Lack of mycothiol and ergothioneine induces different protective mechanisms in Mycobacterium smegmatis

Mycobacterium smegmatis contains the low molecular weight thiols, mycothiol (MSH) and ergothioneine (ESH). Examination of transposon mutants disrupted in mshC and egtA, involved in the biosynthesis of MSH and ESH respectively, demonstrated that both mutants were sensitive to oxidative, alkylating, and metal stress. However, the mshC mutant exhibited significantly more protein carbonylation and lipid peroxidation than wildtype, while the egtA mutant had less protein and lipid damage than wildtype. We further show that Ohr, KatN, and AhpC, involved in protection against oxidative stress, are upregulated in the egtA mutant. In the mshC mutant, an Usp and a putative thiol peroxidase are upregulated. In addition, mutants lacking MSH also contained higher levels of Coenzyme F420 as compared to wildtype and two Coenzyme F420 dependent enzymes were found to be upregulated. These results indicate that lack of MSH and ESH result in induction of different mechanisms for protecting against oxidative stress.     

Siderophore synthesis in Magnaporthe grisea is essential for vegetative growth,conidiation and resistance to oxidative stress

The plant pathogenic fungus Magnaporthe grisea excretes siderophores of the coprogen-type for iron acquisition and uses ferricrocin for intracellular iron storage. In the present report we characterize mutants with defects in extracellular siderophore biosynthesis. Deletion of the M. grisea SSM2 gene, which encodes a non-ribosomal peptide synthetase, resulted in a loss of the production of all coprogens. The mutant strains had a reduced growth rate, produced fewer conidia and were more sensitive to oxidative stress. Ferricrocin production was not affected. Upon deletion of M. grisea OMO1, a gene predicted to encode an l-ornithine-N⁵-monooxygenase, no siderophores of any type were detected, the strain was aconidial, growth rate was reduced and sensitivity to oxidative stress was increased. Abundance of several proteins was affected in the mutants. The Δssm2 and Δomo1 mutant phenotypes were complemented by supplementation of the medium with siderophores or reintroduction of the respective genes.     

Glutathione Deficiency Leads to Riboflavin Oversynthesis in the Yeast Pichia guilliermondii

The Pichia guilliermondii GSH1 and GSH2 genes encoding Saccharomyces cerevisiae homologues of glutathione (GSH) biosynthesis enzymes, γ-glutamylcysteine synthetase and glutathione synthetase, respectively, were cloned and deleted. Constructed P. guilliermondii Δgsh1 and Δgsh2 mutants were GSH auxotrophs, displayed significantly decreased cellular GSH+GSSG levels and sensitivity to tert-butyl hydroperoxide, hydrogen peroxide, and cadmium ions. In GSH-deficient synthetic medium, growths of Δgsh1 and Δgsh2 mutants were limited to 3–4 and 5–6 cell divisions, respectively. Under these conditions Δgsh1 and Δgsh2 mutants possessed 365 and 148 times elevated riboflavin production, 10.7 and 2.3 times increased cellular iron content, as well as 6.8 and 1.4 fold increased ferrireductase activity, respectively, compared to the wild-type strain. Glutathione addition to the growth medium completely restored the growth of both mutants and decreased riboflavin production, cellular iron content, and ferrireductase activity to the level of the parental strain. Cysteine also partially restored the growth of the Δgsh2 mutants, while methionine or dithiothreitol could not restore the growth neither of the Δgsh1, nor of the Δgsh2 mutants. Besides, it was shown that in GSH presence riboflavin production by both Δgsh1 and Δgsh2 mutants, similarly to that of the wild-type strain, depended on iron concentration in the growth medium. Furthermore, in GSH-deficient synthetic medium P. guilliermondii Δgsh2 mutant cells, despite iron overload, behaved like iron-deprived wild-type cells. Thus, in P. guilliermondii yeast, glutathione is required for proper regulation of both riboflavin and iron metabolism.     

