Intracellular Spermidine Research Articles

Polyamine-Enriched Exosomes from Leishmania donovani Drive Host Macrophage Polarization via Immunometabolism Reprogramming.

Leishmania donovani (Ld) promastigotes secrete exosomes that are crucial in host-pathogen interactions and intercellular communication by carrying parasite-specific molecules. Although the composition of cargos in Leishmania exosomes is known, the effects of the unique metabolic repertoire on immunometabolism rewiring of macrophage polarization are poorly understood. Interestingly, we found the enrichment of polyamines (PAs) such as spermidine and putrescine in the Ld-exosomes. Herein, we investigate the critical polycationic molecules and their crucial role in parasite survival. Our study shows that PA inhibition or depletion significantly impairs parasite growth and fitness, particularly in drug-resistant strains. Furthermore, we aimed to elucidate the impact of PAs-enriched Ld-exosomes on host macrophages. The data demonstrated that macrophages efficiently internalized these exosomes, leading to heightened phagocytic activity and infectivity. In addition, internalized Ld-exosomes induced M2 macrophage polarization characterized by elevated Arginase-1 expression and activity. The increased expression of the solute carrier gene (SLC3A2) and elevated intracellular spermidine levels suggest that Ld-exosomes contribute to the host PAs pool and create an anti-inflammatory milieu. These findings highlight the essential role of PAs-enriched Ld-exosomes in parasite survival and establishing a pro-parasitic environment in the host macrophage.

Effects of Spermine Synthase Deficiency in Mesenchymal Stromal Cells Are Rescued by Upstream Inhibition of Ornithine Decarboxylase.

Despite the well-known relevance of polyamines to many forms of life, little is known about how polyamines regulate osteogenesis and skeletal homeostasis. Here, we report a series of in vitro studies conducted with human-bone-marrow-derived pluripotent stromal cells (MSCs). First, we show that during osteogenic differentiation, mRNA levels of most polyamine-associated enzymes are relatively constant, except for the catabolic enzyme spermidine/spermine N1-acetyltransferase 1 (SAT1), which is strongly increased at both mRNA and protein levels. As a result, the intracellular spermidine to spermine ratio is significantly reduced during the early stages of osteoblastogenesis. Supplementation of cells with exogenous spermidine or spermine decreases matrix mineralization in a dose-dependent manner. Employing N-cyclohexyl-1,3-propanediamine (CDAP) to chemically inhibit spermine synthase (SMS), the enzyme catalyzing conversion of spermidine into spermine, also suppresses mineralization. Intriguingly, this reduced mineralization is rescued with DFMO, an inhibitor of the upstream polyamine enzyme ornithine decarboxylase (ODC1). Similarly, high concentrations of CDAP cause cytoplasmic vacuolization and alter mitochondrial function, which are also reversible with the addition of DFMO. Altogether, these studies suggest that excess polyamines, especially spermidine, negatively affect hydroxyapatite synthesis of primary MSCs, whereas inhibition of polyamine synthesis with DFMO rescues most, but not all of these defects. These findings are relevant for patients with Snyder-Robinson syndrome (SRS), as the presenting skeletal defects-associated with SMS deficiency-could potentially be ameliorated by treatment with DFMO.

Identification and Characterization of Novel Small-Molecule SMOX Inhibitors.

The major intracellular polyamines spermine and spermidine are abundant and ubiquitous compounds that are essential for cellular growth and development. Spermine catabolism is mediated by spermine oxidase (SMOX), a highly inducible flavin-dependent amine oxidase that is upregulated during excitotoxic, ischemic, and inflammatory states. In addition to the loss of radical scavenging capabilities associated with spermine depletion, the catabolism of spermine by SMOX results in the production of toxic byproducts, including H2O2 and acrolein, a highly toxic aldehyde with the ability to form adducts with DNA and inactivate vital cellular proteins. Despite extensive evidence implicating SMOX as a key enzyme contributing to secondary injury associated with multiple pathologic states, the lack of potent and selective inhibitors has significantly impeded the investigation of SMOX as a therapeutic target. In this study, we used a virtual and physical screening approach to identify and characterize a series of hit compounds with inhibitory activity against SMOX. We now report the discovery of potent and highly selective SMOX inhibitors 6 (IC50 0.54 μM, Ki 1.60 μM) and 7 (IC50 0.23 μM, Ki 0.46 μM), which are the most potent SMOX inhibitors reported to date. We hypothesize that these selective SMOX inhibitors will be useful as chemical probes to further elucidate the impact of polyamine catabolism on mechanisms of cellular injury.

