Akira Ogita

Amplification of Vacuole-targeting Fungicidal Activity of Antibacterial Antibiotic Polymyxin B by Allicin, an Allyl Sulfur Compound from Garlic

A cationic antibacterial peptide, polymyxin B (PMB), was evaluated as an antifungal antibiotic against various yeasts and filamentous fungi when used in combination with allicin, an allyl sulfur compound from garlic. Allicin was not lethal but could markedly amplify the fungicidal activity of PMB, which was weakly detected with the increase in the plasma membrane permeability in Saccharomyces cerevisiae. Their combined actions caused a dynamic structural damage to the yeast vacuole as judged by the disappearance of its swollen spherical architecture. The vacuole-targeting activity of PMB was similarly amplified in medium with t-butyl hydroperoxide as a substitute for the action of allicin. These findings suggest that the allicin-mediated lipoperoxide production in fungal plasma membrane is the cause of the enhancement in the cellular uptake of PMB as well as its action against the vacuole.

Antifungal thiopeptide cyclothiazomycin B1 exhibits growth inhibition accompanying morphological changes via binding to fungal cell wall chitin.

Cyclothiazomycin B1 (CTB1) is an antifungal cyclic thiopeptide isolated from the culture broth of Streptomyces sp. HA 125-40. CTB1 inhibited the growth of several filamentous fungi including plant pathogens along with swelling of hyphae and spores. The antifungal activity of CTB1 was weakened by hyperosmotic conditions, and hyphae treated with CTB1 burst under hypoosmotic conditions, indicating increased cell wall fragility. CTB1-sensitive fungal species contain high levels of cell wall chitin and/or chitosan. Unlike nikkomycin Z, a competitive inhibitor of chitin synthase (CHS), CTB1 did not inhibit CHS activity. Although CTB1 inhibited CHS biosynthesis, the same result was also obtained with a non-specific proteins inhibitor, cycloheximide, which did not reduce cell wall rigidity. These results indicate that the primary target of CTB1 is not CHS, and we concluded that CTB1 antifungal activity was independent of this sole inhibition. We found that CTB1 bound to chitin but did not bind to β-glucan and chitosan. The results of the present study suggest that CTB1 induces cell wall fragility by binding to chitin, which forms the fungal cell wall. The antifungal activity of CTB1 could be explained by this chitin-binding ability.

The vacuole-targeting fungicidal activity of amphotericin B against the pathogenic fungus Candida albicans and its enhancement by allicin.

In this study, the vacuole disruptive activity was evaluated as a cause of amphotericin B (AmB) lethality against the pathogenic fungus Candida albicans in terms of its enhancement by allicin, an allyl-sulfur compound from garlic. Vacuole disruption was observed in parallel to AmB-induced cell death when the antibiotic was used at a lethal concentration and at a non-lethal concentration in combination with allicin. Allicin did not enhance AmB-induced cell death and the accompanying vacuole disruption when the cells were incubated with exogenous ergosterol for its enrichment in the vacuole. The vacuoles isolated from intact cells could be directly disrupted by the action of AmB to the same extent in the absence and presence of allicin, whereas the organelles isolated from ergosterol-enriched cells were resistant to its direct disruptive action. AmB was similarly incorporated into the fungal cytoplasm in cells with or without ergosterol enrichment, supporting the fact that AmB-induced vacuole disruption depends on its direct disruptive action on the organelle. In agreement with these findings, allicin was found to inhibit ergosterol transport from the plasma membrane to the cytoplasm, which is considered to be a cellular protective response to AmB-induced vacuole disruption in S. cerevisiae. Our study suggests that AmB lethality against C. albicans depends at least in part on its vacuole disruptive activity under the physiological condition permissive for invasive growth of the fungus.

Enhancement of the fungicidal activity of amphotericin B by allicin: effects on intracellular ergosterol trafficking.

