD was not due to a substantial proliferation of mitochondria, as RES treatment induced a 10% increase in mitochondrial abundance (Citrate Synthase activity). The potential neuroprotective properties of MnSOD have been well established, and we demonstrate that a dietary delivery of RES is able to increase the expression and activity of this enzyme in vivo.     

Selective superoxide generation within mitochondria by the targeted redox cycler MitoParaquat

Superoxide is the proximal reactive oxygen species (ROS) produced by the mitochondrial respiratory chain and plays a major role in pathological oxidative stress and redox signaling. While there are tools to detect or decrease mitochondrial superoxide, none can rapidly and specifically increase superoxide production within the mitochondrial matrix. This lack impedes progress, making it challenging to assess accurately the roles of mitochondrial superoxide in cells and in vivo. To address this unmet need, we synthesized and characterized a mitochondria-targeted redox cycler, MitoParaquat (MitoPQ) that comprises a triphenylphosphonium lipophilic cation conjugated to the redox cycler paraquat. MitoPQ accumulates selectively in the mitochondrial matrix driven by the membrane potential. Within the matrix, MitoPQ produces superoxide by redox cycling at the flavin site of complex I, selectively increasing superoxide production within mitochondria. MitoPQ increased mitochondrial superoxide in isolated mitochondria and cells in culture ~a thousand-fold more effectively than untargeted paraquat. MitoPQ was also more toxic than paraquat in the isolated perfused heart and in Drosophila in vivo. MitoPQ enables the selective generation of superoxide within mitochondria and is a useful tool to investigate the many roles of mitochondrial superoxide in pathology and redox signaling in cells and in vivo.     

Mitochondrial Dysfunction Confers Resistance to Multiple Drugs in Caenorhabditis elegans

In a previous genetic screen for Caenorhabditis elegans mutants that survive in the presence of an antimitotic drug, hemiasterlin, we identified eight strong mutants. Two of these were found to be resistant to multiple toxins, and in one of these we identified a missense mutation in phb-2, which encodes the mitochondrial protein prohibitin 2. Here we identify two additional mutations that confer drug resistance, spg-7 and har-1, also in genes encoding mitochondrial proteins. Other mitochondrial mutants, isp-1, eat-3, and clk-1, were also found to be drug-resistant. Respiratory complex inhibitors, FCCP and oligomycin, and a producer of reactive oxygen species (ROS), paraquat, all rescued wild-type worms from hemiasterlin toxicity. Worms lacking mitochondrial superoxide dismutase (MnSOD) were modestly drug-resistant, and elimination of MnSOD in the phb-2, har-1, and spg-7 mutants enhanced resistance. The antioxidant N-acetyl-l-cysteine prevented mitochondrial inhibitors from rescuing wild-type worms from hemiasterlin and sensitized mutants to the toxin, suggesting that a mechanism sensitive to ROS is necessary to trigger drug resistance in C. elegans. Using genetics, we show that this drug resistance requires pkc-1, the C. elegans ortholog of human PKCε.     

Significant longevity-extending effects of EGCG on Caenorhabditis elegans under stress

Epigallocatechin gallate (EGCG), a main active ingredient of green tea, is believed to be beneficial in association with anticarcinogenesis, antiobesity, and blood pressure reduction. Here we report that EGCG extended Caenorhabditis elegans longevity under stress. Under heat stress (35°C), EGCG improved the mean longevity by 13.1% at 0.1 μg/ml, 8.0% at 1.0 μg/ml, and 11.8% at 10.0 μg/ml. Under oxidative stress, EGCG could improve the mean longevity of C. elegans by 172.9% at 0.1 μg/ml, 177.7% at 1.0 μg/ml, and 88.5% at 10.0 μg/ml. However, EGCG could not extend the life span of C. elegans under normal culture conditions. Further studies demonstrated that the significant longevity-extending effects of EGCG on C. elegans could be attributed to its in vitro and in vivo free radical-scavenging effects and its up-regulating effects on stress-resistance-related proteins, including superoxide dismutase-3 (SOD-3) and heat shock protein-16.2 (HSP-16.2), in transgenic C. elegans with SOD-3∷green fluorescent protein (GFP) and HSP-16.2∷GFP expression. Quantitative real-time PCR results showed that the up-regulation of aging-associated genes such as daf-16, sod-3, and skn-1 could also contribute to the stress resistance attributed to EGCG. As the death rate of a population is closely related to the mortality caused by external stress, it could be concluded that the survival-enhancing effects of EGCG on C. elegans under stress are very important for antiaging research.     

