Min-Kyu Kwak

Cyclic dipeptides from lactic acid bacteria inhibit proliferation of the influenza a virus

We isolated Lactobacillus plantarum LBP-K10 from the traditional Korean fermented food kimchi. When organic acids were removed, the culture filtrate of this isolate showed high antiviral activity (measured using a plaque-forming assay) against the influenza A (H3N2) virus. Two fractions that were active against influenza A virus were purified from the culture filtrate using a C18 column with high-performance liquid chromatography. These active fractions were crystallized and identified to be the cyclic dipeptides cis-cyclo (l-Leu-l-Pro) and cis-cyclo(l-Phe-l-Pro) using gas chromatography-mass spectrometry; this identification was confirmed by X-ray crystallography. These cyclic dipeptides were identified in the culture filtrate of other lactic acid bacteria, including Lactobacillus spp., Leuconostoc spp., Weissella spp., and Lactococcus lactis.

Cyclic Dipeptides from Lactic Acid Bacteria Inhibit the Proliferation of Pathogenic Fungi

Lactobacillus plantarum LBP-K10 was identified to be the most potent antifungal strain from Korean traditional fermented vegetables. The culture filtrate of this strain showed remarkable antifungal activity against Ganoderma boninense. Five fractions from the culture filtrate were observed to have an inhibitory effect against G. boninense. Also, the electron ionization and chemical ionization indicated that these compounds might be cyclic dipeptides. Of the five active fractions, two fractions showed the most significant anti-Ganoderma activity, and one of these fractions inhibited the growth of Candida albicans. These compounds were identified to be cis-cyclo(l-Val-l-Pro) and cis-cyclo(l-Phe-l-Pro), as confirmed by X-ray crystallography.

NAD+-linked alcohol dehydrogenase 1 regulates methylglyoxal concentration in Candida albicans

We purified a fraction that showed NAD+‐linked methylglyoxal dehydrogenase activity, directly catalyzing methylglyoxal oxidation to pyruvate, which was significantly increased in glutathione‐depleted Candida albicans. It also showed NADH‐linked methylglyoxal‐reducing activity. The fraction was identified as a NAD+‐linked alcohol dehydrogenase (ADH1) through mass spectrometric analyses. In ADH1‐disruptants of both the wild type and glutathione‐depleted cells, the intracellular methylglyoxal concentration increased significantly; defects in growth, differentiation, and virulence were observed; and G2‐phase arrest was induced.

Polyamines regulate cell growth and cellular methylglyoxal in high-glucose medium independently of intracellular glutathione

Polyamines can presumably inhibit protein glycation, when associated with the methylglyoxal inevitably produced during glycolysis. Herein, we hypothesized a nonenzymatic interaction between putrescine and methylglyoxal in putrescine‐deficient or ‐overexpressing Dictyostelium cells in high‐glucose medium, which can control methylglyoxal production. Putrescine was essentially required for growth rescue accompanying methylglyoxal detoxification when cells underwent growth defect and cell cycle G1‐arrest when supplemented with high glucose. Furthermore, methylglyoxal regulation by putrescine seemed to be a parallel pathway independent of the changes in cellular glutathione content in high‐glucose medium. Consequently, we suggest that Dictyostelium cells need polyamines for normal growth and cellular methylglyoxal regulation.

Candida albicans erythroascorbate peroxidase regulates intracellular methylglyoxal and reactive oxygen species independently of d‐erythroascorbic acid

Candida albicans d‐erythroascorbate peroxidase (EAPX1), which can catalyze the oxidation of d‐erythroascorbic acid (EASC) to water, was observed to be inducible in EAPX1‐deficient and EAPX1‐overexpressing cells via activity staining. EAPX1‐deficient cells have remarkably increased intracellular reactive oxygen species and methylglyoxal independent of the intracellular EASC content. The increased methylglyoxal caused EAPX1‐deficient cells to activate catalase‐peroxidase and cytochrome c peroxidase, which led to defects in cell growth, viability, mitochondrial respiration, filamentation and virulence. These findings indicate that EAPX1 mediates cell differentiation and virulence by regulating intracellular methylglyoxal along with oxidative stresses, regardless of endogenous EASC biosynthesis or alternative oxidase expression.

