Nitrosative Stress Response Research Articles

Trace Elements in Alzheimer's Disease and Dementia: The Current State of Knowledge.

Changes in trace element concentrations are being wildly considered when it comes to neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease. This study aims to present the role that trace elements play in the central nervous system. Moreover, we reviewed the mechanisms involved in their neurotoxicity. Low zinc concentrations, as well as high levels of copper, manganese, and iron, activate the signalling pathways of the inflammatory, oxidative and nitrosative stress response. Neurodegeneration occurs due to the association between metals and proteins, which is then followed by aggregate formation, mitochondrial disorder, and, ultimately, cell death. In Alzheimer's disease, low Zn levels suppress the neurotoxicity induced by β-amyloid through the selective precipitation of aggregation intermediates. High concentrations of copper, iron and manganese cause the aggregation of intracellular α-synuclein, which results in synaptic dysfunction and axonal transport disruption. Parkinson's disease is caused by the accumulation of Fe in the midbrain dopaminergic nucleus, and the pathogenesis of multiple sclerosis derives from Zn deficiency, leading to an imbalance between T cell functions. Aluminium disturbs the homeostasis of other metals through a rise in the production of oxygen reactive forms, which then leads to cellular death. Selenium, in association with iron, plays a distinct role in the process of ferroptosis. Outlining the influence that metals have on oxidoreduction processes is crucial to recognising the pathophysiology of neurodegenerative diseases and may provide possible new methods for both their avoidance and therapy.

Characterization ofantifungal properties of lipopeptide-producing Bacillus velezensis strains and their proteome-based response to the phytopathogens, Diaporthe spp.

Introduction: B. velezensis strains are of interest in agricultural applications due to their beneficial interactions with plants, notable through their antimicrobial activity. The biocontrol ability of two new lipopeptides-producing B. velezensis strains ES1-02 and EFSO2-04, against fungal phytopathogens of Diaporthe spp., was evaluated and compared with reference strains QST713 and FZB42. All strains were found to be effective against the plant pathogens, with the new strains showing comparable antifungal activity to QST713 and slightly lower activity than FZB42. Methods: Lipopeptides and their isoforms were identified by high-performance thin-layer chromatography (HPTLC) and mass spectrometric measurements. The associated antifungal influences were determined in direct in vitro antagonistic dual culture assays, and the inhibitory growth effects on Diaporthe spp. as representatives of phytopathogenic fungi were determined. The effects on bacterial physiology of selected B. velezensis strains were analyzed by mass spectrometric proteomic analyses using nano-LC-MS/MS. Results and Discussion: Lipopeptide production analysis revealed that all strains produced surfactin, and one lipopeptide of the iturin family, including bacillomycin L by ES1-02 and EFSO2-04, while QST713 and FZB42 produced iturin A and bacillomycin D, respectively. Fengycin production was however only detected in the reference strains. As a result of co-incubation of strain ES1-02 with the antagonistic phytopathogen D. longicolla, an increase in surfactin production of up to 10-fold was observed, making stress induction due to competitors an attractive strategy for surfactin bioproduction. An associated global proteome analysis showed a more detailed overview about the adaptation and response mechanisms of B. velezensis, including an increased abundance of proteins associated with the biosynthesis of antimicrobial compounds. Furthermore, higher abundance was determined for proteins associated with oxidative, nitrosative, and general stress response. In contrast, proteins involved in phosphate uptake, amino acid transport, and translation were decreased in abundance. Altogether, this study provides new insights into the physiological adaptation of lipopeptide-producing B. velezensis strains, which show the potential for use as biocontrol agents with respect to phytopathogenic fungi.

Radical-Driven Methane Formation in Humans Evidenced by Exogenous Isotope-Labeled DMSO and Methionine.