In Saccharomyces cerevisiae, ergosterol was visible in the plasma membrane of untreated cells and was enriched in the vacuole membrane in response to amphotericin B (AmB) treatment at a non-lethal concentration. The simultaneous addition of allicin was inhibitory to AmB-induced ergosterol enrichment in the vacuole membrane, resulting in increased sensitivity of the organelles to the disruptive action of AmB. Allicin was also inhibitory to ergosterol enrichment in the vacuole membrane that was achieved by its external addition to cells. The combined fungicidal activity of AmB and allicin was suppressed together with suppression of vacuole membrane damage in cells where ergosterol had been fully enriched in the vacuole membrane. AmB caused direct disruptive damage of the isolated vacuoles, but the antibiotic was apparently less effective in disrupting the organelles that were isolated after ergosterol enrichment. These findings suggest that allicin enhances AmB-induced vacuole membrane damage by inhibiting ergosterol trafficking from the plasma membrane to the vacuole membrane.

Synergistic fungicidal activities of amphotericin B and N-methyl-N"-dodecylguanidine: a constituent of polyol macrolide antibiotic niphimycin.

The synergy between the alkylguanidinium chain of niphimycin (NM), a polyol macrolide antibiotic, and polyene macrolide amphotericin B (AmB) without such an alkyl side chain was examined using N-methyl-N″-alkylguanidines as its synthetic analogs. Among the analogs, N-methyl-N″-dodecylguanidine (MC12) most strongly inhibited the growth of Saccharomyces cerevisiae cells and those of other fungal strains in synergy with AmB. MC12 itself was not lethal but the analog could be a cause of a rapid cell death progression of yeast cells in the presence of AmB at a nonlethal concentration. Their combined actions resulted in the generation of NM-like fungicidal activity that depended on plasma membrane disability and cellular reactive oxygen species production. We also found an aberrant vacuolar morphogenesis and an associated vacuolar membrane disability in cells treated simultaneously with MC12 and AmB, as in the case of NM-treated cells. These findings support the idea that the alkylguanidinium chain plays a major role in the fungicidal activity of NM in cooperation with the polyol lactone ring as its enhancer.

Enhancing Effects on Vacuole-Targeting Fungicidal Activity of Amphotericin B

Invasive fungal infections are major threats for immunocompromised patients as well as for those undergoing cancer chemotherapy. Amphotericin B (AmB), a classical antifungal drug with a polyene macrolide structure, is widely used for the control of serious fungal infections. However, the clinical use of this antifungal drug is limited by its side effects and the emergence of drug-resistant strains. AmB lethality has been generally attributed to alterations in plasma membrane ion permeability due to its specific binding to plasma membrane ergosterol. Recent studies with Saccharomyces cerevisiae and Candida albicans reveal the vacuole disruptive action as another cause of AmB lethality on the basis of marked amplification of its activity in combination with allicin, an allyl-sulfur compound from garlic. The enhancing effect of allicin is dependent on the inhibition of ergosterol-trafficking from the plasma membrane to the vacuole membrane, which is considered to be a cellular response to protect against disintegration of the vacuole membrane. The polyol macrolide niphimycin (NM) also possesses vacuole-targeting fungicidal activity, which is greater than that of AmB and nystatin. The alkyl side chain attached to the macrolide ring of NM is considered to possess an allicin-like inhibitory effect on the intracellular trafficking of ergosterol. The vacuole-targeting fungicidal activity was additionally detected with a bactericidal cyclic peptide polymyxin B (PMB), and was markedly enhanced when administered together with allicin, monensin, or salinomycin. The synergistic fungicidal activities of AmB and allicin may have significant implications for the development of vacuole-targeting chemotherapy against fungal infections.

Alliinase from Ensifer adhaerens and Its Use for Generation of Fungicidal Activity

A bacterium Ensifer adhaerens FERM P-19486 with the ability of alliinase production was isolated from a soil sample. The enzyme was purified for characterization of its general properties and evaluation of its application in on-site production of allicin-dependent fungicidal activity. The bacterial alliinase was purified 300-fold from a cell-free extract, giving rise to a homogenous protein band on polyacrylamide gel electrophoresis. The bacterial alliinase (96 kDa) consisted of two identical subunits (48 kDa), and was most active at 60°C and at pH 8.0. The enzyme stoichiometrically converted (-)-alliin ((-)-S-allyl-L-cysteine sulfoxide) to form allicin, pyruvic acid, and ammonia more selectively than (+)-alliin, a naturally occurring substrate for plant alliinase ever known. The C-S lyase activity was also detected with this bacterial enzyme when S-alkyl-L-cysteine was used as a substrate, though such a lyase activity is absolutely absent in alliinase of plant origin. The enzyme generated a fungicidal activity against Saccharomyces cerevisiae in a time- and a dose-dependent fashion using alliin as a stable precursor. Alliinase of Ensifer adhaerens FERM P-19486 is the enzyme with a novel type of substrate specificity, and thus considered to be beneficial when used in combination with garlic enzyme with respect to absolute conversion of (±)-alliin to allicin.

