Functional identification of bacterial spermine, thermospermine, norspermine, norspermidine, spermidine, and N1-aminopropylagmatine synthases.

Spermine synthase is an aminopropyltransferase that adds an aminopropyl group to the essential polyamine spermidine to form tetraamine spermine, needed for normal human neural development, plant salt and drought resistance, and yeast CoA biosynthesis. We functionally identify for the first time bacterial spermine synthases, derived from phyla Bacillota, Rhodothermota, Thermodesulfobacteriota, Nitrospirota, Deinococcota, and Pseudomonadota. We also identify bacterial aminopropyltransferases that synthesize the spermine same mass isomer thermospermine, from phyla Cyanobacteriota, Thermodesulfobacteriota, Nitrospirota, Dictyoglomota, Armatimonadota, and Pseudomonadota, including the human opportunistic pathogen Pseudomonas aeruginosa. Most of these bacterial synthases were capable of synthesizing spermine or thermospermine from the diamine putrescine and so possess also spermidine synthase activity. We found that most thermospermine synthases could synthesize tetraamine norspermine from triamine norspermidine, that is, they are potential norspermine synthases. This finding could explain the enigmatic source of norspermine in bacteria. Some of the thermospermine synthases could synthesize norspermidine from diamine 1,3-diaminopropane, demonstrating that they are potential norspermidine synthases. Of 18 bacterial spermidine synthases identified, 17 were able to aminopropylate agmatine to form N1-aminopropylagmatine, including the spermidine synthase of Bacillus subtilis, a species known to be devoid of putrescine. This suggests that the N1-aminopropylagmatine pathway for spermidine biosynthesis, which bypasses putrescine, may be far more widespread than realized and may be the default pathway for spermidine biosynthesis in species encoding L-arginine decarboxylase for agmatine production. Some thermospermine synthases were able to aminopropylate N1-aminopropylagmatine to form N12-guanidinothermospermine. Our study reveals an unsuspected diversification of bacterial polyamine biosynthesis and suggests a more prominent role for agmatine.

Exploiting Spermidine N-Hydroxycinnamoyltransferase Diversity and Substrate Promiscuity to Produce Various Trihydroxycinnamoyl Spermidines and Analogues in Engineered Yeast.

Trihydroxycinnamoyl spermidines (THCSpd) are plant specialized metabolites with promising pharmacological activities as antifungals, antibacterial, antiviral, and antidepressant drugs. However, their characterization and potential pharmaceutical exploitation are greatly impaired by the sourcing of these compounds, restricted to the pollen of core Eudicot plant species. In this work, we developed a precursor-directed biosynthesis of THCSpd in yeast using a dual enzymatic system based on 4-coumarate-CoA ligases (4CL) and spermidine N-hydroxycinnamoyltransferases (SHT). The system relies on the yeast endogenous spermidine pool and only requires hydroxycinnamic acids as exogenous precursors. By exploring 4CL isoforms and SHT diversity among plants, we have driven the production of 8 natural THCSpd, using single or mixed hydroxycinnamic acid precursors. Substrate promiscuities of 4CL and SHT were genuinely exploited to produce 8 new-to-nature THCSpd from exotic hydroxycinnamic and dihydrohydroxycinnamic acids, together with 3 new-to-nature THCSpd containing halogenated hydroxycinnamoyl moieties. In this work, we established a versatile and modular biotechnological production platform allowing the tailor-made THCSpd synthesis, constituting pioneer metabolic engineering for access to these valuable natural products.

Biosynthesis of a Novel Bioactive Metabolite of Spermidine from Bacillus amyloliquefaciens: Gene Mining, Sequence Analysis, and Combined Expression.

Spermidine is a biologically active polyamine with extensive application potential in functional foods. However, previously reported spermidine titers by biosynthesis methods are relatively low, which hinders its industrial application. To improve the spermidine titer, key genes affecting the spermidine production were mined to modify Bacillus amyloliquefaciens. Genes of S-adenosylmethionine decarboxylase (speD) and spermidine synthase (speE) from different microorganisms were expressed and compared in B. amyloliquefaciens. Therein, the speD from Escherichia coli and speE from Saccharomyces cerevisiae were confirmed to be optimal for spermidine synthesis, respectively. Gene and amino acid sequence analysis further confirmed the function of speD and speE. Then, these two genes were co-expressed to generate a recombinant strain B. amyloliquefaciens HSAM2(PDspeD-SspeE) with a spermidine titer of 105.2 mg/L, improving by 11.0-fold compared with the control (HSAM2). Through optimization of the fermentation medium, the spermidine titer was increased to 227.4 mg/L, which was the highest titer among present reports. Moreover, the consumption of the substrate S-adenosylmethionine was consistent with the accumulation of spermidine, which contributed to understanding its synthesis pattern. In conclusion, two critical genes for spermidine synthesis were obtained, and an engineering B. amyloliquefaciens strain was constructed for enhanced spermidine production.