Methane (CH4), which is produced endogenously in animals and plants, was recently suggested to play a role in cellular physiology, potentially influencing the signaling pathways and regulatory mechanisms involved in nitrosative and oxidative stress responses. In addition, it was proposed that the supplementation of CH4 to organisms may be beneficial for the treatment of several diseases, including ischemia, reperfusion injury, and inflammation. However, it is still unclear whether and how CH4 is produced in mammalian cells without the help of microorganisms, and how CH4 might be involved in physiological processes in humans. In this study, we produced the first evidence of the principle that CH4 is formed non-microbially in the human body by applying isotopically labeled methylated sulfur compounds, such as dimethyl sulfoxide (DMSO) and methionine, as carbon precursors to confirm cellular CH4 formation. A volunteer applied isotopically labeled (2H and 13C) DMSO on the skin, orally, and to blood samples. The monitoring of stable isotope values of CH4 convincingly showed the conversion of the methyl groups, as isotopically labeled CH4 was formed during all experiments. Based on these results, we considered several hypotheses about endogenously formed CH4 in humans, including physiological aspects and stress responses involving reactive oxygen species (ROS). While further and broader validation studies are needed, the results may unambiguously serve as a proof of concept for the endogenous formation of CH4 in humans via a radical-driven process. Furthermore, these results might encourage follow-up studies to decipher the potential physiological role of CH4 and its bioactivity in humans in more detail. Of particular importance is the potential to monitor CH4 as an oxidative stress biomarker if the observed large variability of CH4 in breath air is an indicator of physiological stress responses and immune reactions. Finally, the potential role of DMSO as a radical scavenger to counteract oxidative stress caused by ROS might be considered in the health sciences. DMSO has already been investigated for many years, but its potential positive role in medical use remains highly uncertain.

Modeling Selenoprotein Se-Nitrosation: Synthesis of a Se-Nitrososelenocysteine with Persistent Stability.

The Se-nitrosation in selenoproteins such as glutathione peroxidase and thioredoxin reductase to produce Se-nitrososelenocysteines (Sec-SeNOs) has been proposed to play crucial roles in signaling processes mediated by reactive nitrogen species and nitrosative-stress responses, although chemical evidence for the formation of Sec-SeNOs has been elusive not only in proteins but also in small-molecule systems. Herein, we report the first synthesis of a Sec-SeNO by employing a selenocysteine model system that bears a protective molecular cradle. The Sec-SeNO was characterized using 1H and 77Se nuclear magnetic resonance as well as ultraviolet/visible spectroscopy and found to have persistent stability at room temperature in solution. The reaction processes involving the Sec-SeNO provide experimental information that serves as a chemical basis for elucidating the reaction mechanisms involving the SeNO species in biological functions, as well as in selenol-catalyzed NO generation from S-nitrosothiols.

Stabilisation of the RirA [4Fe-4S] cluster results in loss of iron-sensing function.

RirA is a global iron regulator in diverse Alphaproteobacteria that belongs to the Rrf2 superfamily of transcriptional regulators, which can contain an iron-sulfur (Fe-S) cluster. Under iron-replete conditions, RirA contains a [4Fe-4S] cluster, enabling high-affinity binding to RirA-regulated operator sequences, thereby causing the repression of cellular iron uptake. Under iron deficiency, one of the cluster irons dissociates, generating an unstable [3Fe-4S] form that subsequently degrades to a [2Fe-2S] form and then to apo RirA, resulting in loss of high-affinity DNA-binding. The cluster is coordinated by three conserved cysteine residues and an unknown fourth ligand. Considering the lability of one of the irons and the resulting cluster fragility, we hypothesized that the fourth ligand may not be an amino acid residue. To investigate this, we considered that the introduction of an amino acid residue that could coordinate the cluster might stabilize it. A structural model of RirA, based on the Rrf2 family nitrosative stress response regulator NsrR, highlighted residue 8, an Asn in the RirA sequence, as being appropriately positioned to coordinate the cluster. Substitution of Asn8 with Asp, the equivalent, cluster-coordinating residue of NsrR, or with Cys, resulted in proteins that contained a [4Fe-4S] cluster, with N8D RirA exhibiting spectroscopic properties very similar to NsrR. The variant proteins retained the ability to bind RirA-regulated DNA, and could still act as repressors of RirA-regulated genes in vivo. However, they were significantly more stable than wild-type RirA when exposed to O2 and/or low iron. Importantly, they exhibited reduced capacity to respond to cellular iron levels, even abolished in the case of the N8D version, and thus were no longer iron sensing. This work demonstrates the importance of cluster fragility for the iron-sensing function of RirA, and more broadly, how a single residue substitution can alter cluster coordination and functional properties in the Rrf2 superfamily of regulators.

