A search for acrolein scavengers among food components.

Brain stroke is a major cause of being bedridden for elderly people, and preventing stroke is important for maintaining quality of life (QOL). Acrolein is a highly reactive aldehyde and causes tissue damage during stroke. Decreasing acrolein toxicity ameliorates tissue injury during brain stroke. In this study, we tried to identify food components which decrease acrolein toxicity. We found that 2-furanmethanethiol, cysteine methyl and ethyl esters, alliin, lysine and taurine decreased acrolein toxicity. These compounds neutralized acrolein by direct interaction. However, the interaction between acrolein and taurine was not so strong. Approximately 30 mM taurine was necessary to interact with 10 µM acrolein, and 2 g/kg taurine was necessary to decrease the size of mouse brain infarction. Taurine also slightly increased polyamine contents, which are involved in decrease in the acrolein toxicity. Mitochondrial potential damage by acrolein was also protected by taurine. Our results indicate that daily intake of foods containing 2-furanmethanethiol, cysteine methyl and ethyl esters, alliin, lysine and taurine may prevent severe injury in brain stroke and improve the quality of life for elderly people.

Transient Receptor Potential Ankyrin 1 (TRPA1) Channel Mediates Acrolein Cytotoxicity in Human Lung Cancer Cells.

Transient receptor potential ankyrin 1 (TRPA1) is a nonselective ion channel implicated in thermosensation and inflammatory pain. It has been reported that expression of the TRPA1 channel is induced by cigarette smoke extract. Acrolein found in cigarette smoke is highly toxic and known as an agonist of the TRPA1 channel. However, the role of TRPA1 in the cytotoxicity of acrolein remains unclear. Here, we investigated whether the TRPA1 channel is involved in the cytotoxicity of acrolein in human lung cancer A549 cells. The IC50 of acrolein in A549 cells was 25 µM, and acrolein toxicity increased in a concentration- and time-dependent manner. When the effect of acrolein on TRPA1 expression was examined, the expression of TRPA1 in A549 cells was increased by treatment with 50 µM acrolein for 24 h or 500 µM acrolein for 30 min. AP-1, a transcription factor, was activated in the cells treated with 50 µM acrolein for 24 h, while induction of NF-κB and HIF-1α was observed in the cells treated with 500 µM acrolein for 30 min. These results suggest that acrolein induces TRPA1 expression by activating these transcription factors. Overexpression of TRPA1 in A549 cells increased acrolein sensitivity and the level of protein-conjugated acrolein (PC-Acro), while knockdown of TRPA1 in A549 cells or treatment with a TRPA1 antagonist caused tolerance to acrolein. These findings suggest that acrolein induces the TRPA1 channel and that an increase in TRPA1 expression promotes the cytotoxicity of acrolein.

Alkaline Stress Causes Changes in Polyamine Biosynthesis in Thermus thermophilus.

An extreme thermophile, Thermus thermophilus, produces 16 different polyamines including long-chain and branched-chain polyamines. The composition and content of polyamines in the thermophile cells change not only with growth temperature but also with pH changes. In particular, cell growth decreased greatly at alkaline medium together with significant changes in the composition and content of polyamines. The amounts of tetraamines (spermine and its homologs) markedly decreased at alkaline pH. Thus, we knocked out the speE gene, which is involved in the biosynthesis of tetraamines, and changes of composition of polyamines with pH changes in the mutant cells were studied. Cell growth in the ΔspeE strain was decreased compared with that of the wild-type strain for all pHs, suggesting that tetraamines are important for cell proliferation. Interestingly, the amount of spermidine decreased and that of putrescine increased in wild-type cells at elevated pH, although T. thermophilus lacks a putrescine synthesizing pathway. In addition, polyamines possessing a diaminobutane moiety, such as spermine, decreased greatly at high pH. We assessed whether the speB gene encoding aminopropylagmatine ureohydrolase (TtSpeB) is directly involved in the synthesis of putrescine. The catalytic assay of the purified enzyme indicated that TtSpeB accepts agmatine as its substrate and produces putrescine due to the change in substrate specificity at high pH. These results suggest that pH stress was exacerbated upon intracellular depletion of polyamines possessing a diaminobutane moiety induced by unusual changes in polyamine biosynthesis under high pH conditions.

