Yuto Kamei

MC21-A, a Bactericidal Antibiotic Produced by a New Marine Bacterium, Pseudoalteromonas phenolica sp. nov. O-BC30T, against Methicillin-Resistant Staphylococcus aureus

ABSTRACT We previously reported a new marine bacterium, Pseudoalteromonas phenolica sp. nov. O-BC30T, which produced a bactericidal antibiotic against methicillin-resistant Staphylococcus aureus (MRSA). In the present study, we purified an anti-MRSA substance (MC21-A) from the methanol extract of the cells of P. phenolica O-BC30T and analyzed its chemical structure. MC21-A was determined to be 3,3′,5,5′-tetrabromo-2,2′-biphenyldiol by spectrometric analyses. Its anti-MRSA activity against 10 clinical isolates of MRSA was comparable to that of vancomycin (MC21-A MICs, 1 to 2 μg/ml; vancomycin MICs, <0.25 to 2 μg/ml). This substance was also high active against Enterococcus serolicida, Enterococcus faecium, and Enterococcus faecalis but was less active against Streptococcus spp. A time-kill study also demonstrated that MC21-A was bactericidal and that its killing rate was much higher than that of vancomycin. The postantibiotic effect (PAE) of MC21-A against a clinical MRSA isolate, strain E 31243, was also comparable to that of vancomycin (MC21-A PAEs, 1.46 to 1.65 h; vancomycin PAEs, 0.84 to 1.43 h). However, a lysis experiment demonstrated that this substance failed to lyse MRSA cells. This substance also did not lyse human erythrocytes. A SYTOX Green staining experiment implied that this substance permeabilized the cell membrane of MRSA as its mode of action. When its activities against a hypersensitive Escherichia coli mutant (KO 1489) and wild-type strains were tested, MC21-A exhibited higher levels of activity against the former. Furthermore, MC21-A was not cytotoxic to human normal fibroblast, rat pheochromocytoma, and Vero cells at concentrations up to 50 μg/ml. These results suggest that MC21-A might be useful as a lead compound in the development of new types of anti-MRSA substances with modes of action different from those of vancomycin and teicoplanin.

Antibacterial activity of 2,4-diacetylphloroglucinol produced by Pseudomonas sp. AMSN isolated from a marine alga, against vancomycin-resistant Staphylococcus aureus

Antibacterial activity of 2,4-diacetylphloroglucinol (DAPG) was evaluated against 23 vancomycin-resistant Staphylococcus aureus (VRSA) strains isolated from several Asian and European countries, Brazil, South Africa and USA, and against vancomycin-resistant Enterococcus spp (VRE) genotypes A, B and C. DAPG was active against a wide range of VRSA isolates as well as vancomycin hetero-resistant S. aureus (h-VRSA) at MIC 4 mg/l. This substance also had moderate activity against both VRE-A and -B at MIC 8 mg/l, but not against VRE-C at up to 16 mg/l. The activity of DAPG did not directly correlate with levels of vancomycin resistance in VRSA and VRE. These results suggest that DAPG might be useful in developing new antibiotics against VRSA.

Bioactive substances produced by marine isolates of Pseudomonas.

Pseudomonas is a genus of non-fermentative gram-negative Gammaproteobacteria found both on land and in the water. Many terrestrial isolates of this genus have been studied extensively. While many produce bioactive substances, enzymes, and biosurfactants, other Pseudomonas isolates are used for biological control of plant diseases and bioremediation. In contrast, only a few marine isolates of this genus have been described that produce novel bioactive substances. The chemical structures of the bioactive substances from marine Pseudomonas are diverse, including pyroles, pseudopeptide pyrrolidinedione, phloroglucinol, phenazine, benzaldehyde, quinoline, quinolone, phenanthren, phthalate, andrimid, moiramides, zafrin and bushrin. Some of these bioactive compounds are antimicrobial agents, and dibutyl phthalate and di-(2-ethylhexyl) phthalate have been reported to be cathepsin B inhibitors. In addition to being heterogeneous in terms of their structures, the antibacterial substances produced by Pseudomonas also have diverse mechanisms of action: some affect the bacterial cell membrane, causing bacterial cell lysis, whereas others act as acetyl-CoA carboxylase and nitrous oxide synthesis inhibitors. Marine Pseudomonas spp. have been isolated from a wide range of marine environments and are a potential untapped source for medically relevant bioactive substances.