S-glutathionylation of fructose-1,6-bisphosphate aldolase confers nitrosative stress tolerance on yeast cells via a metabolic switch.

Nitric oxide as a signaling molecule exerts cytotoxicity known as nitrosative stress at its excess concentrations. In the yeast Saccharomyces cerevisiae, the cellular responses to nitrosative stress and their molecular mechanisms are not fully understood. Here, focusing on the posttranslational modifications that are associated with nitrosative stress response, we show that nitrosative stress increased the protein S-glutathionylation level in yeast cells. Our proteomic and immunochemical analyses demonstrated that the fructose-1,6-bisphosphate aldolase Fba1 underwent S-glutathionylation at Cys112 in response to nitrosative stress. The enzyme assay using a recombinant Fba1 demonstrated that S-glutathionylation at Cys112 inhibited the Fba1 activity. Moreover, we revealed that the cytosolic glutaredoxin Grx1 reduced S-glutathionylation of Fba1 and then recovered its activity. The intracellular contents of fructose-1,6-bisphosphate and 6-phosphogluconate, which are a substrate of Fba1 and an intermediate of the pentose phosphate pathway (PPP), respectively, were increased in response to nitrosative stress, suggesting that the metabolic flow was switched from glycolysis to PPP. The cellular level of NADPH, which is produced in PPP and functions as a reducing force for nitric oxide detoxifying enzymes, was also elevated under nitrosative stress conditions, but this increase was canceled by the amino acid substitution of Cys112 to Ser in Fba1. Furthermore, the viability of yeast cells expressing Cys112Ser-Fba1 was significantly lower than that of the wild-type cells under nitrosative stress conditions. These results indicate that the inhibition of Fba1 by its S-glutathionylation changes metabolism from glycolysis to PPP to increase NADPH production, leading to nitrosative stress tolerance in yeast cells.

Interrelation between Stress Management and Secretion Systems of Ralstonia solanacearum: An In Silico Assessment.

Ralstonia solanacearum (Rs), the causative agent of devastating wilt disease in several major and minor economic crops, is considered one of the most destructive bacterial plant pathogens. However, the mechanism(s) by which Rs counteracts host-associated environmental stress is still not clearly elucidated. To investigate possible stress management mechanisms, orthologs of stress-responsive genes in the Rs genome were searched using a reference set of known genes. The genome BLAST approach was used to find the distributions of these orthologs within different Rs strains. BLAST results were first confirmed from the KEGG Genome database and then reconfirmed at the protein level from the UniProt database. The distribution pattern of these stress-responsive factors was explored through multivariate analysis and STRING analysis. STRING analysis of stress-responsive genes in connection with different secretion systems of Rs was also performed. Initially, a total of 28 stress-responsive genes of Rs were confirmed in this study. STRING analysis revealed an additional 7 stress-responsive factors of Rs, leading to the discovery of a total of 35 stress-responsive genes. The segregation pattern of these 35 genes across 110 Rs genomes was found to be almost homogeneous. Increasing interactions of Rs stress factors were observed in six distinct clusters, suggesting six different types of stress responses: membrane stress response (MSR), osmotic stress response (OSR), oxidative stress response (OxSR), nitrosative stress response (NxSR), and DNA damage stress response (DdSR). Moreover, a strong network of these stress responses was observed with type 3 secretion system (T3SS), general secretory proteins (GSPs), and different types of pili (T4P, Tad, and Tat). To the best of our knowledge, this is the first report on overall stress response management by Rs and the potential connection with secretion systems.

S-Nitrosylation of the virulence regulator AphB promotes Vibrio cholerae pathogenesis.

