Presence of Fusobacterium nucleatum in relation to patient survival and an acidic environment in oesophagogastric junction and gastric cancers.

BACKGROUND: Fusobacterium nucleatum inhabits the oral cavity and affects the progression of gastrointestinal cancer. Our prior findings link F. nucleatum to poor prognosis in oesophageal squamous cell carcinoma via NF-κB pathway. However, its role in oesophagogastric junction and gastric adenocarcinoma remains unexplored. We investigated whether F. nucleatum influences these cancers, highlighting its potential impact. METHODS: Two cohorts of EGJ and gastric adenocarcinoma patients (438 from Japan, 380 from the USA) were studied. F. nucleatum presence was confirmed by qPCR, FISH, and staining. Patient overall survival (OS) was assessed based on F. nucleatum positivity. EGJ and gastric adenocarcinoma cell lines were exposed to F. nucleatum to study molecular and phenotypic effects, validated in xenograft mouse model. RESULTS: In both cohorts, F. nucleatum-positive EGJ or gastric adenocarcinoma patients had notably shorter OS. F. nucleatum positivity decreased in more acidic tumour environments. Cancer cell lines with F. nucleatum showed enhanced proliferation and NF-κB activation. The xenograft model indicated increased tumour growth and NF-κB activation in F. nucleatum-treated cells. Interestingly, co-occurrence of F. nucleatum and Helicobacter pylori, a known risk factor, was rare. CONCLUSIONS: F. nucleatum can induce the NF-κB pathway in EGJ and gastric adenocarcinomas, leading to tumour progression and poor prognosis.

Supersulfides suppress type-â and type-â ¡ interferon responses by blocking JAK/STAT signaling in macrophages.

Interferons (IFNs) are cytokines produced and secreted by immune cells when viruses, tumor cells, and so forth, invade the body. Their biological effects are diverse, including antiviral, cell growth-inhibiting, and antitumor effects. The main subclasses of interferons include type-I (e.g., IFN-α and IFN-ß) and type-II (IFN-γ), which activate intracellular signals by binding to type-I and type-II IFN receptors, respectively. We have previously shown that when macrophages are treated with supersulfide donors, which have polysulfide structures in which three or more sulfur atoms are linked within the molecules, IFN-ß-induced cellular responses, including signal transducer and activator of transcription 1 (STAT1) phosphorylation and inducible nitric oxide synthase (iNOS) expression, were strongly suppressed. However, the subfamily specificity of the suppression of IFN signals by supersulfides and the mechanism of this suppression are unknown. This study demonstrated that supersulfide donor N-acetyl-L-cysteine tetrasulfide (NAC-S2) can inhibit IFN signaling in macrophages stimulated not only with IFN-α/ß but also with IFN-γ. Our data suggest that NAC-S2 blocks phosphorylation of Janus kinases (JAKs), thereby contributes to the inhibition of phosphorylation of STAT1. Under the current experimental conditions, hydrogen sulfide (H2S) donor NaHS failed to inhibit IFN signaling. Similar to NAC-S2, carbohydrate-based supersulfide donor thioglucose tetrasulfide (TGS4) was capable of strongly inhibiting tumor necrosis factor-αproduction, iNOS expression, and nitric oxide production from macrophages stimulated with lipopolysaccharide. Further understanding of molecular mechanisms how supersulfide donors exhibit their inhibitory actions towards JAK/STAT signaling is necessary basis for development of supersulfide-based therapeutic strategy against autoimmune disorders with dysregulated IFN signaling.

Proposal of Helicobacter higonensis sp. nov. isolated from a human clinical specimen, and emended description of Helicobacter valdiviensis Collado, 2014.