Polyamines regulate gene expression by stimulating translation of histone acetyltransferase mRNAs.

Polyamines regulate gene expression in Escherichia coli by translationally stimulating mRNAs encoding global transcription factors. In this study, we focused on histone acetylation, one of the mechanisms of epigenetic regulation of gene expression, to attempt to clarify the role of polyamines in the regulation of gene expression in eukaryotes. We found that activities of histone acetyltransferases in both the nucleus and cytoplasm decreased significantly in polyamine-reduced mouse mammary carcinoma FM3A cells. Although protein levels of histones H3 and H4 did not change in control and polyamine-reduced cells, acetylation of histones H3 and H4 was greatly decreased in the polyamine-reduced cells. Next, we used control and polyamine-reduced cells to identify histone acetyltransferases whose synthesis is stimulated by polyamines. We found that polyamines stimulate the translation of histone acetyltransferases GCN5 and HAT1. Accordingly, GCN5- and HAT1-catalyzed acetylation of specific lysine residues on histones H3 and H4 was stimulated by polyamines. Consistent with these findings, transcription of genes required for cell proliferation was enhanced by polyamines. These results indicate that polyamines regulate gene expression by enhancing the expression of the histone acetyltransferases GCN5 and HAT1 at the level of translation. Mechanistically, polyamines enhanced the interaction of microRNA-7648-5p (miR-7648-5p) with the 5'-UTR of GCN5 mRNA, resulting in stimulation of translation due to the destabilization of the double-stranded RNA (dsRNA) between the 5'-UTR and the ORF of GCN5 mRNA. Because HAT1 mRNA has a short 5'-UTR, polyamines may enhance initiation complex formation directly on this mRNA.

Ischemic stroke disrupts the endothelial glycocalyx through activation of proHPSE via acrolein exposure.

Infiltration of peripheral immune cells after blood-brain barrier dysfunction causes severe inflammation after a stroke. Although the endothelial glycocalyx, a network of membrane-bound glycoproteins and proteoglycans that covers the lumen of endothelial cells, functions as a barrier to circulating cells, the relationship between stroke severity and glycocalyx dysfunction remains unclear. In this study, glycosaminoglycans, a component of the endothelial glycocalyx, were studied in the context of ischemic stroke using a photochemically induced thrombosis mouse model. Decreased levels of heparan sulfate and chondroitin sulfate and increased activity of hyaluronidase 1 and heparanase (HPSE) were observed in ischemic brain tissues. HPSE expression in cerebral vessels increased after stroke onset and infarct volume greatly decreased after co-administration of N-acetylcysteine + glycosaminoglycan oligosaccharides as compared with N-acetylcysteine administration alone. These results suggest that the endothelial glycocalyx was injured after the onset of stroke. Interestingly, scission activity of proHPSE produced by immortalized endothelial cells and HEK293 cells transfected with hHPSE1 cDNA were activated by acrolein (ACR) exposure. We identified the ACR-modified amino acid residues of proHPSE using nano LC-MS/MS, suggesting that ACR modification of Lys139 (6-kDa linker), Lys107, and Lys161, located in the immediate vicinity of the 6-kDa linker, at least in part is attributed to the activation of proHPSE. Because proHPSE, but not HPSE, localizes outside cells by binding with heparan sulfate proteoglycans, ACR-modified proHPSE represents a promising target to protect the endothelial glycocalyx.

Functional roles of polyamines and their metabolite acrolein in eukaryotic cells.