Vibrio cholerae is the etiologic agent of the severe human diarrheal disease cholera. To colonize mammalian hosts, this pathogen must defend against host-derived toxic compounds, such as nitric oxide (NO) and NO-derived reactive nitrogen species (RNS). RNS can covalently add an NO group to a reactive cysteine thiol on target proteins, a process called protein S-nitrosylation, which may affect bacterial stress responses. To better understand how V. cholerae regulates nitrosative stress responses, we profiled V. cholerae protein S-nitrosylation during RNS exposure. We identified an S-nitrosylation of cysteine 235 of AphB, a LysR-family transcription regulator that activates the expression of tcpP, which activates downstream virulence genes. Previous studies show that AphB C235 is sensitive to O2 and reactive oxygen species (ROS). Under microaerobic conditions, AphB formed dimer and directly repressed transcription of hmpA, encoding a flavohemoglobin that is important for NO resistance of V. cholerae. We found that tight regulation of hmpA by AphB under low nitrosative stress was important for V. cholerae optimal growth. In the presence of NO, S-nitrosylation of AphB abolished AphB activity, therefore relieved hmpA expression. Indeed, non-modifiable aphBC235S mutants were sensitive to RNS in vitro and drastically reduced colonization of the RNS-rich mouse small intestine. Finally, AphB S-nitrosylation also decreased virulence gene expression via debilitation of tcpP activation, and this regulation was also important for V. cholerae RNS resistance in vitro and in the gut. These results suggest that the modulation of the activity of virulence gene activator AphB via NO-dependent protein S-nitrosylation is critical for V. cholerae RNS resistance and colonization.

Bioactivity of Inhaled Methane and Interactions With Other Biological Gases.

A number of studies have demonstrated explicit bioactivity for exogenous methane (CH4), even though it is conventionally considered as physiologically inert. Other reports cited in this review have demonstrated that inhaled, normoxic air-CH4 mixtures can modulate the in vivo pathways involved in oxidative and nitrosative stress responses and key events of mitochondrial respiration and apoptosis. The overview is divided into two parts, the first being devoted to a brief review of the effects of biologically important gases in the context of hypoxia, while the second part deals with CH4 bioactivity. Finally, the consequence of exogenous, normoxic CH4 administration is discussed under experimental hypoxia- or ischaemia-linked conditions and in interactions between CH4 and other biological gases, with a special emphasis on its versatile effects demonstrated in pulmonary pathologies.

ArfX2 GTPase Regulates Trafficking From the Trans-Golgi to Lysosomes and Is Necessary for Liver Abscess Formation in the Protozoan Parasite Entamoeba histolytica.

Entamoeba histolytica is the causative agent of amoebic dysentery and liver abscess in humans. The parasitic lifestyle and the virulence of the protist require elaborate biological processes, including vesicular traffic and stress management against a variety of reactive oxygen and nitrogen species produced by the host immune response. Although the mechanisms for intracellular traffic of representative virulence factors have been investigated at molecular levels, it remains poorly understood whether and how intracellular traffic is involved in the defense against reactive oxygen and nitrogen species. Here, we demonstrate that EhArfX2, one of the Arf family of GTPases known to be involved in the regulation of vesicular traffic, was identified by comparative transcriptomic analysis of two isogenic strains: an animal-passaged highly virulent HM-1:IMSS Cl6 and in vitro maintained attenuated avirulent strain. EhArfX2 was identified as one of the most highly upregulated genes in the highly virulent strain. EhArfX2 was localized to small vesicle-like structures and largely colocalized with the marker for the trans-Golgi network SNARE, EhYkt6, but neither with the endoplasmic reticulum (ER)-resident chaperon, EhBip, nor the cis-Golgi SNARE, EhSed5, and Golgi-luminal galactosyl transferase, EhGalT. Expression of the dominant-active mutant form of EhArfX2 caused an increase in the number of lysosomes, while expression of the dominant-negative mutant led to a defect in lysosome formation and cysteine protease transport to lysosomes. Expression of the dominant-negative mutant in the virulent E. histolytica strain caused a reduction of the size of liver abscesses in a hamster model. This defect in liver abscess formation was likely at least partially attributed to reduced resistance to nitrosative, but not oxidative stress in vitro. These results showed that the EhArfX2-mediated traffic is necessary for the nitrosative stress response and virulence in the host.

Transcriptomics Analysis Uncovers Transient Ceftazidime Tolerance in Burkholderia Biofilms.