Apoptosis as a mechanism for removal of mutated cells of Saccharomyces cerevisiae: The role of Grx2 under cadmium exposure

Cadmium is a strong mutagen that acts by inhibiting DNA mismatch repair, while its toxic effect seems to be related to an indirect oxidative stress that involves glutathione (GSH) mobilization. Among the roles of GSH is the protection of proteins against oxidative damage, by forming reversible mixed disulfides with cysteine residues, a process known as protein glutathionylation and catalyzed by glutaredoxins (Grx). In this current study, Saccharomyces cerevisiae cells deficient in GRX2, growing in 80 μM CdSO₄, showed high mitochondrial mutagenic rate, determined by frequency of mutants that had lost mitochondrial function (petite mutants), high tolerance and lower apoptosis induction. The mutant strain also showed decreased levels of glutathionylated-protein after cadmium exposure, which might difficult the signaling to apoptosis, leading to increased mutagenic rates. Taken together, these results suggest that Grx2 is involved with the apoptotic death induced by cadmium, a form of cellular suicide that might lead of removal of mutated cells.     

Proline Biosynthesis Is Required for Endoplasmic Reticulum Stress Tolerance in Saccharomyces cerevisiae

The amino acid proline is uniquely involved in cellular processes that underlie stress response in a variety of organisms. Proline is known to minimize protein aggregation, but a detailed study of how proline impacts cell survival during accumulation of misfolded proteins in the endoplasmic reticulum (ER) has not been performed. To address this we examined in Saccharomyces cerevisiae the effect of knocking out the PRO1, PRO2, and PRO3 genes responsible for proline biosynthesis. The null mutants pro1, pro2, and pro3 were shown to have increased sensitivity to ER stress relative to wild-type cells, which could be restored by proline or the corresponding genetic complementation. Of these mutants, pro3 was the most sensitive to tunicamycin and was rescued by anaerobic growth conditions or reduced thiol reagents. The pro3 mutant cells have higher intracellular reactive oxygen species, total glutathione, and a NADP⁺/NADPH ratio than wild-type cells under limiting proline conditions. Depletion of proline biosynthesis also inhibits the unfolded protein response (UPR) indicating proline protection involves the UPR. To more broadly test the role of proline in ER stress, increased proline biosynthesis was shown to partially rescue the ER stress sensitivity of a hog1 null mutant in which the high osmolality pathway is disrupted.     

Nucleoside diphosphate kinase-1 regulates hyphal development via the transcriptional regulation of catalase in Neurospora crassa

A nucleoside diphosphate kinase-1-disrupted (ndk-1^RIP-1) mutant was observed to be defective in aerial hyphal and conidial development. In this study, two types of hyphae, fine and thick, were observed in wild-type (Wt) strains. However, only fine-type hyphae were observed in the ndk-1^RIP-1 mutants. The ndk-1^RIP-1 mutants were stimulated by oxidative stress and constitutively expressed an antioxidant enzyme catalase (CAT)-3. Furthermore the ndk-1^RIP-1 mutants could form thick hyphae by oxidative stress and a disruption of cat-3. These results suggest that the loss of thick hyphae in the ndk-1^RIP-1 mutants may be caused by the over-expression of cat-3.     

Yeast Genome-Wide Expression Analysis Identifies a Strong Ergosterol and Oxidative Stress Response during the Initial Stages of an Industrial Lager Fermentation

Genome-wide expression analysis of an industrial strain of Saccharomyces cerevisiae during the initial stages of an industrial lager fermentation identified a strong response from genes involved in the biosynthesis of ergosterol and oxidative stress protection. The induction of the ERG genes was confirmed by Northern analysis and was found to be complemented by a rapid accumulation of ergosterol over the initial 6-h fermentation period. From a test of the metabolic activity of deletion mutants in the ergosterol biosynthesis pathway, it was found that ergosterol is an important factor in restoring the fermentative capacity of the cell after storage. Additionally, similar ERG10 and TRR1 gene expression patterns over the initial 24-h fermentation period highlighted a possible interaction between ergosterol biosynthesis and the oxidative stress response. Further analysis showed that erg mutants producing altered sterols were highly sensitive to oxidative stress-generating compounds. Here we show that genome-wide expression analysis can be used in the commercial environment and was successful in identifying environmental conditions that are important in industrial yeast fermentation.     