We have previously isolated a gram-negative microaerophilic strain, PAGU2000T from a patient presenting with a fever in Kumamoto Prefecture, Japan. The present study aimed to comprehensively analyze the taxonomy of the isolated strain using a polyphasic approach. The 16S rRNA gene sequence analysis indicated that the strain was a member of enterohepatic Helicobacter. The strain PAGU2000T shared a 97.5% 16S rRNA gene nucleotide identity with Helicobacter valdiviensis, and this taxonomic position was confirmed by phylogenetic analysis of the GyrA amino acid sequences. The proposed strain PAGU2000T has a 1.482 Mbp chromosome with a DNA G + C content of 31.3 mol% and encodes 1520 predicted coding sequences. The average nucleotide identity between the strain PAGU2000T and type strain of H. valdiviensis was 70.3%, which was lower than the recommended threshold of 95% for species delineation. The strain PAGU2000T was a motile, non-spore-forming, and spiral-shaped bacterium, exhibiting catalase and oxidase activities but not urease and nitrate reduction. This study demonstrates that the isolate represents a novel species within enterohepatic Helicobacter, for which the name Helicobacter higonensis is proposed (type strain: PAGU2000T = GTC 16811T = LMG 33095T). In this study, we describe the phenotypic and morphological features of this strain and propose an emended description of some biochemical traits of H. valdiviensis.

[Redox Biology Involved in the Toxicity of Enterohemorrhagic Escherichia coli Toxin SubAB].

AB5 toxins of pathogenic bacteria enter host cells and utilize the retrograde trafficking pathway to translocate to the cytoplasm and exert its pathogenesis. Cholera toxin and Shiga toxin reach the endoplasmic reticulum (ER), and the A subunit undergoes redox regulation by ER proteins to become active fragments, which pass through the ER membrane and translocate to the cytoplasm. By acting on molecular targets in the cytoplasm, the normal function of host cells are disrupted, causing diseases. ER chaperone proteins such as protein disulfide isomerase (PDI) and binding immunoglobulin protein (BiP) induce conformational changes triggered by the reduction of disulfide bonds in the A subunit. This is thought to be dependent on cysteine thiol-mediated redox regulation, but the detailed mechanism remains unclear. On the other hand, subtilase cytotoxin (SubAB), produced by enterohemorrhagic Escherichia coli (EHEC), localizes to the ER without translocating to the cytoplasm and cleaves BiP as a substrate. Therefore, it is thought that ER stress-based cytotoxicity and intestinal bleeding occur without translocating to the cytoplasm. We reported that PDI is involved in BiP cleavage through SubAB localization to the ER. Like other AB5 toxins, this indicates the involvement of redox regulation via chaperone proteins in the ER, but also suggests that SubAB does not translocate to the cytoplasm because it cleaves BiP. Although there are few reports on the redox state of ER protein thiols, it is suggested that polysulfidation, which is discussed in this symposium, may be involved.

Alkyl gallates inhibit serine O-acetyltransferase in bacteria and enhance susceptibility of drug-resistant Gram-negative bacteria to antibiotics.

A principal concept in developing antibacterial agents with selective toxicity is blocking metabolic pathways that are critical for bacterial growth but that mammalian cells lack. Serine O-acetyltransferase (CysE) is an enzyme in many bacteria that catalyzes the first step in l-cysteine biosynthesis by transferring an acetyl group from acetyl coenzyme A (acetyl-CoA) to l-serine to form O-acetylserine. Because mammalian cells lack this l-cysteine biosynthesis pathway, developing an inhibitor of CysE has been thought to be a way to establish a new class of antibacterial agents. Here, we demonstrated that alkyl gallates such as octyl gallate (OGA) could act as potent CysE inhibitors in vitro and in bacteria. Mass spectrometry analyses indicated that OGA treatment markedly reduced intrabacterial levels of l-cysteine and its metabolites including glutathione and glutathione persulfide in Escherichia coli to a level similar to that found in E. coli lacking the cysE gene. Consistent with the reduction of those antioxidant molecules in bacteria, E. coli became vulnerable to hydrogen peroxide-mediated bacterial killing in the presence of OGA. More important, OGA treatment intensified susceptibilities of metallo-ß-lactamase-expressing Gram-negative bacteria (E. coli and Klebsiella pneumoniae) to carbapenem. Structural analyses showed that alkyl gallate bound to the binding site for acetyl-CoA that limits access of acetyl-CoA to the active site. Our data thus suggest that CysE inhibitors may be used to treat infectious diseases caused by drug-resistant Gram-negative bacteria not only via direct antibacterial activity but also by enhancing therapeutic potentials of existing antibiotics.

Helicobacter kumamotonensis sp. nov., isolated from human clinical specimens.