Burkholderia pseudomallei is an etiological agent of melioidosis, a severe community-acquired infectious disease. B.pseudomallei strain K96243 is sensitive to the drug ceftazidime (CAZ), but has been shown to exhibit transient CAZ tolerance when in a biofilm form. To investigate an observed shift in gene expression profile during CAZ tolerance condition and to better understand the mechanistic aspects of this transient tolerance, RNA-sequencing was performed on B.pseudomallei K96243 from the following three states: planktonic, biofilm, and planktonic shedding. Results indicated that the expression of 651 genes (10.97%) were significantly changed in both biofilm (resistant) and planktonic shedding (sensitive) cells in comparison to the planktonic state. The top four highly expressed genes identified in both states are associated with nitrosative stress response (BPSL2368), Fe-S homeostasis (BPSL2369), and nitrate respiration (BPSS1154 and BPSS1158). Additionally, five orthologous genes, BPSL2370-BPSL2374, implicated in Fe-S cluster biogenesis, and another gene, BPSL2863, involved in DNA-binding of the stress protein ferritin, were shown to increase expression by RT-qPCR. The shift in gene expression was especially prominent at the late stages of biofilm growth (72 and 96 h), specifically in the biofilm-challenged CAZ survivor cells. This suggested that in response to stress in a biofilm, differential expression of these genes may support development of the CAZ tolerance in Burkholderia. The application of iron chelator deferoxamine (DFO) to the biofilm caused a significant reduction in biofilm formation and associated CAZ tolerance. Therefore, the shift in Fe-S metabolism when B.pseudomallei is in a biofilm may help stabilize the levels of reactive oxygen species (ROS), thereby limiting tolerance to CAZ.

A Nitric Oxide-Responsive Transcriptional Regulator NsrR Cooperates With Lrp and CRP to Tightly Control the hmpA Gene in Vibrio vulnificus.

Nitric oxide (NO) is an important antimicrobial effector produced by the host innate immune system to counteract invading pathogens. To survive and establish a successful infection, a fulminating human pathogen Vibrio vulnificus expresses the hmpA gene encoding an NO dioxygenase in an NO-responsive manner. In this study, we identified an Rrf2-family transcriptional regulator NsrR that is predicted to contain the Fe-S cluster coordinated by three cysteine residues. Transcriptome analysis showed that NsrR controls the expression of multiple genes potentially involved in nitrosative stress responses. Particularly, NsrR acts as a strong repressor of hmpA transcription and relieves the repression of hmpA upon exposure to NO. Notably, nsrR and hmpA are transcribed divergently, and their promoter regions overlap with each other. Molecular biological analyses revealed that NsrR directly binds to this overlapping promoter region, which is alleviated by loss of the Fe-S cluster, leading to the subsequent derepression of hmpA under nitrosative stress. We further found that a leucine-responsive regulatory protein (Lrp) negatively regulates hmpA in an NsrR-dependent manner by directly binding to the promoter region, presumably resulting in a DNA conformation change to support the repression by NsrR. Meanwhile, a cyclic AMP receptor protein (CRP) positively regulates hmpA probably through repression of nsrR and lrp by directly binding to each promoter region in a sequential cascade. Altogether, this collaborative regulation of NsrR along with Lrp and CRP enables an elaborate control of hmpA transcription, contributing to survival under host-derived nitrosative stress and thereby the pathogenesis of V. vulnificus.

Interplay of two transcription factors for recruitment of the chromatin remodeling complex modulates fungal nitrosative stress response

Nitric oxide (NO) is a diffusible signaling molecule that modulates animal and plant immune responses. In addition, reactive nitrogen species derived from NO can display antimicrobial activities by reacting with microbial cellular components, leading to nitrosative stress (NS) in pathogens. Here, we identify FgAreB as a regulator of the NS response in Fusarium graminearum, a fungal pathogen of cereal crops. FgAreB serves as a pioneer transcription factor for recruitment of the chromatin-remodeling complex SWI/SNF at the promoters of genes involved in the NS response, thus promoting their transcription. FgAreB plays important roles in fungal infection and growth. Furthermore, we show that a transcription repressor (FgIxr1) competes with the SWI/SNF complex for FgAreB binding, and negatively regulates the NS response. NS, in turn, promotes the degradation of FgIxr1, thus enhancing the recruitment of the SWI/SNF complex by FgAreB.