Lipidomic Analysis Links Mycobactin Synthase K to Iron Uptake and Virulence in M. tuberculosis

The prolonged survival of Mycobacterium tuberculosis (M. tb) in the host fundamentally depends on scavenging essential nutrients from host sources. M. tb scavenges non-heme iron using mycobactin and carboxymycobactin siderophores, synthesized by mycobactin synthases (Mbt). Although a general mechanism for mycobactin biosynthesis has been proposed, the biological functions of individual mbt genes remain largely untested. Through targeted gene deletion and global lipidomic profiling of intact bacteria, we identify the essential biochemical functions of two mycobactin synthases, MbtK and MbtN, in siderophore biosynthesis and their effects on bacterial growth in vitro and in vivo. The deletion mutant, ΔmbtN, produces only saturated mycobactin and carboxymycobactin, demonstrating an essential function of MbtN as the mycobactin dehydrogenase, which affects antigenicity but not iron uptake or M. tb growth. In contrast, deletion of mbtK ablated all known forms of mycobactin and its deoxy precursors, defining MbtK as the essential acyl transferase. The mbtK mutant showed markedly reduced iron scavenging and growth in vitro. Further, ΔmbtK was attenuated for growth in mice, demonstrating a non-redundant role of hydroxamate siderophores in virulence, even when other M. tb iron scavenging mechanisms are operative. The unbiased lipidomic approach also revealed unexpected consequences of perturbing mycobactin biosynthesis, including extreme depletion of mycobacterial phospholipids. Thus, lipidomic profiling highlights connections among iron acquisition, phospholipid homeostasis, and virulence, and identifies MbtK as a lynchpin at the crossroads of these phenotypes.     

Mycobacterium tuberculosis has diminished capacity to counteract redox stress induced by elevated levels of endogenous superoxide

Mycobacterium tuberculosis (Mtb) has evolved protective and detoxification mechanisms to maintain cytoplasmic redox balance in response to exogenous oxidative stress encountered inside host phagocytes. In contrast, little is known about the dynamic response of this pathogen to endogenous oxidative stress generated within Mtb. Using a noninvasive and specific biosensor of cytoplasmic redox state of Mtb, we for first time discovered a surprisingly high sensitivity of this pathogen to perturbation in redox homeostasis induced by elevated endogenous reactive oxygen species (ROS). We synthesized a series of hydroquinone-based small molecule ROS generators and found that ATD-3169 permeated mycobacteria to reliably enhance endogenous ROS including superoxide radicals. When Mtb strains including multidrug-resistant (MDR) and extensively drug-resistant (XDR) patient isolates were exposed to this compound, a dose-dependent, long-lasting, and irreversible oxidative shift in intramycobacterial redox potential was detected. Dynamic redox potential measurements revealed that Mtb had diminished capacity to restore cytoplasmic redox balance in comparison with Mycobacterium smegmatis (Msm), a fast growing nonpathogenic mycobacterial species. Accordingly, Mtb strains were extremely susceptible to inhibition by ATD-3169 but not Msm, suggesting a functional linkage between dynamic redox changes and survival. Microarray analysis showed major realignment of pathways involved in redox homeostasis, central metabolism, DNA repair, and cell wall lipid biosynthesis in response to ATD-3169, all consistent with enhanced endogenous ROS contributing to lethality induced by this compound. This work provides empirical evidence that the cytoplasmic redox poise of Mtb is uniquely sensitive to manipulation in steady-state endogenous ROS levels, thus revealing the importance of targeting intramycobacterial redox metabolism for controlling TB infection.     

Assessment of chronological lifespan dependent molecular damages in yeast lacking mitochondrial antioxidant genes

The free radical theory of aging states that oxidative damage to biomolecules causes aging and that antioxidants neutralize free radicals and thus decelerate aging. Mitochondria produce most of the reactive oxygen species, but at the same time have many antioxidant enzymes providing protection from these oxidants. Expecting that cells without mitochondrial antioxidant genes would accumulate higher levels of oxidative damage and, therefore, will have a shorter lifespan, we analyzed oxidative damages to biomolecules in young and chronologically aged mutants lacking the mitochondrial antioxidant genes: GRX2, CCP1, SOD1, GLO4, TRR2, TRX3, CCS1, SOD2, GRX5, and PRX1. Among these mutants, ccp1Δ, trx3Δ, grx5Δ, prx1Δ, mutants were sensitive to diamide, and ccs1Δ and sod2Δ were sensitive to both diamide and menadione. Most of the mutants were less viable in stationary phase. Chronologically aged cells produced higher amount of superoxide radical and accumulated higher levels of oxidative damages. Even though our results support the findings that old cells harbor higher amount of molecular damages, no significant difference was observed between wild type and mutant cells in terms of their damage content.     