Herpesvirus-induced spermidine synthesis and eIF5A hypusination for viral episomal maintenance.

Spermidine is essential for cellular growth and acts as a prerequisite of hypusination, a post-translational modification of eukaryotic initiation factor 5A (eIF5A), allowing the translation of polyproline-containing proteins. Here, we show that oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) increases spermidine synthesis and eIF5A hypusination to enhance expression of polyproline-containing latency-associated nuclear antigen (LANA) for viral episomal maintenance. KSHV upregulates intracellular spermidine levels by dysregulating polyamine metabolic pathways in three-dimensional (3D) culture and 2D de novo infection conditions. Increased intracellular spermidine leads to increased eIF5A hypusination, ultimately enhancing LANA expression. In contrast, inhibition of spermidine synthesis or eIF5A hypusination alleviates LANA expression, decreasing viral episomal maintenance and KSHV-infected cell proliferation invitro and invivo, which is reversed by spermidine supplement. This demonstrates that KSHV hijacks spermidine synthesis and eIF5A hypusination pathways to enhance LANA expression for viral episomal maintenance, suggesting polyamine metabolism and eIF5A hypusination as therapeutic targets for KSHV-induced tumorigenesis.

A link between urease and polyamine metabolism in Cryptococcus neoformans

The urease enzyme of Cryptococcus neoformans is linked to different metabolic pathways within the yeast cell, several of which are involved in polyamine metabolism. Cryptococcal biogenic amine production is, however, largely unexplored and is yet to be investigated in relation to urease. The aim of this study was therefore to explore and compare polyamine metabolism in wild-type, urease-negative and urease-reconstituted strains of C. neoformans. Mass spectrometry analysis showed that agmatine and spermidine were the major extra- and intracellular polyamines of C. neoformans and significant differences were observed between 26 and 37 °C. In addition, compared to the wild-type, the relative percentages of extracellular putrescine and spermidine were found to be lower and agmatine higher in cultures of the urease-deficient mutant. The inverse was true for intracellular spermidine and agmatine. Cyclohexylamine was a more potent polyamine inhibitor compared to DL-α-difluoromethylornithine and inhibitory effects were more pronounced at 37 °C than at 26 °C. At both temperatures, the urease-deficient mutant was less susceptible to cyclohexylamine treatment compared to the wild-type. For both inhibitors, growth inhibition was alleviated with polyamine supplementation. This study has provided novel insight into the polyamine metabolism of C. neoformans, highlighting the involvement of urease in biogenic amine production.

Evaluating the Engineered Saccharomyces cerevisiae With High Spermidine Contents for Increased Tolerance to Lactic, Succinic, and Malic Acids and Increased Xylose Fermentation

Saccharomyces cerevisiae is a promising candidate for production of organic acids as it is more tolerant to these acids than the prokaryotes. However, the large-scale production of organic acids from lignocellulosic biomass is limited by their accumulation in the growth medium and inability of xylose fermentation by S. cerevisiae. Here we showed that high intracellular spermidine (SPD) contents confers enhanced tolerance to lactic, succinic, and malic acids in S. cerevisiae. Specifically, in the presence of 20 g/L malic acid, the maximum specific growth rate and dry cell weight of a S. cerevisiae with two fold higher SPD content were 40% and 36% higher than those of the control strain. When a xylose assimilation pathway was introduced into an engineered strain with high SPD content, the resulting S. cerevisiae strain exhibited 23∼47% higher xylose consumption rate and 6∼16% higher ethanol productivity than those of the control strain during the four times of repeated-batch fermentations using a mixture of glucose and xylose as carbon sources. These results suggest that the strain developed in this study would serve as a platform strain for production of organic acids.