Kidney nitrosative stress response to acute ischemia‐reperfusion injury is present only in the external medulla and independent of sex in adult rats

Despite the morphological and physiological differences between male and female kidneys., and the differences in the pathophysiological response to ischemia-reperfusion injury at different experimental models (genetic, subcellular, cellular, animals and humans), they're still controversies between if these responses are depended to nitric oxide - nitrosative stress vía, and the site in the renal parenchyma which are predominant. METHODS We analyzed (Image G program and Wilcoxon test in JMP software) the level of nitrosylated protein (fluorescent microscopy, DHPEF-filter, 20x objective, 0.5 slight exposure, antibodies AB61392, revealed with antibody-CF488A, diluted 1:1000) in 10-µm slices of left-kidney subjected to 45 min ischemia and 24 h reperfusion, both male (n=5 w= 120-150g) and female Wistar adult rats (n=9, w=120-150g). The right kidney of the same animals was the control kidney (no ischemia). RESULTS The level of nitrosylated protein were similar between groups for the internal medulla (ischemic kidney male= 115.24±10.73, ischemic kidney female= 130.97 ± 2.54, control kidney male= 107.72 ± 3.41, control kidney female= 112.53 ± 3.31, p=ns) and the glomeruli (ischemic kidney male= 122.88 ± 16.13, ischemic kidney female= 117.79 ± 9.26, control kidney male= 101.58 ± 13.75, control kidney female= 98.26 ± 5.75, p=ns). For the external medulla, there was an increase in the level of nitrosylated protein after the treatment (45 min ischemia, 24 h reperfusion), in both male and females (ischemic kidney male= 121.81 ± 10.97, ischemic kidney female= 125.96 ± 3.19) compared with control kidney (male= 93.75 ± 4.94, female= 99.78 ± 2.29, p=0.0004 ischemic vs control female kidney, p=0.0069 ischemic vs controlled male kidney, p=0.01 ischemic male vs control female kidney), but without differences between sexes. CONCLUSION The level of nitrosative protein was similar in control kidney (without ischemia) in both sexes, which represents a nitrosative basal level necessary for physiological responses and independent of the protein level sex-differences reported in the literature (SOD, eNOS and iNOS, among others). Also, the level of nitrosative protein was increased in 29% in males and 26% in females only in the external medulla, without differences in the other sections of renal parenchyma. The results of this work showed similarities in epigenetic changes secondary to flow interruption of the whole renal pedicle in female and male rats only in the external medulla, but not a behavior pattern in the papilla or cortex. In-situ study of this phenomenon, in contrast to other techniques that require tissue homogenization (like flow cytometry or western blot), allowed for a precise quantitative signal measurement in each region.

Anna Karenina and the subgingival microbiome associated with periodontitis

BackgroundAlthough localized aggressive periodontitis (LAP), generalized aggressive periodontitis (GAP), and chronic periodontitis (CP) are microbially driven diseases, our inability to separate disease-specific associations from those common to all three forms of periodontitis has hampered biomarker discovery. Therefore, we aimed to map the genomic content of, and the biological pathways encoded by, the microbiomes associated with these clinical phenotypes. We also estimated the extent to which these biomes are governed by the Anna Karenina principle (AKP), which states that eubiotic communities are similar between individuals while disease-associated communities are highly individualized.MethodsWe collected subgingival plaque from 25 periodontally healthy individuals and diseased sites of 59 subjects with stage 3 periodontitis and used shotgun metagenomics to characterize the aggregate of bacterial genes.ResultsBeta-dispersion metrics demonstrated that AKP was most evident in CP, followed by GAP and LAP. We discovered broad dysbiotic signatures spanning the three phenotypes, with over-representation of pathways that facilitate life in an oxygen-poor, protein- and heme-rich, pro-oxidant environment and enhance capacity for attachment and biofilm formation. Phenotype-specific indicators were more readily evident in LAP microbiome than GAP or CP. Genes that enable acetate-scavenging lifestyle, utilization of alternative nutritional sources, oxidative and nitrosative stress responses, and siderophore production were unique to LAP. An attenuation of virulence-related functionalities and stress response from LAP to GAP to CP was apparent. We also discovered that clinical phenotypes of disease resolved variance in the microbiome with greater clarity than the newly established grading system. Importantly, we observed that one third of the metagenome of LAP is unique to this phenotype while GAP shares significant functional and taxonomic features with both LAP and CP, suggesting either attenuation of an aggressive disease or an early-onset chronic disease.ConclusionWithin the limitations of a small sample size and a cross-sectional study design, the distinctive features of the microbiomes associated with LAP and CP strongly persuade us that these are discrete disease entities, while calling into question whether GAP is a separate disease, or an artifact induced by cross-sectional study designs. Further studies on phenotype-specific microbial genes are warranted to explicate their role in disease etiology.542UkY-RA6fKgB5kXgVxbtVideo

Nano-Boehmite Induced Oxidative and Nitrosative Stress Responses in Vigna radiata L.