DNA supercoiling differences in bacteria result from disparate DNA gyrase activation by polyamines.

DNA supercoiling is essential for all living cells because it controls all processes involving DNA. In bacteria, global DNA supercoiling results from the opposing activities of topoisomerase I, which relaxes DNA, and DNA gyrase, which compacts DNA. These enzymes are widely conserved, sharing >91% amino acid identity between the closely related species Escherichia coli and Salmonella enterica serovar Typhimurium. Why, then, do E. coli and Salmonella exhibit different DNA supercoiling when experiencing the same conditions? We now report that this surprising difference reflects disparate activation of their DNA gyrases by the polyamine spermidine and its precursor putrescine. In vitro, Salmonella DNA gyrase activity was sensitive to changes in putrescine concentration within the physiological range, whereas activity of the E. coli enzyme was not. In vivo, putrescine activated the Salmonella DNA gyrase and spermidine the E. coli enzyme. High extracellular Mg2+ decreased DNA supercoiling exclusively in Salmonella by reducing the putrescine concentration. Our results establish the basis for the differences in global DNA supercoiling between E. coli and Salmonella, define a signal transduction pathway regulating DNA supercoiling, and identify potential targets for antibacterial agents.

Mechanistic and biological characterisation of novel N5-substituted paullones targeting the biosynthesis of trypanothione in Leishmania.

Trypanothione synthetase (TryS) produces N1,N8-bis(glutathionyl)spermidine (or trypanothione) at the expense of ATP. Trypanothione is a metabolite unique and essential for survival and drug-resistance of trypanosomatid parasites. In this study, we report the mechanistic and biological characterisation of optimised N5-substituted paullone analogues with anti-TryS activity. Several of the new derivatives retained submicromolar IC50 against leishmanial TryS. The binding mode to TryS of the most potent paullones has been revealed by means of kinetic, biophysical and molecular modelling approaches. A subset of analogues showed an improved potency (EC50 0.5-10 µM) and selectivity (20-35) against the clinically relevant stage of Leishmania braziliensis (mucocutaneous leishmaniasis) and L. infantum (visceral leishmaniasis). For a selected derivative, the mode of action involved intracellular depletion of trypanothione. Our findings shed light on the molecular interaction of TryS with rationally designed inhibitors and disclose a new set of compounds with on-target activity against different Leishmania species.

Spermine oxidase mediates Helicobacter pylori-induced gastric inflammation, DNA damage, and carcinogenic signaling.

Helicobacter pylori infection is the main risk factor for the development of gastric cancer, the third leading cause of cancer death worldwide. H. pylori colonizes the human gastric mucosa and persists for decades. The inflammatory response is ineffective in clearing the infection, leading to disease progression that may result in gastric adenocarcinoma. We have shown that polyamines are regulators of the host response to H. pylori, and that spermine oxidase (SMOX), which metabolizes the polyamine spermine into spermidine plus H2O2, is associated with increased human gastric cancer risk. We now used a molecular approach to directly address the role of SMOX, and demonstrate that Smox-deficient mice exhibit significant reductions of gastric spermidine levels and H. pylori-induced inflammation. Proteomic analysis revealed that cancer was the most significantly altered functional pathway in Smox-/- gastric organoids. Moreover, there was also less DNA damage and ß-catenin activation in H. pylori-infected Smox-/- mice or gastric organoids, compared to infected wild-type animals or gastroids. The link between SMOX and ß-catenin activation was confirmed in human gastric organoids that were treated with a novel SMOX inhibitor. These findings indicate that SMOX promotes H. pylori-induced carcinogenesis by causing inflammation, DNA damage, and activation of ß-catenin signaling.

Prevention of Atherosclerosis by the Induction of Microbial Polyamine Production in the Intestinal Lumen.