A Gram-stain-negative, spiral bacterium (PAGU 1991T) was isolated from the blood of a patient with diffuse large B-cell lymphoma. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate was very closely related to Helicobacter equorum LMG 23362T (99.1 % similarity), originally isolated from a faecal sample from a healthy horse. PAGU 1991T was also very closely related to PAGU 1750 in our strain library (=CCUG 41437) with 99.7 % similarity. Additional phylogenetic analyses based on the 23S rRNA gene sequence and GyrA amino acid sequence further supported the close relationship between the two human isolates (PAGU 1991T and PAGU 1750) and the horse strain. However, a phylogenetic analysis based on 16S rRNA showed that the two human isolates formed a lineage that was distinct from the horse strain (less than 99.2 % similarity). In silico whole-genome comparisons based on digital DNA-DNA hybridization, average nucleotide identity based on blast and orthologous average nucleotide identity using usearch between the two human isolates and the type strain of H. equorum showed values of less than 52.40, 93.47, and 93.50 %, respectively, whereas those between the two human isolates were 75.8, 97.2, and 97.2 %, respectively. These data clearly demonstrated that the two human isolates formed a single species, distinct from H. equorum. Morphologically, the human isolates could be distinguished by the type of flagella; the human isolates showed a bipolar sheathed flagellum, whereas that of H. equorum was monopolar. Biochemically, the human isolate was characterized by growth at 42 °C under microaerobic conditions and nitrate reduction unability. We conclude that the two human isolates, obtained from geographically and temporally distinct sources, were a novel species, for which we propose the name Helicobacter kumamotonensis sp. nov., with the type strain PAGU 1991T (=GTC 16810T=CCUG 75774T).

Structural Determination of the Nanocomplex of Borate with Styrene-Maleic Acid Copolymer-Conjugated Glucosamine Used as a Multifunctional Anticancer Drug.

The development of effective anticancer drugs is essential for chemotherapy that specifically targets cancer tissues. We recently synthesized a multifunctional water-soluble anticancer polymer drug consisting of styrene-maleic acid copolymer (SMA) conjugated with glucosamine and boric acid (BA) (SGB complex). It demonstrated about 10 times higher tumor-selective accumulation compared with accumulation in normal tissues because of the enhanced permeability and retention effect, and it inhibited tumor growth via glycolysis inhibition, mitochondrial damage, and thermal neutron irradiation. Gaining insight into the anticancer effects of this SGB complex requires a determination of its structure. We therefore investigated the chemical structure of the SGB complex by means of nuclear magnetic resonance, infrared (IR) spectroscopy, and liquid chromatography-mass spectrometry. To establish the chemical structure of the SGB complex, we synthesized a simple model compound─maleic acid-glucosamine (MAG) conjugate─by using a maleic anhydride (MA) monomer unit instead of the SMA polymer. We obtained two MAG-BA complexes (MAGB) with molecular weights of 325 and 343 after the MAG reaction with BA. We confirmed, by using IR spectroscopy, that MAGB formed a stable complex via an amide bond between MA and glucosamine and that BA bound to glucosamine via a diol bond. As a result of this chemical design, identified via analysis of MAGB, the SGB complex can release BA and demonstrate toxicity to cancer cells through inhibition of lactate secretion in mild hypoxia that mimics the tumor microenvironment. For clinical application of the SGB complex, we confirmed that this complex is stable in the presence of serum. These findings confirm that our design of the SGB complex has various advantages in targeting solid cancers and exerting therapeutic effects when combined with neutron irradiation.

New insights into the regulatory roles of glutathione in NLRP3-inflammasome-mediated immune and inflammatory responses.

Glutathione (GSH) is the most abundant non-protein thiol (-SH) in mammalian cells. Its synthesis and metabolism serve to maintain cellular reduction-oxidation (redox) homeostasis, which is important for multiple cellular processes including proliferation, differentiation and death. An accumulating body of evidence suggests that the essential roles of GSH extended far beyond its oxidant and electrophile scavenger activities and regulatory role in the lifespan of cells. Recent findings revealed that altered GSH levels are closely associated with a wide range of pathologies including bacterial and viral infections, neurodegenerative diseases and autoimmune disorders, all of which are also characterized by aberrant activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome. As a result of these findings, GSH was assigned a central role in influencing the activation of the NLRP3 inflammasome. To expand on our recent advances in understanding this process, we discuss here the emerging roles of GSH in activation of the NLRP3 inflammasome, and the therapeutic potential of GSH in its associated pathologies.

