Fungal Cell Death Research Articles

リポソーマル アムホテリシンＢ

Liposomal amphotericin B (AmBisome) is a DDS (drug delivery system) formulation of amphotericin B (AMPH-B), and has been developed in an attempt to reduce the toxicity of AMPH-B while retaining its therapeutic efficacy. AMPH-B has been the "gold standard" of antifungal therapy over the past four decades. It has a broad spectrum of fungicidal activity against a number of clinically important pathogens including Aspergillus and Candida. The mechanism of action of AMPH-B involves binding to ergosterol, the principal sterol in fungal cell membranes. Binding to ergosterol causes an increase in fungal membrane permeability, electrolyte leakage, and cell death. AMPH-B has affinity for cholesterol in mammalian membranes, which leads to severe side-effects including kidney damage. AmBisome is a unilamellar vesicle composed of AMPH-B and phospholipid. Upon administration, AmBisome remains intact in the blood and distributes to the tissues where fungal infection may occur, and is disrupted after attachment to the outside of fungal cells, resulting in fungal cell death. AmBisome and AMPH-B show similar in vitro and in vivo antifungal activity and clinical efficacy. However, AmBisome has less infusion-related toxicity and nephrotoxicity than AMPH-B.

Fungal elicitor-induced cell death in Taxus chinensis suspension cells is mediated by ethylene and polyamines

The involvements of ethylene and polyamines in cell death induced by fungal elicitor from Asperillus niger in Taxus chinensis suspension cells were studied. Exogenous fungal elicitor stimulated cell death and enhanced ethylene production and polyamine putrescine, spermidine and spermine concentrations compared with the control. Cell death, ethylene production and putrescine concentration induced by elicitor were increased by the precursor of ethylene biosynthesis 1-aminocyclopropane-1-carboxylic acid (ACC), while were overcome by ACC together with the inhibitor of ACC synthase aminoethoxyvinylycin. In contrast, fungal elicitor-induced cell death was decreased by, and ethylene production and putrescine concentration were enhanced by the inhibitor of ethylene action silver thiosulphate. Since the biosynthetic pathways of ethylene and polyamines are linked through S-adenosylmethionine (SAM), this promoted further probation into the roles of polyamines in cell death. Exogenous polyamines putrescine, spermidine and spermine all reduced elicitor-induced ethylene production and cell death. Blocking endogenous polyamine concentrations with methylglyoxal ( bis-guanylhydrazone) stimulated elicitor-induced ethylene production and cell death. These results indicated that ethylene promoted while polyamines alleviated elicitor-induced cell death.

Sublethal injury and resuscitation of Candida albicans after amphotericin B treatment.

Amphotericin B treatment was previously shown to inhibit Candida albicans reproduction and reduce the fluorescence of vitality-specific dyes without causing a corresponding increase in the fluorescence of the mortality-specific dyes bis-(1,3-dibutylbarbituric acid)trimethine oxonol and SYBR Green I. In the present study, we have confirmed these results and have shown that the numbers of CFU are reduced by 99.9% by treatment with 0.5 micro g of amphotericin B per ml for 10 h at 35 degrees C. This reduction was not due to fungal cell death. First, the level of reduction of the tetrazolium salt 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide increased in the presence of concentrations of amphotericin B that caused greater than 90% reductions in the numbers of CFU. Second, fungal cells treated with amphotericin B at a concentration of 0.5 micro g/ml were resuscitated by further incubation at 22 degrees C for 15 h in the continued presence of amphotericin B. Third, recovery of the ability to replicate was prevented by sequential treatment with 20 micro g of miconazole per ml, which also increased the fluorescence of mortality-specific dyes to near the maximal levels achieved with 0.9 micro g of amphotericin B per ml. Sequential treatment with fluconazole and flucytosine did not increase the levels of staining with the mortality-specific dyes. Itraconazole was less effective than ketoconazole, which was less effective than miconazole. The practice of equating the loss of the capacity of C. albicans to form colonies with fungal cell death may give incorrect results in assays with amphotericin B, and the results of assays with caution with other antifungal agents that are lipophilic or that possess significant postantifungal effects may need to be interpreted.