(R,R)-1,12-Dimethylspermine can mitigate abnormal spermidine accumulation in Snyder–Robinson syndrome

Snyder-Robinson syndrome (SRS) is an X-linked intellectual disability syndrome caused by a loss-of-function mutation in the spermine synthase (SMS) gene. Primarily affecting males, the main manifestations of SRS include osteoporosis, hypotonic stature, seizures, cognitive impairment, and developmental delay. Because there is no cure for SRS, treatment plans focus on alleviating symptoms rather than targeting the underlying causes. Biochemically, the cells of individuals with SRS accumulate excess spermidine, whereas spermine levels are reduced. We recently demonstrated that SRS patient-derived lymphoblastoid cells are capable of transporting exogenous spermine and its analogs into the cell and, in response, decreasing excess spermidine pools to normal levels. However, dietary supplementation of spermine does not appear to benefit SRS patients or mouse models. Here, we investigated the potential use of a metabolically stable spermine mimetic, (R,R)-1,12-dimethylspermine (Me2SPM), to reduce the intracellular spermidine pools of SRS patient-derived cells. Me2SPM can functionally substitute for the native polyamines in supporting cell growth while stimulating polyamine homeostatic control mechanisms. We found that both lymphoblasts and fibroblasts from SRS patients can accumulate Me2SPM, resulting in significantly decreased spermidine levels with no adverse effects on growth. Me2SPM administration to mice revealed that Me2SPM significantly decreases spermidine levels in multiple tissues. Importantly, Me2SPM was detectable in brain tissue, the organ most affected in SRS, and was associated with changes in polyamine metabolic enzymes. These findings indicate that the (R,R)-diastereomer of 1,12-Me2SPM represents a promising lead compound in developing a treatment aimed at targeting the molecular mechanisms underlying SRS pathology.

Analysis of crystalline and solution states of ligand-free spermidine N-acetyltransferase (SpeG) from Escherichia coli.

Spermidine N-acetyltransferase (SpeG) transfers an acetyl group from acetyl-coenzyme A to an N-terminal amino group of intracellular spermidine. This acetylation inactivates spermidine, reducing the polyamine toxicity that tends to occur under certain chemical and physical stresses. The structure of the SpeG protein from Vibrio cholerae has been characterized: while the monomer possesses a structural fold similar to those of other Gcn5-related N-acetyltransferase superfamily members, its dodecameric structure remains exceptional. In this paper, structural analyses of SpeG isolated from Escherichia coli are described. Like V. cholerae SpeG, E.coli SpeG forms dodecamers, as revealed by two crystal structures of the ligand-free E. coli SpeG dodecamer determined at 1.75 and 2.9 Å resolution. Although both V. cholerae SpeG and E.coli SpeG can adopt an asymmetric open dodecameric state, solution analysis showed that the oligomeric composition of ligand-free E. coli SpeG differs from that of ligand-free V.cholerae SpeG. Based on these data, it is proposed that the equilibrium balance of SpeG oligomers in the absence of ligands differs from one species to another and thus might be important for SpeG function.

Novel Synthetic Polyamines Have Potent Antimalarial Activities in vitro and in vivo by Decreasing Intracellular Spermidine and Spermine Concentrations.

Twenty-two compounds belonging to several classes of polyamine analogs have been examined for their ability to inhibit the growth of the human malaria parasite Plasmodium falciparum in vitro and in vivo. Four lead compounds from the thiourea sub-series and one compound from the urea-based analogs were found to be potent inhibitors of both chloroquine-resistant (Dd2) and chloroquine-sensitive (3D7) strains of Plasmodium with IC50 values ranging from 150 to 460 nM. In addition, the compound RHW, N1,N7-bis (3-(cyclohexylmethylamino) propyl) heptane-1,7-diamine tetrabromide was found to inhibit Dd2 with an IC50 of 200 nM. When RHW was administered to P. yoelii-infected mice at 35 mg/kg for 4 days, it significantly reduced parasitemia. RHW was also assayed in combination with the ornithine decarboxylase inhibitor difluoromethylornithine, and the two drugs were found not to have synergistic antimalarial activity. Furthermore, these inhibitors led to decreased cellular spermidine and spermine levels in P. falciparum, suggesting that they exert their antimalarial activities by inhibition of spermidine synthase.

Familial Sinus Node Disease Caused by a Gain of GIRK (G-Protein Activated Inwardly Rectifying K+ Channel) Channel Function.