Visualization analysis of the vacuole-targeting fungicidal activity of amphotericin B against the parent strain and an ergosterol-less mutant of Saccharomyces cerevisiae.

Here, we sought to investigate the vacuole-targeting fungicidal activity of amphotericin B (AmB) in the parent strain and AmB-resistant mutant of Saccharomyces cerevisiae and elucidate the mechanisms involved in this process. Our data demonstrated that the vacuole-targeting fungicidal activity of AmB was markedly enhanced by N-methyl-N″-dodecylguanidine (MC12), a synthetic analogue of the alkyl side chain in niphimycin, as represented by the synergy in their antifungal activities against parent cells of S. cerevisiae. Indifference was observed only with Δerg3 cells, indicating that the replacement of ergosterol with episterol facilitated their resistance to the combined lethal actions of AmB and MC12. Dansyl-labelled amphotericin B (AmB-Ds) was concentrated into normal rounded vacuoles when parent cells were treated with AmB-Ds alone, even at a non-lethal concentration. The additional supplementation of MC12 resulted in a marked loss of cell viability and vacuole disruption, as judged by the fluorescence from AmB-Ds scattered throughout the cytoplasm. In Δerg3 cells, AmB-Ds was scarcely detected in the cytoplasm, even with the addition of MC12, reflecting its failure to normally incorporate across the plasma membrane into the vacuole. Thus, this study supported the hypothesis that ergosterol is involved in the mobilization of AmB into the vacuolar membrane so that AmB-dependent vacuole disruption can be fully enhanced by cotreatment with MC12.

Synergistic fungicidal activities of polymyxin B and ionophores, and their dependence on direct disruptive action of polymyxin B on fungal vacuole

Polymyxin B (PMB) acts selectively on Gram-negative bacteria by electrostatic and hydrophobic interactions with anionic cell envelope components such as phospholipids and lipopolysaccharides. In this study, PMB was shown to exhibit marked fungicidal activity against yeasts and filamentous fungi in combination with ionophores such as salinomycin (SAM) and monensin (MON), which can selectively interact with monovalent cations. Ca2+-selective ionophores, A23187 and ionomycin, were absolutely ineffective in enhancing the fungicidal activity of PMB. SAM and MON increased the rate of cellular uptake of PMB possibly in favor of its intracellular action on the organelle. PMB could indeed directly disrupt the spherical membrane-enclosed architecture of the isolated vacuoles equally in the absence and presence of the ionophores. The loss of energy barrier for transmembrane transport of monovalent cations is considered to be a cause of enhanced incorporation of larger cationic compounds such as PMB across fungal plasma membrane.

Morphological changes of the filamentous fungus Mucor mucedo and inhibition of chitin synthase activity induced by anethole.

trans‐Anethole (anethole), a major component of anise oil, has a broad antimicrobial spectrum with antimicrobial activity relatively weaker than those of well‐known antibiotics, and significantly enhances the antifungal activity of polygodial and dodecanol against the bakers yeast Saccharomyces cerevisiae and human pathogenic yeast Candida albicans. However, the antifungal mechanism of anethole is unresolved. Anethole demonstrated antifungal activity against the filamentous fungus, Mucor mucedo IFO 7684, accompanied by hyphal morphological changes such as swollen hyphae at the tips. Its minimum growth inhibitory concentration was 0.625 mm. A hyperosmotic condition (1.2 m sorbitol) restricted the induction of morphological changes, while hypoosmotic treatment (distilled water) induced bursting of hyphal tips and leakage of cytoplasmic constituents. Furthermore, anethole dose‐dependently inhibited chitin synthase (CHS) activity in permeabilized hyphae in an uncompetitive manner. These results suggest that the morphological changes of M. mucedo could be explained by the fragility of cell walls caused by CHS inhibition. Copyright