Wide spectrum and increasing use of nano-sized aluminum oxyhydroxide (boehmite, nBhm) particles have left a risk of their environmental exposure and possible toxic impacts. However, little is known about their toxicity. Current investigation was aimed to assess the toxic impacts of nBhm on Vigna radiata L., a model plant. Seedlings of V. radiata were exposed to 0 – 12 mM nBhm for ten days, and nBhm reduced the biomass production in a dose-dependent manner, with the greatest impacts under 12 mM nBhm causing 16% and 4% reduction in root and shoot dry weight (DW), respectively. Higher dose of nBhm damaged the cellular membrane stability and chlorophyll contents. The levels of major reactive oxygen species including superoxide radical, hydrogen peroxide and hydroxyl radical were increased by 2.65, 2.45 and 2.50 times more in leaves and 2.31, 2.55 and 5.71 times more in roots, respectively, in plants subjected to 12 mM nBhm treatment. Spectrophotometry and in-gel assays confirmed that the nBhm triggered antioxidative machinery with up-regulation of superoxide dismutase, catalase and peroxidase enzymes. Concurrent accumulation of non-enzymatic antioxidants was also recorded in the form of proline and ascorbic acid. The nBhm exposed plants showed higher nitric oxide production with 12 mM nBhm treatment causing 1.91- and 1.49-fold hike in nitric oxide content of leaves and roots, respectively. Interestingly, roots and leaves behaved contrastingly in terms of nitrate reductase activity in response to the nBhm treatment. The hierarchical clustering and principal component analysis revealed the closeness of the stress-indicating attributes towards higher nBhm concentrations. Collectively, nBhm exerted negative impacts on V. radiata in a dose-dependent manner, with higher toxicity at the concentration ≥ 8 mM, mediated through oxidative and nitrosative bursts.

Experimental evolution reveals nitrate tolerance mechanisms in Desulfovibrio vulgaris.

Elevated nitrate in the environment inhibits sulfate reduction by important microorganisms of sulfate-reducing bacteria (SRB). Several SRB may respire nitrate to survive under elevated nitrate, but how SRB that lack nitrate reductase survive to elevated nitrate remains elusive. To understand nitrate adaptation mechanisms, we evolved 12 populations of a model SRB (i.e., Desulfovibrio vulgaris Hildenborough, DvH) under elevated NaNO3 for 1000 generations, analyzed growth and acquired mutations, and linked their genotypes with phenotypes. Nitrate-evolved (EN) populations significantly (p < 0.05) increased nitrate tolerance, and whole-genome resequencing identified 119 new mutations in 44 genes of 12 EN populations, among which six functional gene groups were discovered with high mutation frequencies at the population level. We observed a high frequency of nonsense or frameshift mutations in nitrosative stress response genes (NSR: DVU2543, DVU2547, and DVU2548), nitrogen regulatory protein C family genes (NRC: DVU2394-2396, DVU2402, and DVU2405), and nitrate cluster (DVU0246-0249 and DVU0251). Mutagenesis analysis confirmed that loss-of-functions of NRC and NSR increased nitrate tolerance. Also, functional gene groups involved in fatty acid synthesis, iron regulation, and two-component system (LytR/LytS) known to be responsive to multiple stresses, had a high frequency of missense mutations. Mutations in those gene groups could increase nitrate tolerance through regulating energy metabolism, barring entry of nitrate into cells, altering cell membrane characteristics, or conferring growth advantages at the stationary phase. This study advances our understanding of nitrate tolerance mechanisms and has important implications for linking genotypes with phenotypes in DvH.