Inherited forms of sinus node dysfunction (SND) clinically include bradycardia, sinus arrest, and chronotropic incompetence and may serve as disease models to understand sinus node physiology and impulse generation. Recently, a gain-of-function mutation in the G-protein gene GNB2 led to enhanced activation of the GIRK (G-protein activated inwardly rectifying K+ channel). Thus, human cardiac GIRK channels are important for heart rate regulation and subsequently, genes encoding their subunits Kir3.1 and Kir3.4 ( KCNJ3 and KCNJ5) are potential candidates for inherited SND in human. We performed a combined approach of targeted sequencing of KCNJ3 and KCNJ5 in 52 patients with idiopathic SND and subsequent whole exome sequencing of additional family members in a genetically affected patient. A putative novel disease-associated gene variant was functionally analyzed by voltage-clamp experiments using various heterologous cell expression systems (Xenopus oocytes, CHO cells, and rat atrial cardiomyocytes). In a 3-generation family with SND we identified a novel variant in KCNJ5 which leads to an amino acid substitution (p.Trp101Cys) in the first transmembrane domain of the Kir3.4 subunit of the cardiac GIRK channel. The identified variant cosegregated with the disease in the family and was absent in the Exome Variant Server and Exome Aggregation Consortium databases. Expression of mutant Kir3.4 (±native Kir3.1) in different heterologous cell expression systems resulted in increased GIRK currents ( IK,ACh) and a reduced inward rectification which was not compensated by intracellular spermidine. Moreover, in silico modeling of heterotetrameric mutant GIRK channels indicates a structurally altered binding site for spermine. For the first time, an inherited gain-of-function mutation in the human GIRK3.4 causes familial human SND. The increased activity of GIRK channels is likely to lead to a sustained hyperpolarization of pacemaker cells and thereby reduces heart rate. Modulation of human GIRK channels may pave a way for further treatment of cardiac pacemaking.

Polyamine Biosynthetic Pathway as a Drug Target for Osteosarcoma Therapy.

Osteosarcoma (OS) is the most common bone tumor in children. Polyamines (PAs) are ubiquitous cations involved in many cell processes including tumor development, invasion and metastasis. In other pediatric cancer models, inhibition of the PA biosynthesis pathway with ornithine decarboxylase (ODC) inhibitor alpha-difluoromethylornithine (DFMO) results in decreased cell proliferation and differentiation. In OS, the PA pathway has not been evaluated. DFMO is an attractive, orally administered drug, is well tolerated, can be given for prolonged periods, and is already used in pediatric patients. Three OS cell lines were used to study the cellular effects of PA inhibition with DFMO: MG-63, U-2 OS and Saos-2. Effects on proliferation were analyzed by cell count, flow cytometry-based cell cycle analysis and RealTime-Glo™ MT Cell Viability assays. Intracellular PA levels were measured with high-performance liquid chromatography (HPLC). Western blot analysis was used to evaluate cell differentiation. DFMO exposure resulted in significantly decreased cell proliferation in all cell lines. After treatment, intracellular spermidine levels were drastically decreased. Cell cycle arrest at G2/M was observed in U-2 OS and Saos-2. Cell differentiation was most prominent in MG-63 and U-2 OS as determined by increases in the terminal differentiation markers osteopontin and collagen 1a1. Cell proliferation continued to be suppressed for several days after removal of DFMO. Based on our findings, DFMO is a promising new adjunct to current osteosarcoma therapy in patients at high risk of relapse, such as those with poor necrosis at resection or those with metastatic or recurrent osteosarcoma. It is a well-tolerated oral drug that is currently in phase II clinical trials in pediatric neuroblastoma patients as a maintenance therapy. The same type of regimen may also improve outcomes in osteosarcoma patients in whom there have been essentially no medical advances in the last 30 years.

Synergistic drug combination GC7/DFMO suppresses hypusine/spermidine-dependent eIF5A activation and induces apoptotic cell death in neuroblastoma.

The eukaryotic initiation factor 5A (eIF5A), which contributes to several crucial processes during protein translation, is the only protein that requires activation by a unique post-translational hypusine modification. eIF5A hypusination controls cell proliferation and has been linked to cancer. eIF5A hypusination requires the enzymes deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase and uniquely depends on the polyamine (PA) spermidine as the sole substrate. Ornithine decarboxylase (ODC) is the rate-limiting enzyme in PA biosynthesis. Both ODC and PAs control cell proliferation and are frequently dysregulated in cancer. Since only spermidine can activate eIF5A, we chose the hypusine-PA nexus as a rational target to identify new drug combinations with synergistic antiproliferative effects. We show that elevated mRNA levels of the two target enzymes DHPS and ODC correlate with poor prognosis in a large cohort of neuroblastoma (NB) tumors. The DHPS inhibitor GC7 (N1-guanyl-1,7-diaminoheptane) and the ODC inhibitor α-difluoromethylornithine (DFMO) are target-specific and in combination induced synergistic effects in NB at concentrations that were not individually cytotoxic. Strikingly, while each drug alone at higher concentrations is known to induce p21/Rb- or p27/Rb-mediated G1 cell cycle arrest, we found that the drug combination induced caspase 3/7/9, but not caspase 8-mediated apoptosis, in NB cells. Hypusinated eIF5A levels and intracellular spermidine levels correlated directly with drug treatments, signifying specific drug targeting effects. This two-pronged GC7/DFMO combination approach specifically inhibits both spermidine biosynthesis and post-translational, spermidine-dependent hypusine-eIF5A activation, offering an exciting clue for improved NB drug therapy.