Changes in the Sterol Composition of the Plasma Membrane Affect Membrane Potential,Salt Tolerance and the Activity of Multidrug Resistance Pumps in Saccharomyces cerevisiae

We investigated the impact of the deletions of genes from the final steps in the biosynthesis of ergosterol (ERG6, ERG2, ERG3, ERG5, ERG4) on the physiological function of the Saccharomyces cerevisiae plasma membrane by a combination of biological tests and the diS-C₃(3) fluorescence assay. Most of the erg mutants were more sensitive than the wild type to salt stress or cationic drugs, their susceptibilities were proportional to the hyperpolarization of their plasma membranes. The different sterol composition of the plasma membrane played an important role in the short-term and long-term processes that accompanied the exposure of erg strains to a hyperosmotic stress (effect on cell size, pH homeostasis and survival of yeasts), as well as in the resistance of cells to antifungal drugs. The pleiotropic drug-sensitive phenotypes of erg strains were, to a large extent, a result of the reduced efficiency of the Pdr5 efflux pump, which was shown to be more sensitive to the sterol content of the plasma membrane than Snq2p. In summary, the erg4Δ and erg6Δ mutants exhibited the most compromised phenotypes. As Erg6p is not involved in the cholesterol biosynthetic pathway, it may become a target for a new generation of antifungal drugs.     

Evaluation of a Brucella melitensis mutant deficient in O-polysaccharide export system ATP-binding protein as a rough vaccine candidate

Rough Brucella mutants have been sought as vaccine candidates that do not interfere with the conventional serological diagnosis of brucellosis. In this study, a rough mutant of Brucella melitensis was generated by the disruption of the wzt gene, which encodes the O-polysaccharide (O-PS) export system ATP-binding protein. In vivo, the mutant 16MΔwzt was attenuated and conferred a level of protection against B. melitensis 16M challenge similar to that conferred by the vaccine strain B. melitensis M5 in mice. In pregnant sheep, the mutant 16MΔwzt did not induce abortion. In vitro, 16MΔwzt was more susceptible to polymyxin B and complement-mediated killing than B. melitensis 16M was. Most importantly, although 16MΔwzt had a rough phenotype, it was able to synthesize O-PS and did not induce detectable specific antibodies in sheep. These results suggested that 16MΔwzt deserved to further systematic evaluation as a vaccine for target animal hosts due to its promising features.     

Phenethyl isothiocyanate induces DNA damage-associated G2/M arrest and subsequent apoptosis in oral cancer cells with varying p53 mutations

Phenethyl isothiocyanate (PEITC) is a naturally occurring cruciferous vegetable-derived compound that inhibits cell growth and induces apoptosis in oral cancer cells. However, the exact mechanism of PEITC action has not been fully elucidated. This study investigated the molecular mechanism and anticancer potential of PEITC in oral squamous cell carcinoma (OSCC) cells with various p53 statuses. PEITC inhibited the growth of OC2, SCC4, and SCC25 cells (functional p53 mutants) in a dose-dependent manner with low toxicity to normal cells. Treatment with PEITC induced reactive oxygen species production, nitric oxide generation, and GSH depletion and triggered DNA damage response as evidenced by flow cytometry, 8-OHdG formation, and comet assay. Furthermore, the subsequent activation of ATM, Chk2, and p53 as well as the increased expression of downstream proteins p21 and Bax resulted in a G2/M phase arrest by inhibiting Cdc25C, Cdc2, and cyclin B1. The PEITC-induced apoptotic cell death, following a diminished mitochondrial transmembrane potential, reduced the expression of Bcl-2 and Mcl-1, released mitochondrial cytochrome c, and activated caspase 3 and PARP cleavage. The p53 inhibitor pifithrin-α and the antioxidants N-acetylcysteine and glutathione (GSH) protected the cells from PEITC-mediated apoptosis. However, mito-TEMPO, catalase, apocynin, and L-NAME did not prevent PEITC-induced cell death, suggesting that PEITC induced G2/M phase arrest and apoptosis in oral cancer cells via a GSH redox stress and oxidative DNA damage-induced ATM–Chk2–p53-related pathway. These results provide new insights into the critical roles of both GSH redox stress and p53 in the regulation of PEITC-induced G2/M cell cycle arrest and apoptosis in OSCCs.     