Fusobacterium nucleatum promotes esophageal squamous cell carcinoma progression via the NOD1/RIPK2/NF-κB pathway.

Fusobacterium nucleatum, found in the oral cavity, influences the progression of gastrointestinal cancers. Additionally, our previous results suggested that F. nucleatum is associated with poor patient prognosis in esophageal squamous cell carcinoma (ESCC). However, the mechanism by which F. nucleatum affects aggressive tumor behavior has yet to be elucidated. We have conducted this clinical, in vitro, and in vivo study to clarify the mechanism of ESCC progression induced by F. nucleatum. Transmission electron microscopy revealed that F. nucleatum invaded and occupied ESCC cells and impacted gene and protein expression. Comprehensive mRNA expression and pathway enrichment analyses of F. nucleatum-treated ESCC cells identified the "NF-κB" and "NOD-like receptor" signaling pathways as enriched. We confirmed the relationship between the presence of F. nucleatum and NF-κB activation in resected ESCC tissues. Furthermore, F. nucleatum-treated ESCC cells demonstrated enhanced growth ability, and NF-κB activation, as well as overexpression of NOD1 and phosphorylated RIPK2. Furthermore, treated cells showed accelerated tumor growth, with NF-κB activation in xenograft models. F. nucleatum invaded ESCC cells and induced the NF-κB pathway through the NOD1/RIPK2 pathway, leading to tumor progression.

Regulation of nitric oxide/reactive oxygen species redox signaling by nNOS splicing variants.

We previously demonstrated different expression patterns of the neuronal nitric oxide synthase (nNOS) splicing variants, nNOS-µ and nNOS-α, in the rat brain; however, their exact functions have not been fully elucidated. In this study, we compared the enzymatic activities of nNOS-µ and nNOS-α and investigated intracellular redox signaling in nNOS-expressing PC12 cells, stimulated with a neurotoxicant, 1-methyl-4-phenylpyridinium ion (MPP+), to enhance the nNOS uncoupling reaction. Using in vitro studies, we show that nNOS-µ produced nitric oxide (NO), as did nNOS-α, in the presence of tetrahydrobiopterin (BH4), an important cofactor for the enzymatic activity. However, nNOS-µ generated more NO and less superoxide than nNOS-α in the absence of BH4. MPP + treatment induced more reactive oxygen species (ROS) production in nNOS-α-expressing PC12 cells than in those expressing nNOS-µ, which correlated with the intracellular production of 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), a downstream messenger of nNOS redox signaling, and apoptosis in these cells. Furthermore, post-treatment with 8-nitro-cGMP aggravated MPP+-induced cytotoxicity via activation of the H-Ras/extracellular signal-regulated kinase signaling pathway. In conclusion, our results provide strong evidence that nNOS-µ exhibits distinctive enzymatic properties of NO/ROS production, contributing to the regulation of intracellular redox signaling, including the downstream production of 8-nitro-cGMP.

ATP exposure stimulates glutathione efflux as a necessary switch for NLRP3 inflammasome activation.

The NLRP3 inflammasome is a multiprotein complex responsible for the maturation of precursor forms of interleukin (IL)-1ß and IL-18 into active proinflammatory cytokines. Increasing evidence suggests that modulation of redox homeostasis contributes to the activation of the NLRP3 inflammasome. However, specific mechanistic details remain unclear. We demonstrate here that ATP exposure evoked a sharp decrease in glutathione (GSH) levels in macrophages, which led to NLRP3 inflammasome activation. We detected an increase in GSH levels in culture supernatants that was comparable to the GSH decrease in macrophages, which suggests that exposure to ATP stimulated GSH efflux. Exogenous addition of P2X7 receptor antagonist, GSH, or the oxidized form GSSG attenuated this efflux. Also, exogenous GSH or GSSG strongly inhibited NLRP3 inflammasome activation in vitro and in vivo. These data suggest that GSH efflux controls NLRP3 inflammasome activation, which may lead to development of novel therapeutic strategies for NLRP3 inflammasome-associated disorders.