Low molecular weight metabolites produced by the intestinal microbiome that have been associated with health and disease as metabolites need to be constantly absorbed from the intestinal lumen and transported to intestinal epithelial cells and blood. Polyamines, especially spermidine and spermine, are bioactive chemicals which promote autophagy and suppress inflammation. The main source of exogenous polyamines is the intestinal lumen, where they are produced by intestinal microbiome. Considering the intestinal microbiome as a manufacturing plant for bioactive substances, we developed a novel hybrid putrescine biosynthesis system strategy, in which the simultaneous intake of Bifidobacterium animalis ssp. lactis LKM512 (Bifal) and arginine (Arg) upregulates the production of the putrescine, a precursor of spermidine and spermine, in the gut by controlling the bacterial metabolism beyond its vast diversity and inter-individual differences. In a clinical trial, healthy individuals with a body mass index near the maximum "healthy" range (25 kg/m3; n = 44) were randomized to consume either normal yogurt containing Bifal and Arg (Bifal + Arg YG) or placebo (normal yogurt) for 12 weeks. The change in reactive hyperemia index determined by EndoPAT from week 0 to 12 in the Bifal + Arg YG group was significantly higher than that in the placebo group, indicating that Bifal + Arg YG intake improved vascular endothelial function. In addition, the concentrations of fecal putrescine and serum spermidine in the Bifal+ Arg YG group were significantly higher than those in the placebo group. These findings suggest that consuming Bifal + Arg YG prevents or reduces atherosclerosis risk by upregulating blood spermidine levels, which subsequently induces autophagy.

A tryparedoxin-coupled biosensor reveals a mitochondrial trypanothione metabolism in trypanosomes.

Trypanosomes have a trypanothione redox metabolism that provides the reducing equivalents for numerous essential processes, most being mediated by tryparedoxin (Tpx). While the biosynthesis and reduction of trypanothione are cytosolic, the molecular basis of the thiol redox homeostasis in the single mitochondrion of these parasites has remained largely unknown. Here we expressed Tpx-roGFP2, roGFP2-hGrx1 or roGFP2 in either the cytosol or mitochondrion of Trypanosoma brucei. We show that the novel Tpx-roGFP2 is a superior probe for the trypanothione redox couple and that the mitochondrial matrix harbors a trypanothione system. Inhibition of trypanothione biosynthesis by the anti-trypanosomal drug Eflornithine impairs the ability of the cytosol and mitochondrion to cope with exogenous oxidative stresses, indicating a direct link between both thiol systems. Tpx depletion abolishes the cytosolic, but only partially affects the mitochondrial sensor response to H2O2. This strongly suggests that the mitochondrion harbors some Tpx and, another, as yet unidentified, oxidoreductase.

Trypanosoma brucei are single-celled parasites that cause human sleeping sickness and animal diseases. Like in other organisms, the parasite contains different compartments, each having several specific roles. The mitochondrion is the compartment that provides most of the energy needed to keep the cell alive. Many cellular processes, such as those that happen in the mitochondrion, produce compounds including hydrogen peroxide that can cause 'oxidative damage'. To counteract this, cells make small molecules called thiols. These thiols provide 'reducing' power to chemically balance out the oxidative damage. Trypanosomes have an unusual thiol system that relies on a molecule called trypanothione. Trypanosoma brucei cells make trypanothione in the cytosol, the fluid which surrounds all cellular compartments; here it is also used up with the help of a protein called tryparedoxin. However, it was not known which thiols are present in the mitochondrion. Ebersoll et al. have now made a molecular sensor that can detect trypanothione. The sensor includes a fluorescent protein, which changes its brightness based on its oxidation state, fused to the tryparedoxin protein. This probe could either be put in the cytosol or mitochondrion of Trypanosoma brucei cells. Treating the cells with hydrogen peroxide changed the fluorescence of the biosensor. Trypanosoma brucei cells without tryparedoxin protein in their cytosol still responded to an oxidative challenge in the mitochondrion. The experiments reveal that trypanosomes do have a mitochondrial trypanothione system. This new fluorescent biosensor will be used to study how other cellular compartments deal with oxidative conditions. The tests will reveal how different compartments communicate with each other to counteract the stress. The sensor could also be used to determine how anti-parasite drugs affect the cells' trypanothione system.

Gamma-glutamylcysteine synthetase and tryparedoxin 1 exert high control on the antioxidant system in Trypanosoma cruzi contributing to drug resistance and infectivity.