Among low molecular weight substances, polyamines (spermidine, spermine and their precursor putrescine) are present in eukaryotic cells at the mM level together with ATP and glutathione. It is expected therefore that polyamines play important roles in cell proliferation and viability. Polyamines mainly exist as a polyamine-RNA complex and regulate protein synthesis. It was found that polyamines enhance translation from inefficient mRNAs. The detailed mechanisms of polyamine stimulation of specific kinds of protein syntheses and the physiological functions of these proteins are described in this review. Spermine is metabolized into acrolein (CH2 = CH-CHO) and hydrogen peroxide (H2O2) by spermine oxidase. Although it is thought that cell damage is mainly caused by reactive oxygen species (O2-, H2O2, and •OH), it was found that acrolein is much more toxic than H2O2. Accordingly, the level of acrolein produced becomes a useful biomarker for several tissue-damage diseases like brain stroke. Thus, the mechanisms of cell toxicity caused by acrolein are described in this review.

Translational Regulation of Clock Genes BMAL1 and REV-ERBα by Polyamines.

Polyamines stimulate the synthesis of specific proteins at the level of translation, and the genes encoding these proteins are termed as the "polyamine modulon". The circadian clock generates daily rhythms in mammalian physiology and behavior. We investigated the role of polyamines in the circadian rhythm using control and polyamine-reduced NIH3T3 cells. The intracellular polyamines exhibited a rhythm with a period of about 24 h. In the polyamine-reduced NIH3T3 cells, the circadian period of circadian clock genes was lengthened and the synthesis of BMAL1 and REV-ERBα was significantly reduced at the translation level. Thus, the mechanism of polyamine stimulation of these protein syntheses was analyzed using NIH3T3 cells transiently transfected with genes encoding enhanced green fluorescent protein (EGFP) fusion mRNA with normal or mutated 5'-untranslated region (5'-UTR) of Bmal1 or Rev-erbα mRNA. It was found that polyamines stimulated BMAL1 and REV-ERBα synthesis through the enhancement of ribosomal shunting during the ribosome shunting within the 5'-UTR of mRNAs. Accordingly, the genes encoding Bmal1 and Rev-erbα were identified as the members of "polyamine modulon", and these two proteins are significantly involved in the circadian rhythm control.

Plasma acrolein level in rheumatoid arthritis increases independently of the disease characteristics.

OBJECTIVE: This study aimed to clarify whether plasma acrolein level actually increases in rheumatoid arthritis (RA) patients, and to elucidate whether any relationship exists between the levels and the RA background variables. METHODS: Plasma levels of protein-conjugated acrolein (PC-Acro) in 84 patients (RA group) and 298 normal individuals (Control group) were measured by enzyme-linked immunosorbent assay procedures. The data were statistically analyzed with Wilcoxon rank-sum test, multiple logistic regression analyses and Spearman's rank correlation coefficient. RESULTS: The RA group showed significantly higher PC-Acro levels than the Control group (median [interquartile range]: 80.5 [63.2-105.2] and 65.9 [58.9-78.1] nmol/ml, respectively). Of background factors giving influence to PC-Acro level in the combination of the two groups, 'diagnosis of RA positive' indicated strong correlation to high PC-Acro level (odds ratio: 2.96; 95% confidence interval: 1.54-5.71). These increases of PC-Acro in the RA patients did not correlate to their disease duration and/or inflammatory variables: PC-Acro level could elevate even in early RA patients showing negative inflammatory findings. CONCLUSION: Plasma levels of PC-Acro increased with RA, but the levels did not correlate with RA background variables. This report provides the basis for further studies of early diagnosis of RA as well as its pathogenesis.

Assessing acrolein for determination of the severity of brain stroke, dementia, renal failure, and Sjögren's syndrome.