Cysteine Hydropersulfide Inactivates ß-Lactam Antibiotics with Formation of Ring-Opened Carbothioic S-Acids in Bacteria.

Hydrogen sulfide (H2S) formed during sulfur metabolism in bacteria has been implicated in the development of intrinsic resistance to antibacterial agents. Despite the conversion of H2S to hydropersulfides greatly enhancing the biochemical properties of H2S such as antioxidant activity, the effects of hydropersulfides on antibiotic resistance have remained unknown. In this work, we investigated the effects of H2S alone or together with cystine to form cysteine hydropersulfide (CysSSH) on the activities of antibacterial agents. By using the disc diffusion test, we found that CysSSH treatment effectively inactivated ß-lactams of the penicillin class (penicillin G and ampicillin) and the carbapenem class (meropenem). These ß-lactams were resistant to treatment with H2S alone or cystine alone. In contrast, cephalosporin class ß-lactams (cefaclor and cefoperazone) and non-ß-lactam antibiotics (tetracycline, kanamycin, erythromycin, and ofloxacin) were stable after CysSSH treatment. Chromatographic and mass spectrometric analyses revealed that CysSSH directly reacted with ß-lactams to form ß-lactam ring-opened carbothioic S-acids (BL-COSH). Furthermore, we demonstrated that certain bacteria (e.g., Escherichia coli and Staphylococcus aureus) efficiently decomposed ß-lactam antibiotics to form BL-COSH, which were transported to the extracellular space. These data suggest that CysSSH-mediated ß-lactam decomposition may contribute to intrinsic bacterial resistance to ß-lactams. BL-COSH may become useful biomarkers for CysSSH-mediated ß-lactam resistance and for investigation of potential antibacterial adjuvants that can enhance the antibacterial activity of ß-lactams by reducing the hydropersulfides in bacteria.

Antioxidative and anti-inflammatory actions of reactive cysteine persulfides.

Cysteine persulfide (CysSSH) and polysulfides (CysS[S] n H, n>1) are cysteine derivatives having sulfane sulfur atoms bound to cysteine thiol. Recent advances in the development of analytical methods for detection and quantification of persulfides and polysulfides have revealed the biological presence, in both prokaryotes and eukaryotes, of persulfide/polysulfide in diverse forms such as CysSSH, glutathione persulfide and protein persulfides. Accumulating evidence has suggested that persulfide/polysulfide species may involve in a variety of biological events such as biosyntheses of sulfur-containing molecules, tRNA modification, regulation of redox-dependent signal transduction, mitochondrial energy metabolism via sulfur respiration, cytoprotection from oxidative stress via their antioxidant activities, and anti-inflammation against Toll-like receptor-mediated inflammatory responses. Development of chemical sulfur donors may facilitate further understanding of physiological and pathophysiological roles of persulfide/polysulfide species, including regulatory roles of these species in immune responses.

Polymer-conjugated glucosamine complexed with boric acid shows tumor-selective accumulation and simultaneous inhibition of glycolysis.

We synthesized unique water-soluble synthetic-polymer, styrene-maleic acid copolymer (SMA) conjugated glucosamine (SG); which formed a stable complex with boric acid (BA). This complex had a mean particle size of 15 nm by light scattering, and single peak in gel permeation chromatography. The particles were taken up by tumor cells five times faster than free BA in vitro and liberated BA at acidic tumor pH (5-7). Liberated BA inhibited glycolysis and resulted in tumor suppression in vivo. Intravenously injected SGB-complex did bind with albumin, and plasma half-life was about 8 h in mice, and accumulated to tumor tissues about 10 times more than in normal organs. IC50 of SGB-complex for HeLa cells under pO2 of 6-9% was about 20 µg/ml (free BA equivalent), 150 times more potent than free BA. Neutron irradiation of human oral cancer cells with SGB-complex resulted in 16 times greater cell-killing than that without SGB-complex. In vivo antitumor effect was evaluated after neutron irradiation only once in SCC VII tumor bearing mice and significant tumor suppression was confirmed. These results indicate that SGB-complex is a unique multifunctional anticancer agent with much more potent activity under low pO2 conditions as in large advanced cancers.

Fusobacterium nucleatum confers chemoresistance by modulating autophagy in oesophageal squamous cell carcinoma.