The C. elegans Opa1 homologue EAT-3 is essential for resistance to free radicals

The C. elegans eat-3 gene encodes a mitochondrial dynamin family member homologous to Opa1 in humans and Mgm1 in yeast. We find that mutations in the C. elegans eat-3 locus cause mitochondria to fragment in agreement with the mutant phenotypes observed in yeast and mammalian cells. Electron microscopy shows that the matrices of fragmented mitochondria in eat-3 mutants are divided by inner membrane septae, suggestive of a specific defect in fusion of the mitochondrial inner membrane. In addition, we find that C. elegans eat-3 mutant animals are smaller, grow slower, and have smaller broodsizes than C. elegans mutants with defects in other mitochondrial fission and fusion proteins. Although mammalian Opa1 is antiapoptotic, mutations in the canonical C. elegans cell death genes ced-3 and ced-4 do not suppress the slow growth and small broodsize phenotypes of eat-3 mutants. Instead, the phenotypes of eat-3 mutants are consistent with defects in oxidative phosphorylation. Moreover, eat-3 mutants are hypersensitive to paraquat, which promotes damage by free radicals, and they are sensitive to loss of the mitochondrial superoxide dismutase sod-2. We conclude that free radicals contribute to the pathology of C. elegans eat-3 mutants.     

C. elegans STI-1, the Homolog of Sti1/Hop, Is Involved in Aging and Stress Response

Environmental and physiological stresses such as heat shock, oxidative stress, heavy metals, and pathogenic conditions induce cellular stress response. This response is often mediated by heat shock proteins that function as molecular chaperones. A stress-inducible cochaperone, Sti1/Hop (Hsp organizer protein), functions as an adaptor protein that simultaneously binds with Hsp70 and Hsp90 to transfer client proteins from Hsp70 to Hsp90. However, the biological role of STI-1 in vivo is poorly understood in metazoans. Here, we report the characterization of the Caenorhabditis elegans homolog of Sti1/Hop, which is approximately 56% identical with human STI-1. C. elegans STI-1 (CeSTI-1) is expressed in the pharynx, intestine, nervous system, and muscle from larvae to adults. Analysis of proteins immunoprecipitated with anti-STI-1 antibody by mass spectrometry revealed that CeSTI-1 can bind with both Hsp70 and Hsp90 homologs like its mammalian counterpart. sti-1 expression is elevated by heat stress, and an sti-1(jh125) null mutant shows decreased fertility under heat stress conditions. These mutants also show abnormally high lethality in extreme heat and may be functioning with DAF-16 in thermotolerance. In addition, sti-1(jh125) mutants have a shortened life span. Our results confirm that CeSTI-1 is a cochaperone protein that may maintain homeostatic functions during episodes of stress and can regulate longevity in nematodes.     

Barley beta‐glucan promotes MnSOD expression and enhances angiogenesis under oxidative microenvironment

Manganese superoxide dismutase (MnSOD), a foremost antioxidant enzyme, plays a key role in angiogenesis. Barley-derived (1.3) β-d-glucan (β-d-glucan) is a natural water-soluble polysaccharide with antioxidant properties. To explore the effects of β-d-glucan on MnSOD-related angiogenesis under oxidative stress, we tested epigenetic mechanisms underlying modulation of MnSOD level in human umbilical vein endothelial cells (HUVECs) and angiogenesis in vitro and in vivo. Long-term treatment of HUVECs with 3% w/v β-d-glucan significantly increased the level of MnSOD by 200% ± 2% compared to control and by 50% ± 4% compared to untreated H₂O₂-stressed cells. β-d-glucan-treated HUVECs displayed greater angiogenic ability. In vivo, 24 hrs-treatment with 3% w/v β-d-glucan rescued vasculogenesis in Tg (kdrl: EGFP) s843Tg zebrafish embryos exposed to oxidative microenvironment. HUVECs overexpressing MnSOD demonstrated an increased activity of endothelial nitric oxide synthase (eNOS), reduced load of superoxide anion (O₂⁻) and an increased survival under oxidative stress. In addition, β-d-glucan prevented the rise of hypoxia inducible factor (HIF)1-α under oxidative stress. The level of histone H4 acetylation was significantly increased by β-d-glucan. Increasing histone acetylation by sodium butyrate, an inhibitor of class I histone deacetylases (HDACs I), did not activate MnSOD-related angiogenesis and did not impair β-d-glucan effects. In conclusion, 3% w/v β-d-glucan activates endothelial expression of MnSOD independent of histone acetylation level, thereby leading to adequate removal of O₂⁻, cell survival and angiogenic response to oxidative stress. The identification of dietary β-d-glucan as activator of MnSOD-related angiogenesis might lead to the development of nutritional approaches for the prevention of ischemic remodelling and heart failure.