It was found recently that acrolein (CH2=CH-CHO), mainly produced from spermine, is more toxic than ROS (reactive oxygen species, O2-·, H2O2, and ·OH). In this review, we describe how the seriousness of brain infarction, dementia, renal failure, and SjÓ§gren's syndrome is correlated with acrolein. In brain infarction and dementia, it was possible to identify incipient patients with high sensitivity and specificity by measuring protein-conjugated acrolein (PC-Acro) in plasma together with IL-6 and CRP in brain infarction and Aß40/42 in dementia. The level of PC-Acro in plasma and saliva correlated with the seriousness of renal failure and SjÓ§gren's syndrome, respectively. Thus, development of acrolein scavenger medicines containing SH-group such as N-acetylcysteine derivatives is important to maintain QOL (quality of life) of the elderly.

A cross-sectional study on metabolic similarities and differences between inpatients with schizophrenia and those with mood disorders.

BACKGROUND: One of the main causes of death in psychiatric patients is cardiovascular diseases which are closely related with lifestyle-related diseases. Psychiatric disorders include schizophrenia and mood disorders, whose symptoms and treatment medicines are different, suggesting that they might have different metabolic disorders. Thus, we studied the differences of lifestyle-related diseases between schizophrenia and mood disorders in Japan. METHODS: This cross-sectional study was performed from 2015 to 2017. Study participants were 189 Japanese hospitalized patients (144 schizophrenia group, 45 mood disorders group) in the department of psychiatry at Kohnodai hospital. We examined physical disorders, metabolic status of glucose and lipid, estimated glomerular filtration rate (eGFR) and brain magnetic resonance imaging. We compared these data between schizophrenia and mood disorders groups using analysis of covariance or logistic regression analysis. In comparisons between inpatients with schizophrenia or mood disorders group and the standard, we quoted 'The National Health and Nutrition Survey in Japan 2015' by Ministry of Health, Labor and Welfare as the standard. RESULTS: eGFR and prevalence of smoking were significantly lower in patients with mood disorder group than those with schizophrenia group by adjustment for age. In comparisons between patients with schizophrenia group or mood disorders group and each standard, the ratio of silent brain infarction (SBI) and cerebral infarction were significantly high in both groups. Schizophrenia group showed significantly higher prevalence of diabetes, low high-density lipoprotein (HDL) cholesterolemia, metabolic syndrome and smoking than the standard. Mood disorders group had significantly high prevalence of low HDL-cholesterolemia compared with the standard. Fasting blood glucose and HbA1c were significantly higher in schizophrenia group and female mood disorders group than the standard. Female mood disorders group had significantly decreased eGFR with increased ratio of eGFR < 60 ml/min than the standard. CONCLUSIONS: Participants of both groups had increased ratio of SBI and cerebral infarction, accompanied with glucose and lipid disorders. Compared with schizophrenia group, mood disorders group showed significantly low eGFR and prevalence of smoking.

AMD1 is essential for ESC self-renewal and is translationally down-regulated on differentiation to neural precursor cells.

The gene expression networks governing embryonic stem cell (ESC) pluripotency are complex and finely regulated during differentiation toward specific lineages. We describe a new role for Amd1 (adenosyl methionine decarboxylase), a key enzyme in the polyamine synthesis pathway, in regulating both ESC self-renewal and differentiation to the neural lineage. Amd1 is highly expressed in ESCs and is translationally down-regulated by the neural precursor cell (NPC)-enriched microRNA miR-762 during NPC differentiation. Overexpression of Amd1 or addition of the polyamine spermine blocks ESC-to-NPC conversion, suggesting Amd1 must be down-regulated to decrease the levels of inhibitory spermine during differentiation. In addition, we demonstrate that high levels of Amd1 are required for maintenance of the ESC state. We show that forced overexpression of Amd1 in ESCs results in maintenance of high Myc levels and a delay in differentiation on removal of LIF. We propose that Amd1 is a major regulator of ESC self-renewal and that its essential role lies in its regulation of Myc levels within the cell.

Cytotoxic Mechanism of Excess Polyamines Functions through Translational Repression of Specific Proteins Encoded by Polyamine Modulon.