Trypanothione (T(SH)2) is the main antioxidant metabolite for peroxide reduction in Trypanosoma cruzi; therefore, its metabolism has attracted attention for therapeutic intervention against Chagas disease. To validate drug targets within the T(SH)2 metabolism, the strategies and methods of Metabolic Control Analysis and kinetic modeling of the metabolic pathway were used here, to identify the steps that mainly control the pathway fluxes and which could be appropriate sites for therapeutic intervention. For that purpose, gamma-glutamylcysteine synthetase (γECS), trypanothione synthetase (TryS), trypanothione reductase (TryR) and the tryparedoxin cytosolic isoform 1 (TXN1) were separately overexpressed to different levels in T. cruzi epimastigotes and their degrees of control on the pathway flux as well as their effect on drug resistance and infectivity determined. Both experimental in vivo as well as in silico analyses indicated that γECS and TryS control T(SH)2 synthesis by 60-74% and 15-31%, respectively. γECS overexpression prompted up to a 3.5-fold increase in T(SH)2 concentration, whereas TryS overexpression did not render an increase in T(SH)2 levels as a consequence of high T(SH)2 degradation. The peroxide reduction flux was controlled for 64-73% by TXN1, 17-20% by TXNPx and 11-16% by TryR. TXN1 and TryR overexpression increased H2O2 resistance, whereas TXN1 overexpression increased resistance to the benznidazole plus buthionine sulfoximine combination. γECS overexpression led to an increase in infectivity capacity whereas that of TXN increased trypomastigote bursting. The present data suggested that inhibition of high controlling enzymes such as γECS and TXN1 in the T(SH)2 antioxidant pathway may compromise the parasite's viability and infectivity.

The Thiol-polyamine Metabolism of Trypanosoma cruzi: Molecular Targets and Drug Repurposing Strategies.

Chagas´ disease continues to be a challenging and neglected public health problem in many American countries. The etiologic agent, Trypanosoma cruzi, develops intracellularly in the mammalian host, which hinders treatment efficacy. Progress in the knowledge of parasite biology and host-pathogen interaction has not been paralleled by the development of novel, safe and effective therapeutic options. It is then urgent to seek for novel therapeutic candidates and to implement drug discovery strategies that may accelerate the discovery process. The most appealing targets for pharmacological intervention are those essential for the pathogen and, whenever possible, absent or significantly different from the host homolog. The thiol-polyamine metabolism of T. cruzi offers interesting candidates for a rational design of selective drugs. In this respect, here we critically review the state of the art of the thiolpolyamine metabolism of T. cruzi and the pharmacological potential of its components. On the other hand, drug repurposing emerged as a valid strategy to identify new biological activities for drugs in clinical use, while significantly shortening the long time and high cost associated with de novo drug discovery approaches. Thus, we also discuss the different drug repurposing strategies available with a special emphasis in their applications to the identification of drug candidates targeting essential components of the thiol-polyamine metabolism of T. cruzi.

Genome sequencing and assessment of plant growth-promoting properties of a Serratia marcescens strain isolated from vermicompost.

BACKGROUND: Plant-bacteria associations have been extensively studied for their potential in increasing crop productivity in a sustainable manner. Serratia marcescens is a species of Enterobacteriaceae found in a wide range of environments, including soil. RESULTS: Here we describe the genome sequencing and assessment of plant growth-promoting abilities of S. marcescens UENF-22GI, a strain isolated from mature cattle manure vermicompost. In vitro, S. marcescens UENF-22GI is able to solubilize P and Zn, to produce indole compounds (likely IAA), to colonize hyphae and counter the growth of two phytopathogenic fungi. Inoculation of maize with this strain remarkably increased seedling growth and biomass under greenhouse conditions. The S. marcescens UENF-22GI genome has 5 Mb, assembled in 17 scaffolds comprising 4662 genes (4528 are protein-coding). No plasmids were identified. S. marcescens UENF-22GI is phylogenetically placed within a clade comprised almost exclusively of non-clinical strains. We identified genes and operons that are likely responsible for the interesting plant-growth promoting features that were experimentally described. The S. marcescens UENF-22GI genome harbors a horizontally-transferred genomic island involved in antibiotic production, antibiotic resistance, and anti-phage defense via a novel ADP-ribosyltransferase-like protein and possible modification of DNA by a deazapurine base, which likely contributes to its competitiveness against other bacteria. CONCLUSIONS: Collectively, our results suggest that S. marcescens UENF-22GI is a strong candidate to be used in the enrichment of substrates for plant growth promotion or as part of bioinoculants for agriculture.