Schiff bases of putrescine with methylglyoxal protect from cellular damage caused by accumulation of methylglyoxal and reactive oxygen species in Dictyostelium discoideum

Polyamines protect protein glycation in cells against the advanced glycation end product precursor methylglyoxal, which is inevitably produced during glycolysis, and the enzymes that detoxify this α-ketoaldehyde have been widely studied. Nonetheless, nonenzymatic methylglyoxal-scavenging molecules have not been sufficiently studied either in vitro or in vivo. Here, we hypothesized reciprocal regulation between polyamines and methylglyoxal modeled in Dictyostelium grown in a high-glucose medium. We based our hypothesis on the reaction between putrescine and methylglyoxal in putrescine-deficient (odc-) or putrescine-overexpressing (odcoe) cells. In these strains, growth and cell cycle were found to be dependent on cellular methylglyoxal and putrescine contents. The odc- cells showed growth defects and underwent G1 phase cell cycle arrest, which was efficiently reversed by exogenous putrescine. Cellular methylglyoxal, reactive oxygen species (ROS), and glutathione levels were remarkably changed in odcoe cells and odc̄ cells. These results revealed that putrescine may act as an intracellular scavenger of methylglyoxal and ROS. Herein, we observed interactions of putrescine and methylglyoxal via formation of a Schiff base complex, by UV-vis spectroscopy, and confirmed this adduct by liquid chromatography with mass spectrometry via electrospray ionization. Schiff bases were isolated, analyzed, and predicted to have molecular masses ranging from 124 to 130. We showed that cellular putrescine-methylglyoxal Schiff bases were downregulated in proportion to the levels of endogenous or exogenous putrescine and glutathione in the odc mutants. The putrescine-methylglyoxal Schiff base affected endogenous metabolite levels. This is the first report showing that cellular methylglyoxal functions as a signaling molecule through reciprocal interactions with polyamines by forming Schiff bases.

Proline-Based Cyclic Dipeptides from Korean Fermented Vegetable Kimchi and from Leuconostoc mesenteroides LBP-K06 Have Activities against Multidrug-Resistant Bacteria

Lactobacillus plantarum and Leuconostoc mesenteroides play a prominent role as functional starters and predominant isolates in the production of various types of antimicrobial compound-containing fermented foods, especially including kimchi. In the case of the bioactive cyclic dipeptides, their racemic diastereomers inhibitory to bacteria and fungi have been suggested to come solely from Lactobacillus spp. of these strains. We previously demonstrated the antifungal and antiviral activities of proline-based cyclic dipeptides, which were fractionated from culture filtrates of Lb. plantarum LBP-K10 originated from kimchi. However, cyclic dipeptides have not been identified in the filtrates, either from cultures or fermented subject matter, driven by Ln. mesenteroides, which have been widely used as starter cultures for kimchi fermentation. Most importantly, the experimental verification of cyclic dipeptide-content changes during kimchi fermentation have also not been elucidated. Herein, the antibacterial fractions, including cyclo(Leu-Pro) and cyclo(Phe-Pro), from Ln. mesenteroides LBP-K06 culture filtrates, which exhibited a typical chromatographic retention behavior (tR), were identified by using semi-preparative high-performance liquid chromatography and gas chromatography-mass spectrometry. Based on this finding, the proline-based cyclic dipeptides, including cyclo(Ser-Pro), cyclo(Tyr-Pro), and cyclo(Leu-Pro), were additionally identified in the filtrates only when fermenting Chinese cabbage produced with Ln. mesenteroides LBP-K06 starter cultures. The detection and isolation of cyclic dipeptides solely in controlled fermented cabbage were conducted under the control of fermentation-process parameters concomitantly with strong CDP selectivity by using a two-consecutive-purification strategy. Interestingly, cyclic dipeptides in the filtrates, when using this strain as a starter, increased with fermentation time. However, no cyclic dipeptides were observed in the filtrates of other fermented products, including other types of kimchi and fermented materials of plant and animal origin. This is the first report to conclusively demonstrate evidence for the existence of antimicrobial cyclic dipeptides produced by Ln. mesenteroides in kimchi. Through filtrates from lactic acid bacterial cultures and from fermented foods, we have also proved a method of combining chromatographic fractionation and mass spectrometry-based analysis for screening cyclic dipeptide profiling, which may allow evaluation of the fermented dairy foods from a new perspective.

Candida albicans glutathione reductase downregulates Efg1-mediated cyclic AMP/protein kinase A pathway and leads to defective hyphal growth and virulence upon decreased cellular methylglyoxal content accompanied by activating alcohol dehydrogenase and glycolytic enzymes

BACKGROUND Glutathione reductase maintains the glutathione level in a reduced state. As previously demonstrated, glutathione is required for cell growth/division and its biosynthesizing-enzyme deficiency causes methylglyoxal accumulation. However, experimental evidences for reciprocal relationships between Cph1-/Efg1-mediated signaling pathway regulation and methylglyoxal production exerted by glutathione reductase on yeast morphology remain unclear. METHODS Glutathione reductase (GLR1) disruption/overexpression were performed to investigate aspects of pathological/morphological alterations in Candida albicans. These assumptions were proved by observations of cellular susceptibility to oxidants and thiols, and measurements of methylglyoxal and glutathione content in hyphal-inducing conditions mainly through the activity of GLR1-overexpressing cells. Additionally, the transcriptional/translational levels of bioenergetic enzymes and dimorphism-regulating protein kinases were examined in the strain. RESULTS The GLR1-deficient strain was non-viable when GLR1 expression under the control of a CaMAL2 promoter was conditionally repressed, despite partial rescue of growth by exogenous thiols. During filamentation, non-growing hyphal GLR1-overexpressing cells exhibited resistance against oxidants and cellular methylglyoxal was significantly decreased, which concomitantly increased expressions of genes encoding energy-generating enzymes, including fructose-1,6-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, and alcohol dehydrogenase (ADH1), with remarkable repression of Efg1-signaling cascades. CONCLUSIONS This is the first report that GLR1-triggered Efg1-mediated signal transduction repression strictly reduces dimorphic switching and virulence by maintaining the basal level of methylglyoxal following the enhanced gene expressions of glycolytic enzymes and ADH1. GENERAL SIGNIFICANCE The Efg1 downregulatory mechanism by GLR1 expression has possibilities to involve in other complex network of signal pathways. Understanding how GLR1 overexpression affects multiple signaling pathways can help identify attractive targets for antifungal drugs.