Excessive accumulation of polyamines causes cytotoxicity, including inhibition of cell growth and a decrease in viability. We investigated the mechanism of cytotoxicity caused by spermidine accumulation under various conditions using an Escherichia coli strain deficient in spermidine acetyltransferase (SAT), a key catabolic enzyme in controlling polyamine levels. Due to the excessive accumulation of polyamines by the addition of exogenous spermidine to the growth medium, cell growth and viability were markedly decreased through translational repression of specific proteins [RMF (ribosome modulation factor) and Fis (rRNA transcription factor) etc.] encoded by members of polyamine modulon, which are essential for cell growth and viability. In particular, synthesis of proteins that have unusual locations of the Shine-Dalgarno (SD) sequence in their mRNAs was inhibited. In order to elucidate the molecular mechanism of cytotoxicity by the excessive accumulation of spermidine, the spermidine-dependent structural change of the bulged-out region in the mRNA at the initiation site of the rmf mRNA was examined using NMR analysis. It was suggested that the structure of the mRNA bulged-out region is affected by excess spermidine, so the SD sequence of the rmf mRNA cannot approach initiation codon AUG.

Effects of polyamines on protein synthesis and growth of Escherichia coli.

The polyamines (PA) putrescine, spermidine, and spermine have numerous roles in the growth of both prokaryotic and eukaryotic cells. For example, it is well known that putrescine and spermidine are strongly involved in proliferation and viability of Escherichia coli cells. Studies of polyamine functions and distributions in E. coli cells have revealed that polyamines mainly exist as an RNA-polyamine complex. Polyamines stimulate the assembly of 30S ribosomal subunits and thereby increase general protein synthesis 1.5- to 2.0-fold. Moreover, these studies have shown that polyamines stimulate synthesis of 20 different proteins at the level of translation, which are strongly involved in cell growth and viability. The genes encoding these 20 different proteins were termed as the "polyamine modulon." We here review the mechanism of activation of 30S ribosomal subunits and stimulation of specific proteins. Other functions of polyamines in E. coli are also described.

MINDY1 Is a Downstream Target of the Polyamines and Promotes Embryonic Stem Cell Self-Renewal.

Embryonic stem cells have the ability to self-renew or differentiate and these processes are under tight control. We previously reported that the polyamine regulator AMD1 is critical for embryonic stem cell self-renewal. The polyamines putrescine, spermidine, and spermine are essential organic cations that play a role in a wide array of cellular processes. Here, we explore the essential role of the polyamines in the promotion of self-renewal and identify a new stem cell regulator that acts downstream of the polyamines: MINDY1. MINDY1 protein levels are high in embryonic stem cells (ESCs) and are dependent on high polyamine levels. Overexpression of MINDY1 can promote ESC self-renewal in the absence of the usually essential cytokine Leukemia Inhibitory Factor (LIF). MINDY1 protein is prenylated and this modification is required for its ability to promote self-renewal. We go on to show that Mindy1 RNA is targeted for repression by mir-710 during Neural Precursor cell differentiation. Taken together, these data demonstrate that high polyamine levels are required for ESC self-renewal and that they function, in part, through promotion of high MINDY1 levels. Stem Cells 2018;36:1170-1178.

Polyamines stimulate the CHSY1 synthesis through the unfolding of the RNA G-quadruplex at the 5'-untraslated region.