Sargachromenol, a novel nerve growth factor-potentiating substance isolated from Sargassum macrocarpum, promotes neurite outgrowth and survival via distinct signaling pathways in PC12D cells

We previously found that the methanol extract of a marine brown alga, Sargassum macrocarpum showed marked nerve growth factor (NGF)-dependent neurite outgrowth promoting activity to PC12D cells. The active substance purified was elucidated to be sargachromenol. The median effective dose (ED50) was 9 microM against PC12D cells in the presence of 10 ng/ml NGF, although it showed no neurotrophic effect on its own. Pretreatment of cells with protein kinase A (PKA) inhibitor or U0126 substantially suppressed the sargachromenol-enhanced neurite outgrowth from PC12D cells, suggesting that the activation of cyclic AMP-mediated protein kinase and mitogen-activated protein (MAP) kinase 1/2 was apparently required for the action of sargachromenol. On the other hand, sargachromenol significantly promoted the survival of neuronal PC12D cells at 0-50 ng/ml NGF in serum-free medium. Neither PKA inhibitor nor U0126 could inhibit the survival supporting effect of sargachromenol, whereas wortmannin significantly blocked the sargachromenol-induced survival supporting effect on neuronal PC12D cells, suggesting that sargachromenol rescued neuronal PC12D cells by activating phosphatidylinositol-3 kinase. These results demonstrate that sargachromenol promotes neuronal differentiation of PC12D cells and supports the survival of neuronal PC12D cells via two distinct signaling pathways.

Pheophytin a, a low molecular weight compound found in the marine brown alga Sargassum fulvellum, promotes the differentiation of PC12 cells

We identified and characterized a neurodifferentiation compound from the marine brown alga Sargassum fulvellum collected from the Japanese coastline. Several instrumental analyses revealed the compound to be pheophytin a. Pheophytin a did not itself promote neurite outgrowth of PC12 cells. However, when PC12 cells were treated with a low concentration of pheophytin a (3.9 μg/ml) in the presence of a low level of nerve growth factor (10 ng/ml), the compound produced neurite outgrowth similar to that produced by a high level of nerve growth factor (50 ng/ml). Pheophytin a also enhanced signal transduction in the mitogen‐activated protein kinase signaling pathway, which is also induced by nerve growth factor. The effect of pheophytin a on neurite outgrowth of PC12 cells was completely blocked by U0126, a representative mitogen‐activated protein kinase kinase inhibitor.

Lysis of methicillin-resistant Staphylococcus aureus by 2,4-diacetylphloroglucinol produced by Pseudomonas sp. AMSN isolated from a marine alga

Previously 2,4-diacetylphloroglucinol (DAPG) produced by Pseudomonas sp. AMSN isolated from a marine alga, had demonstrated a high level of anti-methicillin-resistant Staphylococcus aureus (MRSA) comparable with that of vancomycin. In this study, this substance had bacteriolytic activity against MRSA at 1 microg/ml as well as similar activity against Vibrio parahaemolyticus at 24 microg/ml that suggests a novel antibacterial mode of action by this substance. Heat and pH stability tests showed it to be stable at temperatures ranging from 35 to 70 degrees C and at pHs ranging from 2-7. It was not acutely toxic to mice at levels up to 100 mg/kg.

An Antifungal Protein from the Marine Bacterium Streptomyces sp. Strain AP77 Is Specific for Pythium porphyrae, a Causative Agent of Red Rot Disease in Porphyra spp.

ABSTRACT A novel antifungal protein (SAP) was found in the culture supernatant of a marine bacterium, Streptomyces sp. strain AP77, and was purified. This protein was characterized by chemical, biochemical, and biological analyses. By using gel filtration, the molecular mass of SAP was estimated to be 160 kDa. Structural analysis of SAP by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectrometry suggested that SAP is composed of three heterologous protein subunits of 41.7 kDa (SAP1), 21.7 kDa (SAP2), and 18.7 kDa (SAP3) at a molar ratio of 1:1:5 (or 1:1:6). N-terminal amino acid sequence analysis and a homology search revealed that SAP1, SAP2, and SAP3 exhibit 64.3, 68.4, and 86.7% similarity to three Streptomyces coelicolor polypeptides, puromycin resistance protein (Pur8), a conserved hypothetical protein, and bacterioferritin, respectively. The MIC of purified SAP against Pythium porphyrae was determined to be 1.6 μg/disk, whereas no inhibitory effect was observed at concentrations up to 100 μg/disk against most of the fungal and bacterial strains tested; the only exception was relatively strong antifungal activity against Pythium ultimum (MIC, 6.3 μg/disk). In vitro and in vivo toxicity tests demonstrated that SAP showed no toxicity against Porphyra yezoensis cells, human normal dermal fibroblasts, and mice at doses up to 700 μg/ml (for 24 h), 250 μg/ml (for 12 h), and 75 mg/kg (for 35 days), respectively. SAP was labile when it was subjected to a heated-air drying treatment, which is a great advantage in food production procedures. These results indicated that Streptomyces sp. strain AP77 might be useful as a gene source for safe transgenic Porphyra breeding for tolerance to Pythium infection.