Methylglyoxal upregulates Dictyostelium discoideum slug migration by triggering glutathione reductase and methylglyoxal reductase activity

Glutathione (GSH)-deprived Dictyostelium discoideum accumulates methylglyoxal (MG) and reactive oxygen species (ROS) during vegetative growth. However, the reciprocal effects of the production and regulation of these metabolites on differentiation and cell motility are unclear. Based on the inhibitory effects of γ-glutamylcysteine synthetase (gcsA) disruption and GSH reductase (gsr) overexpression on aggregation and culmination, respectively, we overexpressed GSH-related genes encoding superoxide dismutase (Sod2), catalase (CatA), and Gcs, in D. discoideum. Wild-type KAx3 and gcsA-overexpressing (gcsAOE) slugs maintained GSH levels at levels of approximately 2.1-fold less than the reference GSH synthetase-overexpressing mutant; their GSH levels did not correlate with slug migration ability. Through prolonged KAx3 migration by treatment with MG and H2O2, we found that MG increased after the mound stage in this strain, with a 2.6-fold increase compared to early developmental stages; in contrast, ROS were maintained at high levels throughout development. While the migration-defective sod2- and catA-overexpressing mutant slugs (sod2OE and catAOE) decreased ROS levels by 50% and 53%, respectively, these slugs showed moderately decreased MG levels (36.2±5.8 and 40.7±1.6nmolg-1 cells wet weight, P<0.05) compared to the parental strain (54.2±3.5nmolg-1). Importantly, defects in the migration of gcsAOE slugs decreased MG considerably (13.8±4.2nmolg-1, P<0.01) along with a slight decrease in ROS. In contrast to the increase observed in migrating sod2OE and catAOE slugs by treatment with MG and H2O2, the migration of gcsAOE slugs appeared unaffected. This behavior was caused by MG-triggered Gsr and NADPH-linked aldolase reductase activity, suggesting that GSH biosynthesis in gcsAOE slugs is specifically used for MG-scavenging activity. This is the first report showing that MG upregulates slug migration via MG-scavenging-mediated differentiation.

Methylglyoxal synthase regulates cell elongation via alterations of cellular methylglyoxal and spermidine content in Bacillus subtilis

Methylglyoxal regulates cell division and differentiation through its interaction with polyamines. Loss of their biosynthesizing enzyme causes physiological impairment and cell elongation in eukaryotes. However, the reciprocal effects of methylglyoxal and polyamine production and its regulatory metabolic switches on morphological changes in prokaryotes have not been addressed. Here, Bacillus subtilis methylglyoxal synthase (mgsA) and polyamine biosynthesizing genes encoding arginine decarboxylase (SpeA), agmatinase (SpeB), and spermidine synthase (SpeE), were disrupted or overexpressed. Treatment of 0.2mM methylglyoxal and 1mM spermidine led to the elongation and shortening of B. subtilis wild-type cells to 12.38±3.21μm (P<0.05) and 3.24±0.73μm (P<0.01), respectively, compared to untreated cells (5.72±0.68μm). mgsA-deficient (mgsA-) and -overexpressing (mgsAOE) mutants also demonstrated cell shortening and elongation, similar to speB- and speE-deficient (speB- and speE-) and -overexpressing (speBOE and speEOE) mutants. Importantly, both mgsA-depleted speBOE and speEOE mutants (speBOE/mgsA- and speEOE/mgsA-) were drastically shortened to 24.5% and 23.8% of parental speBOE and speEOE mutants, respectively. These phenotypes were associated with reciprocal alterations of mgsA and polyamine transcripts governed by the contents of methylglyoxal and spermidine, which are involved in enzymatic or genetic metabolite-control mechanisms. Additionally, biophysically detected methylglyoxal-spermidine Schiff bases did not affect morphogenesis. Taken together, the findings indicate that methylglyoxal triggers cell elongation. Furthermore, cells with methylglyoxal accumulation commonly exhibit an elongated rod-shaped morphology through upregulation of mgsA, polyamine genes, and the global regulator spx, as well as repression of the cell division and shape regulator, FtsZ.