BACKGROUND: Fusobacterium nucleatum (F. nucleatum) is a gut microbe implicated in gastrointestinal tumorigenesis. Predicting the chemotherapeutic response is critical to developing personalised therapeutic strategies for oesophageal cancer patients. The present study investigated the relationship between F. nucleatum and chemotherapeutic resistance in oesophageal squamous cell carcinoma (ESCC). METHODS: We examined the relationship between F. nucleatum and chemotherapy response in 120 ESCC resected specimens and 30 pre-treatment biopsy specimens. In vitro studies using ESCC cell lines and co-culture assays further uncovered the mechanism underlying chemotherapeutic resistance. RESULTS: ESCC patients with F. nucleatum infection displayed lesser chemotherapeutic response. The infiltration and subsistence of F. nucleatum in the ESCC cells were observed by transmission electron microscopy and laser scanning confocal microscopy. We also observed that F. nucleatum modulates the endogenous LC3 and ATG7 expression, as well as autophagosome formation to induce chemoresistance against 5-FU, CDDP, and Docetaxel. ATG7 knockdown resulted in reversal of F. nucleatum-induced chemoresistance. In addition, immunohistochemical studies confirmed the correlation between F. nucleatum infection and ATG7 expression in 284 ESCC specimens. CONCLUSIONS: F. nucleatum confers chemoresistance to ESCC cells by modulating autophagy. These findings suggest that targeting F. nucleatum, during chemotherapy, could result in variable therapeutic outcomes for ESCC patients.

Preparation of Biodegradable PLGA-Nanoparticles Used for pH-Sensitive Intracellular Delivery of an Anti-inflammatory Bacterial Toxin to Macrophages.

Poly(D,L-lactide-co-glycolic) acid (PLGA) is a synthetic copolymer that has been used to design micro/nanoparticles as a carrier for macromolecules, such as protein and nucleic acids, that can be internalized by the endocytosis pathway. However, it is difficult to control the intracellular delivery to target organelles. Here we report an intracellular delivery system of nanoparticles modified with bacterial cytotoxins to the endoplasmic reticulum (ER) and anti-inflammatory activity of the nanoparticles. Subtilase cytotoxin (SubAB) is a bacterial toxin in certain enterohemorrhagic Escherichia coli (EHEC) strains that cleaves the host ER chaperone BiP and suppresses nuclear factor-kappaB (NF-κB) activation and nitric oxide (NO) generation in macrophages at sub-lethal concentration. PLGA-nanoparticles were modified with oligo histidine-tagged (6 × His-tagged) recombinant SubAB (SubAB-PLGA) through a pH-sensitive linkage, and their translocation to the ER in macrophage cell line J774.1 cells, effects on inducible NO synthase (iNOS), and levels of tumor necrosis factor (TNF)-α cytokine induced by lipopolysaccharide (LPS) were examined. Compared with free SubAB, SubAB-PLGA was significantly effective in BiP cleavage and the induction of the ER stress marker C/EBP homologous protein (CHOP) in J774.1 cells. Furthermore, SubAB-PLGA attenuated LPS-stimulated induction of iNOS and TNF-α. Our findings provide useful information for protein delivery to macrophages and may encourage therapeutic applications of nanoparticles to the treatment of inflammatory diseases.

Enhanced Cellular Polysulfides Negatively Regulate TLR4 Signaling and Mitigate Lethal Endotoxin Shock.

Cysteine persulfide and cysteine polysulfides are cysteine derivatives having sulfane sulfur atoms bound to cysteine thiol. Accumulating evidence has suggested that cysteine persulfides/polysulfides are abundant in prokaryotes and eukaryotes and play important roles in diverse biological processes such as antioxidant host defense and redox-dependent signal transduction. Here, we show that enhancement of cellular polysulfides by using polysulfide donors developed in this study resulted in marked inhibition of lipopolysaccharide (LPS)-initiated macrophage activation. Polysulfide donor treatment strongly suppressed LPS-induced pro-inflammatory responses in macrophages by inhibiting Toll-like receptor 4 (TLR4) signaling. Other TLR signaling stimulants-including zymosan A-TLR2 and poly(I:C)-TLR3-were also significantly suppressed by polysulfur donor treatment. Administration of polysulfide donors protected mice from lethal endotoxin shock. These data indicate that cellular polysulfides negatively regulate TLR4-mediated pro-inflammatory signaling and hence constitute a potential target for inflammatory disorders.