Engineering a spermidine biosynthetic pathway in Clostridium thermocellum results in increased resistance to furans and increased ethanol production.

Polyamines are low molecular weight aliphatic nitrogen compounds found ubiquitously in microorganisms, plants, and animals. Spermidine is a common polyamine that plays a role in stabilizing chromatin, DNA replication, transcription, translation, as well as the regulation of cell growth and apoptosis in eukaryotes. Amines are also associated with defense to a number of environmental stresses including elevated temperature and have been shown to be involved in tolerance to fermentation inhibitors such as furan derivatives and acetic acid in Saccharomyces cerevisiae. While the tolerance and detoxifying mechanisms have been intensively studied, metabolic engineering efforts to construct tolerant and resistant strains have been few. Here we show that exogenously added spermidine confers enhanced tolerance to furans and acetic acid in the Gram-positive bacterium, Clostridium thermocellum. Deletion of the endogenous spermidine synthase resulted in a severe growth defect and hypersensitivity to both furans and acetic acid. Exogenously added spermidine rescued all three phenotypes. Overexpression of the endogenous spermidine synthase resulted in increased tolerance to these compounds without added spermidine. Increased tolerance to these fermentation inhibitors will facilitate the use of C. thermocellum, one of the most cellulolytic of all known bacterial species, for the production of fuels from plant biomass substrates.

Homospermidine biosynthesis in the cyanobacterium Anabaena requires a deoxyhypusine synthase homologue and is essential for normal diazotrophic growth.

Polyamines are primordial, small organic polycations present in almost all cells, but their roles in bacteria are poorly understood. sym-Homospermidine is the dominant polyamine in the filamentous, N2 -fixing, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Synthesis of homospermidine was dependent on speA (encoding arginine decarboxylase), speB (agmatinase) and speY (deoxyhypusine synthase homologue), which in bacteria is an unprecedented pathway. Inactivation of any of these genes impaired diazotrophic growth. Heterocyst differentiation in the speA mutant was blocked at an early step, after induction of the regulatory gene hetR but before production of heterocyst-specific glycolipids (HGL). In contrast, the speY mutant produced HGL and showed slow diazotrophic growth. Analysis of fusions to green fluorescent protein revealed that SpeA (like SpeB previously described) accumulates at higher levels in vegetative cells than in heterocysts, and that SpeY accumulates in vegetative cells but also at significant levels in heterocysts. The homospermidine biosynthetic pathway is therefore active primarily in vegetative cells but the last step can be completed in heterocysts. Our findings indicate an important role for polyamines in the diazotrophic biology of Anabaena. Furthermore, inactivation of a gene cluster (potADB) encoding a polyamine ABC transporter disrupted diazotrophic growth, corroborating the importance of polyamine homeostasis in Anabaena.

Analysis of polyamine biosynthetic- and transport ability of human indigenous Bifidobacterium.

Bifidobacteria are members of the human intestinal microbiota, being numerically dominant in the colon of infants, and also being prevalent in the large intestine of adults. In this study, we measured the concentrations of major polyamines (putrescine, spermidine, and spermine) in cells and culture supernatant of 13 species of human indigenous Bifidobacterium at growing and stationary phase. Except for Bifidobacterium bifidum and Bifidobacterium gallicum, 11 species contained spermidine and/or spermine when grown in Gifu-anaerobic medium (GAM). However, Bifidobacterium scardovii and Bifidobacterium longum subsp. infantis, which contain spermidine when grown in GAM, did not contain spermidine when grown in polyamine-free 199 medium. Of the tested 13 Bifidobacterium species, 10 species showed polyamine transport ability. Combining polyamine concentration analysis in culture supernatant and in cells, with basic local alignment search tool analysis suggested that novel polyamine transporters are present in human indigenous Bifidobacterium. ABBREVIATIONS: Put: putrescine; Spd: spermidine; Spm: spermine; GAM: Gifu anaerobic medium; BHI: brain-heart infusion.

Expression and distribution patterns of spermine, spermidine, and putrescine in rat hair follicle.