Helicobacter pylori does not use spermidine synthase to produce spermidine

Helicobacter pylori is the primary pathogen associated to gastritis and gastric cancer. Growth of H. pylori depends on the availability of spermidine in vivo. Interestingly, the genome of H. pylori contains an incomplete set of genes for the classical pathway of spermidine biosynthesis. It is thus not clear whether some other genes remained in the pathway would have any functions in spermidine biosynthesis. Here, we study spermidine synthase, which is responsible for the final catalytic process in the classical route. Protein sequence alignment reveals that H. pylori SpeE (HpSpeE) lacks key residues for substrate binding. By using isothermal titration calorimetry, we show that purified recombinant HpSpeE does not interact with the putative substrates putrescine and decarboxylated S-adenosylmethionine, and the product spermidine. High performance liquid chromatography analysis further demonstrates that HpSpeE has no detectable in vitro enzymatic activity. Additionally, intracellular spermidine level in speE-null mutant strain is comparable to that in the wild type strain. Collectively, our results suggest that HpSpeE is functionally distinct from spermidine production. H. pylori may instead employ the alternative pathway for spermidine synthesis which is dominantly exploited by other human gut microbes.

Enhanced ethanol fermentation by engineered Saccharomyces cerevisiae strains with high spermidine contents.

Construction of robust and efficient yeast strains is a prerequisite for commercializing a biofuel production process. We have demonstrated that high intracellular spermidine (SPD) contents in Saccharomyces cerevisiae can lead to improved tolerance against various fermentation inhibitors, including furan derivatives and acetic acid. In this study, we examined the potential applicability of the S. cerevisiae strains with high SPD contents under two cases of ethanol fermentation: glucose fermentation in repeated-batch fermentations and xylose fermentation in the presence of fermentation inhibitors. During the sixteentimes of repeated-batch fermentations using glucose as a sole carbon source, the S. cerevisiae strains with high SPD contents maintained higher cell viability and ethanol productivities than a control strain with lower SPD contents. Specifically, at the sixteenth fermentation, the ethanol productivity of a S. cerevisiae strain with twofold higher SPD content was 31% higher than that of the control strain. When the SPD content was elevated in an engineered S. cerevisiae capable of fermenting xylose, the resulting S. cerevisiae strain exhibited much 40-50% higher ethanol productivities than the control strain during the fermentations of synthetic hydrolysate containing high concentrations of fermentation inhibitors. These results suggest that the strain engineering strategy to increase SPD content is broadly applicable for engineering yeast strains for robust and efficient production of ethanol.

Enantiomers of 3-methylspermidine selectively modulate deoxyhypusine synthesis and reveal important determinants for spermidine transport.

Eukaryotic translation initiation factor 5A (eIF5A) is essential for cell proliferation, becoming functionally active only after post-translational conversion of a specific Lys to hypusine [N(ε)-(4-amino-2-hydroxybutyl)lysine]. Deoxyhypusine synthase (DHS) is the rate-limiting enzyme of this two-step process, and the polyamine spermidine is the only natural donor of the butylamine group for this reaction, which is very conserved-hypusine biosynthesis suffers last when the intracellular spermidine pool is depleted. DHS has a very strict substrate specificity, and only a few spermidine analogs are substrates of the enzyme and can support long-term growth of spermidine-depleted cells. Herein, we compared the biological properties of earlier unknown enantiomers of 3-methylspermidine (3-MeSpd) in deoxyhypusine synthesis, in supporting cell growth and in polyamine transport. Long-term treatment of DU145 cells with α-difluoromethylornithine (inhibitor of polyamine biosynthesis) and (R)-3-MeSpd did not cause depletion of hypusinated eIF5A, and the cells were still able to grow, whereas the combination of α-difluoromethylornithine with a racemate or (S)-3-MeSpd caused cessation of cell growth. Noticeably, DHS preferred the (R)- over the (S)-enantiomer as a substrate. (R)-3-MeSpd competed with [(14)C]-labeled spermidine for cellular uptake less efficiently than the (S)-3-MeSpd (Ki = 141 μM vs 19 μM, respectively). The cells treated with racemic 3-MeSpd accumulated intracellularly mainly (S)-3-MeSpd, but not DHS substrate (R)-3-MeSpd, explaining the inability of the racemate to support long-term growth. The distinct properties of 3-MeSpd enantiomers can be exploited in designing polyamine uptake inhibitors, facilitating drug delivery and modulating deoxyhypusine synthesis.