Glycosaminoglycans (GAGs), a group of structurally related acidic polysaccharides, are primarily found as glycan moieties of proteoglycans (PGs). Among these, chondroitin sulfate (CS) and dermatan sulfate, side chains of PGs, are widely distributed in animal kingdom and show structural variations, such as sulfation patterns and degree of epimerization, which are responsible for their physiological functions through interactions with growth factors, chemokines and adhesion molecules. However, structural changes in CS, particularly the ratio of 4-O-sulfation to 6-O-sulfation (4S/6S) and CS chain length that occur during the aging process, are not fully understood. We found that 4S/6S ratio and molecular weight of CS were decreased in polyamine-depleted cells. In addition, decreased levels of chondroitin synthase 1 (CHSY1) and chondroitin 4-O-sulfotransferase 2 proteins were also observed on polyamine depletion. Interestingly, the translation initiation of CHSY1 was suppressed by a highly structured sequence (positions -202 to -117 relative to the initiation codon) containing RNA G-quadruplex (G4) structures in 5'-untranslated region. The formation of the G4s was influenced by the neighboring sequences to the G4s and polyamine stimulation of CHSY1 synthesis disappeared when the formation of the G4s was inhibited by site-directed mutagenesis. These results suggest that the destabilization of G4 structures by polyamines stimulates CHSY1 synthesis and, at least in part, contribute to the maturation of CS chains.

Reduction of Spermidine Content Resulting from Inactivation of Two Arginine Decarboxylases Increases Biofilm Formation in Synechocystis sp. Strain PCC 6803.

The phototropic bacterium Synechocystis sp. strain PCC 6803 is able to adapt its morphology in order to survive in a wide range of harsh environments. Under conditions of high salinity, planktonic cells formed cell aggregates in culture. Further observations using crystal violet staining, confocal laser scanning microscopy, and field emission-scanning electron microscopy confirmed that these aggregates were Synechocystis biofilms. Polyamines have been implicated in playing a role in biofilm formation, and during salt stress the content of spermidine, the major polyamine in Synechocystis, was reduced. Two putative arginine decarboxylases, Adc1 and Adc2, in Synechocystis were heterologously expressed in Escherichia coli and purified. Adc2 had high arginine decarboxylase activity, whereas Adc1 was much less active. Disruption of the adc genes in Synechocystis resulted in decreased spermidine content and formation of biofilms even under nonstress conditions. Based on the characterization of the adc mutants, Adc2 was the major arginine decarboxylase whose activity led to inhibition of biofilm formation, and Adc1 contributed only minimally to the process of polyamine synthesis. Taken together, in Synechocystis the shift from planktonic lifestyle to biofilm formation was correlated with a decrease in intracellular polyamine content, which is the inverse relationship of what was previously reported in heterotroph bacteria.IMPORTANCE There are many reports concerning biofilm formation in heterotrophic bacteria. In contrast, studies on biofilm formation in cyanobacteria are scarce. Here, we report on the induction of biofilm formation by salt stress in the model phototrophic bacterium Synechocystis sp. strain PCC 6803. Two arginine decarboxylases (Adc1 and Adc2) possess function in the polyamine synthesis pathway. Inactivation of the adc1 and adc2 genes leads to biofilm formation even in the absence of salt. The shift from planktonic culture to biofilm formation is regulated by a decrease in spermidine content in Synechocystis This negative correlation between biofilm formation and polyamine content, which is the opposite of the relationship reported in other bacteria, is important not only in autotrophic but also in heterotrophic bacteria.

Protective Effects of Brain Infarction by N-Acetylcysteine Derivatives.

BACKGROUND AND PURPOSE: We recently found that acrolein (CH2=CH-CHO) is more strongly involved in brain infarction compared with reactive oxygen species. In this study, we looked for acrolein scavengers with less side effects. METHODS: Photochemically induced thrombosis model mice were prepared by injection of Rose Bengal. Effects of N-acetylcysteine (NAC) derivatives on brain infarction were evaluated using the public domain National Institutes of Health image program. RESULTS: NAC, NAC ethyl ester, and NAC benzyl ester (150 mg/kg) were administered intraperitoneally at the time of induction of ischemia, or these NAC derivatives (50 mg/kg) were administered 3× at 24-h intervals before induction of ischemia and 1 more administration at the time of induction of ischemia. The size of brain infarction decreased in the order NAC benzyl ester>NAC ethyl ester>NAC in both experimental conditions. Detoxification of acrolein occurred through conjugation of acrolein with glutathione, which was catalyzed by glutathione S-transferases, rather than direct conjugation between acrolein and NAC derivatives. The level of glutathione S-transferases at the locus of brain infarction was in the order of administration of NAC benzyl ester>NAC ethyl ester>NAC>no NAC derivatives, suggesting that NAC derivatives stabilize glutathione S-transferases. CONCLUSIONS: The results indicate that detoxification of acrolein by NAC derivatives is caused through glutathione conjugation with acrolein catalyzed by glutathione S-transferases, which can be stabilized by NAC derivatives. This is a new concept of acrolein detoxification by NAC derivatives.