No expression and distribution patterns of polyamines (PAs), spermine, spermidine, and their precursor putrescine in mammalian hair follicle are available, although polyamines are known to correlate well with hair growth and epidermal tumor genesis. Immunohistochemistry (IHC) using our original two monoclonal antibodies (mAbs) ASPM-29 specific for spermine or spermidine, and APUT-32 specific for putrescine allowed us to detect immunoreactivity for polyamines in hair follicles from normal adult rats. A wide range of immunoreactivity for the total spermine and spermidine was observed in the compartments of hair follicle: The highest degree of immunoreactivity for polyamines was observed in the matrix, in the Huxley's layer, in the deeper Henle's layer, and in the cuticle of the inner root sheath/the hair cuticle, while moderate immunoreactivity existed in the lower-to-mid cortex and the companion layer, followed by lower immunoreactivity in the outer root sheath, including the bulge region and in the deeper medulla, in which the immunoreactivity was also evident in their nuclei. In addition, somewhat surprisingly, with IHC by APUT-32 mAb, we detected significant levels of putrescine in the compartments, in which the immunostaining pattern was the closely similar to that of the total spermine and spermidine. Thus, among these compartments, the cell types of the matrix, the Huxley's layer, the deeper Henle's layer, and the cuticle of the inner root sheath/the hair cuticle seem to have the biologically higher potential in compartments of anagen hair follicle, maybe suggesting that they are involved more critically in the biological event of hair growth. In addition, we noted sharp differences of immunostaining by IHCs between ASPM-29 mAb and APUT-32 mAb in the epidermis cells and fibroblast. ASPM-29 mAb resulted in strong staining in both the cell types, but APUT-32 mAb showed only very light staining in both types. Consequently, the use of the two IHCs could be extremely useful in further studies on hair cycle and epidermal tumor genesis experimentally or clinically.

Determination of S-Adenosylmethionine Decarboxylase Activity in Plants.

The synthesis of spermidine, spermine and thermospermine requires the addition of aminopropyl groups from decarboxylated S-adenosyl-methionine (dSAM). The synthesis of dSAM is catalyzed by S-adenosylmethionine decarboxylase. dSAM levels are usually low, which constitutes a rate-limiting factor in the synthesis of polyamines. In this chapter, we provide a protocol for the determination of SAMDC activity in plants through the detection of radiolabelled CO2 released during the SAMDC reaction.

Buthionine sulfoximine is a multitarget inhibitor of trypanothione synthesis in Trypanosoma cruzi.

Buthionine sulfoximine (BSO) induces decreased glutathione (GSH) and trypanothione [T(SH)2 ] pools in trypanosomatids, presumably because only gamma-glutamylcysteine synthetase (γECS) is blocked. However, some BSO effects cannot be explained by exclusive γECS inhibition; therefore, its effect on the T(SH)2 metabolism pathway in Trypanosoma cruzi was re-examined. Parasites exposed to BSO did not synthesize T(SH)2 even when supplemented with cysteine or GSH, suggesting trypanothione synthetase (TryS) inhibition by BSO. Indeed, recombinant γECS and TryS, but not GSH synthetase, were inhibited by BSO and kinetics and docking analyses on a TcTryS 3D model suggested BSO binding at the GSH site. Furthermore, parasites overexpressing γECS and TryS showed ~ 50% decreased activities after BSO treatment. These results indicated that BSO is also an inhibitor of TryS.

Relative contributions of norspermidine synthesis and signaling pathways to the regulation of Vibrio cholerae biofilm formation.

The polyamine norspermidine is one of the major polyamines synthesized by Vibrionales and has also been found in various aquatic organisms. Norspermidine is among the environmental signals that positively regulate Vibrio cholerae biofilm formation. The NspS/MbaA signaling complex detects extracellular norspermidine and mediates the response to this polyamine. Norspermidine binding to the NspS periplasmic binding protein is thought to inhibit the phosphodiesterase activity of MbaA, increasing levels of the biofilm-promoting second messenger cyclic diguanylate monophosphate, thus enhancing biofilm formation. V. cholerae can also synthesize norspermidine using the enzyme NspC as well as import it from the environment. Deletion of the nspC gene was shown to reduce accumulation of bacteria in biofilms, leading to the conclusion that intracellular norspermidine is also a positive regulator of biofilm formation. Because V. cholerae uses norspermidine to synthesize the siderophore vibriobactin it is possible that intracellular norspermidine is required to obtain sufficient amounts of iron, which is also necessary for robust biofilm formation. The objective of this study was to assess the relative contributions of intracellular and extracellular norspermidine to the regulation of biofilm formation in V. cholerae. We show the biofilm defect of norspermidine synthesis mutants does not result from an inability to produce vibriobactin as vibriobactin synthesis mutants do not have diminished biofilm forming abilities. Furthermore, our work shows that extracellular, but not intracellular norspermidine, is mainly responsible for promoting biofilm formation. We establish that the NspS/MbaA signaling complex is the dominant mediator of biofilm formation in response to extracellular norspermidine, rather than norspermidine synthesized by NspC or imported into the cell.