Osh6 overexpression extends the lifespan of yeast by increasing vacuole fusion

In yeast cells, the vacuole divides and fuses in each round of cell cycle. While mutants defective in vacuole fusion are “wild type” for vegetative growth, most have shortened replicative lifespans under caloric restriction (CR) condition, a manipulation that extends lifespan in wild type cells. To explore whether vacuole fusion extends lifespan, we screened for genes that can complement the fusion defect of selected mutants (erg6Δ, a sterol mutant; nyv1Δ, a mutant involved in the vacuolar SNARE complex and vac8Δ, a vacuolar membrane protein mutant). This screen revealed that Osh6, a member of the oxysterol-binding protein family, can complement the vacuole fusion defect of nyv1Δ, but not erg6Δ or vac8Δ, suggesting that Osh6’s function in vacuole fusion is partly dependent on membrane ergosterol and Vac8. To measure the effect of OSH6 on lifespan, we replaced the endogenous promoter of OSH6 with a shorter version of the ERG6 promoter to obtain P_ERG6-OSH6. This mutant construct significantly extended the replicative lifespan in a wild type background and in a nyv1Δ mutant. Interestingly, P_ERG6-OSH6 cells were more sensitive to drugs that inhibit the activity of the TOR complex 1 (TORC1) than wild type cells. Moreover, a P_ERG6-OSH6 tor1Δ double mutant demonstrated a greatly shortened lifespan, suggesting a genetic interaction between Osh6 and Tor1. Since active TORC1 stimulates vacuole scission and CR downregulates TORC1, Osh6 may link these two pathways by adjusting vacuolar membrane organization to extend lifespan.     

Comparative cytoprotective effects of carbocysteine and fluticasone propionate in cigarette smoke extract-stimulated bronchial epithelial cells

Cigarette smoke extracts (CSE) induce oxidative stress, an important feature in chronic obstructive pulmonary disease (COPD), and oxidative stress contributes to the poor clinical efficacy of corticosteroids in COPD patients. Carbocysteine, an antioxidant and mucolytic agent, is effective in reducing the severity and the rate of exacerbations in COPD patients. The effects of carbocysteine on CSE-induced oxidative stress in bronchial epithelial cells as well as the comparison of these antioxidant effects of carbocysteine with those of fluticasone propionate are unknown. The present study was aimed to assess the effects of carbocysteine (10⁻⁴ M) in cell survival and intracellular reactive oxygen species (ROS) production (by flow cytometry) as well as total glutathione (GSH), heme oxygenase-1 (HO-1), nuclear-related factor 2 (Nrf2) expression and histone deacetylase 2 (HDAC-2) expression/activation in CSE-stimulated bronchial epithelial cells (16-HBE) and to compare these effects with those of fluticasone propionate (10⁻⁸ M). CSE, carbocysteine or fluticasone propionate did not induce cell necrosis (propidium positive cells) or cell apoptosis (annexin V-positive/propidium-negative cells) in 16-HBE. CSE increased ROS production, nuclear Nrf2 and HO-1 in 16-HBE. Fluticasone propionate did not modify intracellular ROS production, GSH and HDCA-2 but reduced Nrf2 and HO-1 in CSE-stimulated 16-HBE. Carbocysteine reduced ROS production and increased GSH, HO-1, Nrf2 and HDAC-2 nuclear expression/activity in CSE-stimulated cells and was more effective than fluticasone propionate in modulating the CSE-mediated effects. In conclusion, the present study provides compelling evidences that the use of carbocysteine may be considered a promising strategy in diseases associated with corticosteroid resistance.     

Negative Regulation of Methyl Jasmonate-Induced Stomatal Closure by Glutathione in Arabidopsis

Glutathione (GSH) has been shown to negatively regulate methyl jasmonate (MeJA)-induced stomatal closure. We investigated the roles of GSH in MeJA signaling in guard cells using an Arabidopsis mutant, cad2-1, that is deficient in the first GSH biosynthesis enzyme, γ-glutamylcysteine synthetase. MeJA-induced stomatal closure and decreased GSH contents in guard cells. Decreasing GSH by the cad2-1 mutation enhanced MeJA-induced stomatal closure. Depletion of GSH by the cad2-1 mutation or increment of GSH by GSH monoethyl ester did not affect either MeJA-induced production of reactive oxygen species (ROS) or MeJA-induced cytosolic alkalization in guard cells. MeJA and abscisic acid (ABA) induced stomatal closure and GSH depletion in atrbohD and atrbohF single mutants but not in the atrbohD atrbohF double mutant. Moreover, exogenous hydrogen peroxide induced stomatal closure but did not deplete GSH in guard cells. These results indicate that GSH affects MeJA signaling as well as ABA signaling and that GSH negatively regulates a signal component other than ROS production and cytosolic alkalization in MeJA signal pathway of Arabidopsis guard cells.