Acrolein toxicity at advanced age: present and future.

It is thought that tissue damage at advanced age is mainly caused by ROS (reactive oxygen species, O2-, H2O2, and ·OH). However, it was found that acrolein (CH2=CH-CHO) is more toxic than ROS, and is mainly produced from spermine (SPM), one of the polyamines, rather than from unsaturated fatty acids. Significant amounts of SPM are present normally as SPM-ribosome complexes, and contribute to protein synthesis. However, SPM was released from ribosomes due to the degradation of ribosomal RNA by ·OH or the binding of Ca2+ to ribosomes, and acrolein was produced from free SPM by polyamine oxidases, particularly by SPM oxidase. Acrolein inactivated several proteins such as GAPDH (glycelaldehyde-3-phosphate dehydrogenase), and also stimulated MMP-9 (matrix metalloproteinase-9) activity. Acrolein-conjugated GAPDH translocated to nucleus, and caused apoptosis like nitrosylated GAPDH. Through acrolein conjugation with several proteins, acrolein causes tissue damage during brain stroke, dementia, renal failure, and primary Sjögren's syndrome. Thus, development of acrolein scavengers with less side effects is very important to maintain QOL (quality of life) of elderly people.

Design, synthesis, and evaluation of polyamine-memantine hybrids as NMDA channel blockers.

N-Methyl-d-aspartate (NMDA) receptors have been implicated in learning and memory, and may also play a central role in various conditions leading to neuronal degradation. NMDA receptor antagonists could therefore be of therapeutic benefit for a number of neurological disorders. We have designed hybrid compounds of polyamines and memantine, both of which function as NMDA channel blockers. The triamine derivative with a guanidine moiety showed more potent antagonistic activity than memantine.

A Novel Putrescine Exporter SapBCDF of Escherichia coli.

Recent research has suggested that polyamines (putrescine, spermidine, and spermine) in the intestinal tract impact the health of animals either negatively or positively. The concentration of polyamines in the intestinal tract results from the balance of uptake and export of the intestinal bacteria. However, the mechanism of polyamine export from bacterial cells to the intestinal lumen is still unclear. In Escherichia coli, PotE was previously identified as a transporter responsible for putrescine excretion in an acidic growth environment. We observed putrescine concentration in the culture supernatant was increased from 0 to 50 µm during growth of E. coli under neutral conditions. Screening for the unidentified putrescine exporter was performed using a gene knock-out collection of E. coli, and deletion of sapBCDF significantly decreased putrescine levels in the culture supernatant. Complementation of the deletion mutant with the sapBCDF genes restored putrescine levels in the culture supernatant. Additionally, the ΔsapBCDF strain did not facilitate uptake of putrescine from the culture supernatant. Quantification of stable isotope-labeled putrescine derived from stable isotope-labeled arginine supplemented in the medium revealed that SapBCDF exported putrescine from E. coli cells to the culture supernatant. It was previously reported that SapABCDF of Salmonella enterica sv. typhimurium and Haemophilus influenzae conferred resistance toantimicrobial peptides; however, the E. coli ΔsapBCDF strain did not affect resistance to antimicrobial peptide LL-37. These results strongly suggest that the natural function of the SapBCDF proteins is the export of putrescine.