Spermidine rescues proximal tubular cells from oxidative stress and necrosis after ischemic acute kidney injury.

Kidney ischemia and reperfusion injury (IRI) is associated with a high mortality rate, which is attributed to tubular oxidative stress and necrosis; however, an effective approach to limit IRI remains elusive. Spermidine, a naturally occurring polyamine, protects yeast cells against aging through the inhibition of oxidative stress and necrosis. In the present study, spermidine supplementation markedly attenuated increases in plasma creatinine concentration and tubular injury score after IRI. In addition, exogenous spermidine potently inhibited oxidative stress, especially lipid peroxidation after IRI in kidneys and exposure to hydrogen peroxide in kidney proximal tubular cells, suppressing plasma membrane disruption and necrosis. Consistent with spermidine supplementation, upregulation of ornithine decarboxylase (ODC) in human kidney proximal tubular cells significantly diminished lipid peroxidation and necrosis induced by hydrogen peroxide-induced injury. Conversely, ODC deficiency significantly enhanced lipid peroxidation and necrosis after exposure to hydrogen peroxide. Finally, small interfering RNA-mediated ODC inhibition induced functional and histological damage in kidneys as well as it increased lipid hydroperoxide levels after IRI. In conclusion, these data suggest that spermidine level determines kidney proximal tubular damage through oxidative stress and necrosis induced by IRI, and this finding provides a novel target for prevention of tubular damage induced by IRI.

[Low-molecular-weight regulators of biogenic polyamine metabolism affect cytokine production and expression of hepatitis С virus proteins in Huh7.5 human hepatocarcinoma cells].

Hepatitis C virus (HCV) induces the expression of the genes of proinflammatory cytokines, the excessive production of which may cause cell death, and contribute to development of liver fibrosis and hepatocarcinoma. The relationship between cytokine production and metabolic disorders in HCV-infected cells remains obscure. The levels of biogenic polyamines, spermine, spermidine, and their precursor putrescine, may be a potential regulator of these processes. The purpose of the present work was to study the effects of the compounds which modulate biogenic polyamines metabolism on cytokine production and HCV proteins expression. Human hepatocarcinoma Huh7.5 cells have been transfected with the plasmids that encode HCV proteins and further incubated with the following low-molecular compounds that affect different stages of polyamine metabolism: (1) difluoromethylornithine (DFMO), the inhibitor of ornithine decarboxylase, the enzyme that catalyzes the biosynthesis of polyamines; (2) N,N'-bis(2,3-butane dienyl)-1,4-diaminobutane (MDL72.527), the inhibitor of proteins involved in polyamine degradation; and (3) synthetic polyamine analog N^(I),N^(II)-diethylnorspermine (DENSpm), an inducer of polyamine degradation enzyme. The intracellular accumulation and secretion of cytokines (IL-6, IL-1ß, TNF-α, and TGF-ß) was assessed by immunocytochemistry and in the immunoenzyme assay, while the cytokine gene expression was studied using reverse transcription and PCR. The effects of the compounds under analysis on the expression of HCV proteins were analyzed using the indirect immunofluorescence with anti-HCV monoclonal antibodies. It has been demonstrated that, in cells transfected with HCV genes, DFMO reduces the production of three out of four tested cytokines, namely, TNF-α and TGF-ß in cells that express HCV core, Е1Е2, NS3, NS5A, and NS5B proteins, and IL-1ß in the cells that express HCV core, Е1Е2, and NS3 proteins. MDL72527 and DENSpm decreased cytokine production to a lesser extent. Incubation with DFMO led to a 28-32% decrease in the number of cells expressing NS5B or NS5A, both of which are key components of the HCV replication complex. The results obtained in the work indicate that a further detailed study of the antiviral activity of DFMO is required in order to assess its potential as an anti-hepatitis C therapeutic agent.