Enhanced tolerance of Saccharomyces cerevisiae to multiple lignocellulose-derived inhibitors through modulation of spermidine contents

Fermentation inhibitors present in lignocellulose hydrolysates are inevitable obstacles for achieving economic production of biofuels and biochemicals by industrial microorganisms. Here we show that spermidine (SPD) functions as a chemical elicitor for enhanced tolerance of Saccharomyces cerevisiae against major fermentation inhibitors. In addition, the feasibility of constructing an engineered S. cerevisiae strain capable of tolerating toxic levels of the major inhibitors without exogenous addition of SPD was explored. Specifically, we altered expression levels of the genes in the SPD biosynthetic pathway. Also, OAZ1 coding for ornithine decarboxylase (ODC) antizyme and TPO1 coding for the polyamine transport protein were disrupted to increase intracellular SPD levels through alleviation of feedback inhibition on ODC and prevention of SPD excretion, respectively. Especially, the strain with combination of OAZ1 and TPO1 double disruption and overexpression of SPE3 not only contained spermidine content of 1.1mg SPD/g cell, which was 171% higher than that of the control strain, but also exhibited 60% and 33% shorter lag-phase period than that of the control strain under the medium containing furan derivatives and acetic acid, respectively. While we observed a positive correlation between intracellular SPD contents and tolerance phenotypes among the engineered strains accumulating different amounts of intracellular SPD, too much SPD accumulation is likely to cause metabolic burden. Therefore, genetic perturbations for intracellular SPD levels should be optimized in terms of metabolic burden and SPD contents to construct inhibitor tolerant yeast strains. We also found that the genes involved in purine biosynthesis and cell wall and chromatin stability were related to the enhanced tolerance phenotypes to furfural. The robust strains constructed in this study can be applied for producing chemicals and advanced biofuels from cellulosic hydrolysates.

Purifying selection against gene conversions between the polyamine transport (TPO) genes of Saccharomyces species.

Saccharomyces species have five TPO genes, TPO1 through TPO5, coding for proteins that are involved in up taking or excreting intracellular spermine, putrescine or spermidine. Here, we investigate the evolutionary fate and functional impacts of gene conversions between these genes. Our results show that gene conversions occurred only between the TPO2 and TPO3 genes of the six Saccharomyces species we studied. They also show that these gene conversions occurred independently in all six species. The facts that they only occur between closely related genes having similar function, and that they are limited to the transmembrane domain of these proteins, suggest that they have little functional impact. These gene conversions therefore likely represent neutral mutations which are not subject to purifying selection.

Spermidine, a sensor for antizyme 1 expression regulates intracellular polyamine homeostasis

Although intracellular polyamine levels are highly regulated, it is unclear whether intracellular putrescine (PUT), spermidine (SPD), or spermine (SPM) levels act as a sensor to regulate their synthesis or uptake. Polyamines have been shown to induce AZ1 expression through a unique +1 frameshifting mechanism. However, under physiological conditions which particular polyamine induces AZ1, and thereby ODC activity, is unknown due to their inter-conversion. In this study we demonstrate that SPD regulates AZ1 expression under physiological conditions in IEC-6 cells. PUT and SPD showed potent induction of AZ1 within 4h in serum-starved confluent cells grown in DMEM (control) medium. Unlike control cells, PUT failed to induce AZ1 in cells grown in DFMO containing medium; however, SPD caused a robust AZ1 induction in these cells. SPM showed very little effect on AZ1 expression in both the control and polyamine-depleted cells. Only SPD induced AZ1 when S-adenosylmethionine decarboxylase (SAMDC) and/or ODC were inhibited. Surprisingly, addition of DENSpm along with DFMO restored AZ1 induction by putrescine in polyamine-depleted cells suggesting that the increased SSAT activity in response to DENSpm converted SPM to SPD, leading to the expression of AZ1. This study shows that intracellular SPD levels controls AZ1 synthesis.