Sargaquinoic acid promotes neurite outgrowth via protein kinase A and MAP kinases-mediated signaling pathways in PC12D cells.

We previously isolated a nerve growth factor (NGF)‐dependent neurite outgrowth promoting substance MC14 (sargaquinoic acid) from a marine brown alga, Sargassum macrocarpum. In the present study, the NGF‐potentiating activity of MC14 to neural differentiation of PC12D cells was investigated in detail. The treatment of cells with 3 μg/ml MC14 in the presence of 1.25–100 ng/ml NGF markedly enhanced the proportion of neurite‐bearing cells compared with the NGF‐only controls. In addition, MC14 significantly elevated the NGF‐induced specific acetylcholinesterase (AchE) activity in PC12D cells, suggesting that MC14 could morphologically and biochemically promote the differentiation of PC12D cells. The mechanism of action of MC14 was further investigated by pharmacological inhibition of several intracellular signaling molecules. Results indicated that the neurite outgrowth promoting activity of MC14 was almost completely blocked by 10 μM PD98059, suggesting that a TrkA‐dependent MAP kinases‐mediated signaling pathway may play a crucial role in modulating the effect of MC14. Besides, the MC14‐enhanced neurite outgrowth was substantially suppressed by the pretreatment with 10 ng/ml protein kinase A (PKA) inhibitor, demonstrating that the adenylate cyclase–PKA signaling cascade was also involved in the action of MC14. In contrast, a PKC inhibitor chelerythrine chloride did not inhibit the neurite outgrowth promoting activity of MC14. Altogether, these results demonstrate that MC14 enhances the neurite outgrowth by cooperating at least two separated signaling pathways, a TrkA–MAP kinases pathway and an adenylate cyclase–PKA pathway, in PC12D cells.

The novel anti-Propionibacterium acnes compound, Sargafuran, found in the marine brown alga Sargassum macrocarpum.

We screened extracts of 342 species of marine algae collected from Japanese coastlines for antibacterial activity against Propionibacterium acnes, and found a novel antibacterial compound, which we named Sargafuran, from the MeOH extract of the marine brown alga, Sargassum macrocarpum. Sargafuran has low cytotoxicity, and the MIC against P. acnes was 15 μg ml−1, showing a broad antibacterial activity against Gram-positive bacteria. A time-kill study showed that Sargafuran was bactericidal and completely killed P. acnes at 4 × MIC by lysing bacterial cells. These results suggest that Sargafuran might be useful as a lead compound to develop new types of anti-P. acnes substances and new skin care cosmetics to prevent or improve acne.

Selective cytotoxicity of marine algae extracts to several human leukemic cell lines

Extracts from 8 species of marine algae which showed selective cytotoxicity in our previous screening program, were further examined for cytotoxic spectra to five human leukemic cell lines. The extract from a red alga, Amphiroa zonata exhibited strong cytotoxicity to all human leukemic cell lines tested and murine leukemic cells L1210 at the final concentrations from 15 to 375 µg ml−1. Then the cytotoxicity was not found in normal human fibroblast HDF and murine normal cells NIH-3T3. The active extract fraction from this alga was soluble in higher polar organic solvents and water and heat-stable. The extract from a brown alga Dilophus okamurae with weak selective cytotoxic activity to L1210 cells exhibited not only strong cytotoxicity to L1210, but also to human leukemic cells, HL60 and MOLT-4 at 50 µg ml-1. While, the extract from a green alga, Cladophoropsis vaucheriaeformis with most selective cytotoxic activity, did not show cytotoxicity to any human leukemic cell lines tested at 50 µg ml-1. However, this extract showed strong cytotoxicity to two human leukemic cell lines and NIH-3T3 at 100 µg ml−1. Thus, it was considered that a red alga, Amphiroa zonata might be suitable natural source for development of anti-cancer agents without side-effect.