Reactive Persulfides from Salmonella Typhimurium Downregulate Autophagy-Mediated Innate Immunity in Macrophages by Inhibiting Electrophilic Signaling.

Reactive persulfides such as cysteine persulfide and glutathione persulfide are produced by bacteria including Salmonella during sulfur metabolism. The biological significance of bacterial reactive persulfides in host-pathogen interactions still warrants investigation. We found that reactive persulfides produced by Salmonella Typhimurium LT2 regulate macrophage autophagy via metabolizing 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), an electrophilic product of reactive oxygen species and nitric oxide signaling. 8-Nitro-cGMP signaling was required for efficient autophagy-mediated clearance of Salmonella from infected macrophages. In the infected cells, 8-nitro-cGMP caused cGMP adduct formation (S-guanylation) of bacterial surface proteins, which triggered recruitment of autophagy-related proteins p62 and LC3-II to the intracellular bacteria. We also found that Salmonella-produced reactive persulfides downregulated this autophagy by decreasing cellular 8-nitro-cGMP content, thereby inhibiting electrophilic signaling. These data reveal a pathogenic role of bacteria-derived reactive persulfides via suppression of anti-bacterial autophagy.

Reactive Cysteine Persulphides: Occurrence, Biosynthesis, Antioxidant Activity, Methodologies, and Bacterial Persulphide Signalling.

Cysteine hydropersulphide (CysSSH) is a cysteine derivative having one additional sulphur atom bound to a cysteinyl thiol group. Recent advances in the development of analytical methods for detection and quantification of persulphides and polysulphides have revealed the biological presence, in both prokaryotes and eukaryotes, of hydropersulphides in diverse forms such as CysSSH, homocysteine hydropersulphide, glutathione hydropersulphide, bacillithiol hydropersulphide, coenzyme A hydropersulphide, and protein hydropersulphides. Owing to the chemical reactivity of the persulphide moiety, biological systems utilize persulphides as important intermediates in the synthesis of various sulphur-containing biomolecules. Accumulating evidence has revealed another important feature of persulphides: their potent reducing activity, which implies that they are implicated in the regulation of redox signalling and antioxidant functions. In this chapter, we discuss the biological occurrence and possible biosynthetic mechanisms of CysSSH and related persulphides, and we include descriptions of recent advances in the analytical methods that have been used to detect and quantitate persulphide species. We also discuss the antioxidant activity of persulphide species that contributes to protecting cells from reactive oxygen species-associated damage, and we examine the signalling roles of CysSSH in bacteria.

Involvement of nitric oxide/reactive oxygen species signaling via 8-nitro-cGMP formation in 1-methyl-4-phenylpyridinium ion-induced neurotoxicity in PC12 cells and rat cerebellar granule neurons.

To investigate the role of nitric oxide (NO)/reactive oxygen species (ROS) redox signaling in Parkinson's disease-like neurotoxicity, we used 1-methyl-4-phenylpyridinium (MPP+) treatment (a model of Parkinson's disease). We show that MPP+-induced neurotoxicity was dependent on ROS from neuronal NO synthase (nNOS) in nNOS-expressing PC12 cells (NPC12 cells) and rat cerebellar granule neurons (CGNs). Following MPP+ treatment, we found production of 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), a second messenger in the NO/ROS redox signaling pathway, in NPC12 cells and rat CGNs, that subsequently induced S-guanylation and activation of H-Ras. Additionally, following MPP+ treatment, extracellular signal-related kinase (ERK) phosphorylation was enhanced. Treatment with a mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor attenuated MPP+-induced ERK phosphorylation and neurotoxicity. In conclusion, we demonstrate for the first time that NO/ROS redox signaling via 8-nitro-cGMP is involved in MPP+-induced neurotoxicity and that 8-nitro-cGMP activates H-Ras/ERK signaling. Our results indicate a novel mechanism underlying MPP+-induced neurotoxicity, and therefore contribute novel insights to the mechanisms underlying Parkinson's disease.