Elevated polyamines in urothelial cells from OAB subjects mediate oxotremorine-evoked rapid intracellular calcium rise and delayed acetylcholine release

Increased polyamine signaling in bladder urothelial cells (BUC) may play a role in the pathophysiology of overactive bladder (OAB). We quantitated intracellular polyamine levels in cultured BUC from OAB and asymptomatic (NB) subjects. We assessed whether polyamines modulated rapid intracellular calcium ([Ca(2+)]i) changes and delayed acetylcholine (ACh) release evoked by oxotremorine (OXO, a muscarinic agonist). BUC were cultured from cystoscopic biopsies. High-performance liquid chromatography (HPLC) quantitated intracellular putrescine, spermidine, and spermine levels. Five-millimeter difluoromethylornithine (DFMO), and one-millimeter methylglyoxalbisguanylhydrazone (MGBG) treatments were used to deplete intracellular polyamines. Ten micrometers of OXO were used to increase [Ca(2+)]i levels (measured by fura 2 microfluorimetry) and trigger extracellular ACh release (measured by ELISA). Polyamine levels were elevated in OAB compared with NB BUC (0.5 ± 0.15 vs. 0.16 ± 0.03 nmol/mg for putrescine, 2.4 ± 0.21 vs. 1.01 ± 0.13 nmol/mg for spermidine, and 1.90 ± 0.27 vs. 0.86 ± 0.26 nmol/mg for spermine; P < 0.05 for all comparisons). OXO evoked greater [Ca(2+)]i rise in OAB (205.10 ± 18.82% increase over baseline) compared with in NB BUC (119.54 ± 13.01%; P < 0.05). After polyamine depletion, OXO evoked [Ca(2+)]i rise decreased in OAB and NB BUC to 43.40 ± 6.45 and 38.82 ± 3.5%, respectively. OXO tended to increase ACh release by OAB vs. NB BUC (9.02 ± 0.1 vs. 7.04 ± 0.09 μM, respectively; P < 0.05). Polyamine depletion reduced ACh release by both OAB and NB BUC. In conclusion, polyamine levels were elevated twofold in OAB BUC. OXO evoked greater increase in [Ca(2+)]i and ACh release in OAB BUC, although these two events may be unrelated. Depletion of polyamines caused OAB BUC to behave similarly to NB BUC.

A New Piece of the Shigella Pathogenicity Puzzle: Spermidine Accumulationby Silencing of the speG Gene

The genome of Shigella, a gram negative bacterium which is the causative agent of bacillary dysentery, shares strong homologies with that of its commensal ancestor, Escherichia coli. The acquisition, by lateral gene transfer, of a large plasmid carrying virulence determinants has been a crucial event in the evolution towards the pathogenic lifestyle and has been paralleled by the occurrence of mutations affecting genes, which negatively interfere with the expression of virulence factors. In this context, we have analysed to what extent the presence of the plasmid-encoded virF gene, the major activator of the Shigella regulon for invasive phenotype, has modified the transcriptional profile of E. coli. Combining results from transcriptome assays and comparative genome analyses we show that in E. coli VirF, besides being able to up-regulate several chromosomal genes, which potentially influence bacterial fitness within the host, also activates genes which have been lost by Shigella. We have focused our attention on the speG gene, which encodes spermidine acetyltransferase, an enzyme catalysing the conversion of spermidine into the physiologically inert acetylspermidine, since recent evidence stresses the involvement of polyamines in microbial pathogenesis. Through identification of diverse mutations, which prevent expression of a functional SpeG protein, we show that the speG gene has been silenced by convergent evolution and that its inactivation causes the marked increase of intracellular spermidine in all Shigella spp. This enhances the survival of Shigella under oxidative stress and allows it to better face the adverse conditions it encounters inside macrophage. This is supported by the outcome of infection assays performed in mouse peritoneal macrophages and of a competitive-infection assay on J774 macrophage cell culture. Our observations fully support the pathoadaptive nature of speG inactivation in Shigella and reveal that the accumulation of spermidine is a key determinant in the pathogenicity strategy adopted by this microrganism.