Stress response, amino acid biosynthesis and pathogenesis genes expressed in Salmonella enterica colonizing tomato shoot and root surfaces

Salmonella enterica can colonize all parts of the tomato plant. Tomatoes have been frequently implicated in salmonellosis outbreaks. In agricultural settings, Salmonella must overcome stress, nutritional and competition barriers to become established on plant surfaces. Knowledge of the genetic mechanisms underlying Salmonella-plant associations is limited, especially when growing epiphytically. A genome-wide transcriptomic analysis of Salmonella Typhimurium (SeT) was conducted with RNA-Seq to elucidate strategies for epiphytic growth on live, intact tomato shoot and root surfaces. Six plasmid-encoded and 123 chromosomal genes were significantly (using Benjamini-Hochberg adjusted p-values) up-regulated; 54 and 110 detected in SeT on shoots and roots, respectively, with 35 common to both. Key signals included NsrR regulon genes needed to mitigate nitrosative stress, oxidative stress genes and host adaptation genes, including environmental stress, heat shock and acid-inducible genes. Several amino acid biosynthesis genes and genes indicative of sulphur metabolism and anaerobic respiration were up-regulated. Some Type III secretion system (T3SS) effector protein genes and their chaperones from pathogenicity island-2 were expressed mostly in SeT on roots. Gene expression in SeT was validated against SeT and also the tomato outbreak strain Salmonella Newport with a high correlation (R2 = 0.813 and 0.874, respectively; both p < 0.001). Oxidative and nitrosative stress response genes, T3SS2 genes and amino acid biosynthesis may be needed for Salmonella to successfully colonize tomato shoot and root surfaces.

Deciphering the Enigmatic Function of Pseudomonas Metallothioneins.

Metallothioneins (MTs) are low molecular weight, Cys-rich proteins that sequester both essential and non-essential metal ions. Despite being highly conserved in the Pseudomonas genus of Gram-negative bacteria, knowledge of their physiological function in this species is scarce. Using the strain P. fluorescens Q2-87 as a model organism, we investigated the role of a conserved MT in zinc homeostasis, cadmium detoxification as well as its implications in stress response. We show that MT expression is only induced in the stationary phase and provides a fitness benefit for long-term starvation survival, while it is not required for metal resistance and acquisition, oxidative or nitrosative stress response, biofilm formation or motility.

YjbH Requires Its Thioredoxin Active Motif for the Nitrosative Stress Response, Cell-to-Cell Spread, and Protein-Protein Interactions in Listeria monocytogenes.

Listeria monocytogenes is a model facultative intracellular pathogen. Tight regulation of virulence proteins is essential for a successful infection, and the gene encoding the annotated thioredoxin YjbH was identified in two forward genetic screens as required for virulence factor production. Accordingly, an L. monocytogenes strain lacking yjbH is attenuated in a murine model of infection. However, the function of YjbH in L. monocytogenes has not been investigated. Here, we provide evidence that L. monocytogenes YjbH is involved in the nitrosative stress response, likely through its interaction with the redox-responsive transcriptional regulator SpxA1. YjbH physically interacted with SpxA1, and our data support a model in which YjbH is a protease adaptor that regulates SpxA1 protein abundance. Whole-cell proteomics identified eight additional proteins whose abundance was altered by YjbH, and we demonstrated that YjbH physically interacted with each in bacterial two-hybrid assays. Thioredoxin proteins canonically require active motif cysteines for function, but thioredoxin activity has not been tested for L. monocytogenes YjbH. We demonstrated that cysteine residues of the YjbH thioredoxin domain active motif are essential for L. monocytogenes sensitivity to nitrosative stress, cell-to-cell spread in a tissue culture model of infection, and several protein-protein interactions. Together, these results demonstrated that the function of YjbH in L. monocytogenes requires its thioredoxin active motif and that YjbH has a role in the posttranslational regulation of several proteins, including SpxA1.IMPORTANCE The annotated thioredoxin YjbH in Listeria monocytogenes has been implicated in virulence, but its function in the cell is unknown. In other bacterial species, YjbH is a protease adaptor that mediates degradation of the transcriptional regulator Spx. Here, we investigated the function of L. monocytogenes YjbH and demonstrated its role in the nitrosative stress response and posttranslational regulation of several proteins with which YjbH physically interacts, including SpxA1. Furthermore, we demonstrated that the cysteine residues of the YjbH thioredoxin active motif are required for the nitrosative stress response, cell-to-cell spread, and some protein-protein interactions. YjbH is widely conserved among Firmicutes, and this work reveals its unique requirement of the thioredoxin-active motif in L. monocytogenes.