The Autolysis of Industrial Filamentous Fungi

ABSTRACT: Fungal autolysis is the natural process of self-digestion of aged hyphal cultures, occurring as a result of hydrolase activity, causing vacuolation and disruption of organelle and cell wall structure. Previously, authors have considered individual aspects of fungal lysis, in terms of either an enzyme, a process or an organism. This review considers both the physiology and morphology of fungal autolysis, with an emphasis on correlations between enzymological profiles and the morphological changes occurring during culture degeneration. The involvement of the main groups of autolytic hydrolases is examined (i.e., proteases, glucanases, and chitinases), in addition to the effects of autolysis on the morphology and products of industrial bioprocesses. We call for a concerted approach to the study of autolysis, as this will be fundamental for research to progress in this field. Increased understanding will allow for greater control of the prevention, or induction of fungal autolysis. Such advances will be applicable in the development of antifungal medicines and enable increased productivity and yields in industrial bioprocesses. Using paradigms in existing model systems, including mammalian cell death and aging in yeast, areas for future study are suggested in order to advance the study of fungal cell death.

A novel jasmonic acid-inducible rice myb gene associates with fungal infection and host cell death.

Endogenous signal molecules such as jasmonic acid (JA) and salicylic acid (SA) play an important role in induced resistance against pathogen infection and insect herbivory. In rice seedlings, JA is an effective inducer of systemic acquired resistance (SAR) against infection of blast fungus (Pyricularia grisea). To gain further insights into JA-mediated defense signaling pathways, we isolated and characterized a pathogen- and JA-induced rice gene (JAmyb) that encodes a Myb transcription factor. The JAmyb gene was induced within 1 day after fungal infection in resistant and susceptible interactions prior to lesion formation. Unlike most defense-related genes that are activated faster and stronger in resistant interactions, JAmyb induction by blast fungus is much higher in susceptible interactions, accompanied by large lesions and extensive tissue damage. Significant induction of JAmyb also was observed during cell death and lesion formation in certain lesion mimic mutants. Interestingly, JAmyb was activated rapidly by JA or wounding, independent of de novo protein synthesis, but not by other endogenous signal molecules such as SA and abscisic acid or SAR inducers such as benzothiadiazole and probenazole. We used SA-deficient transgenic plants to further demonstrate that depletion of SA in rice did not abolish but rather enhanced blast-induced JAmyb expression. These results suggest that JAmyb is related closely to host cell death and is involved in the JA-mediated, SA-independent signaling pathways in rice.

Infection Behavior ofVenturia nashicola, the Cause of Scab on Asian Pears

ABSTRACT The infection of Japanese pear by Venturia nashicola, the cause of scab on Asian pears (Japanese pear, Pyrus pylifolia var. culta; Chinese pear, P. ussuriensis), was examined using light and electron microscopy to determine the mechanism of resistance in pears. Early stages of infection were similar on the susceptible cv. Kosui, the resistant cv. Kinchaku, and the nonhost European pear (P. communis) cv. Flemish Beauty. V. nashicola penetrated only the cuticle layer on pear leaves and formed subcuticular hyphae on all three cultivars. Hyphae were localized in the pectin layer of pear leaves and never penetrated into the cytoplasm of epidermal cells. This restriction of fungal growth suggested that pectinases released by infection hyphae or subcuticular hyphae may be important in infection. Subcuticular hyphae were modified ultrastructurally in the pectin layer of resistant pear cultivars accompanied by fungal cell death. In contrast, fungal cells appeared intact in susceptible pear cultivars, suggesting the existence of resistance mechanisms.

Lovastatin Triggers an Apoptosis-like Cell Death Process in the FungusMucor racemosus

Roze, L. V., and Linz, J. E. 1998. Lovastatin triggers an apoptosis-like cell death process in the fungusMucor racemosusFungal Genetics and Biology25,119–133. The filamentous dimorphic fungusMucor racemosuspossesses threerasgenes, Mras1, 2, and 3, whose expression is correlated to morphogenesis of the fungus. Lovastatin, an indirect inhibitor of protein prenylation, altered the processing of MRas1 protein, blocked the accumulation of MRas3 protein, and caused the MRas1/ p20 protein complex to disappear inM. racemosus.Concurrently it arrested sporangiospore germination, decreased growth rate, caused a loss of cell viability accompanied by cell shrinkage, increased cell density and cytoplasm condensation, and triggered DNA fragmentation, resulting in nucleosomes and nucleosome multimers. The specific morphological and biochemical events seen inMucorcell death, particularly DNA fragmentation, resemble the best known characteristics of classical apoptosis in mammalian cells and prompted us to classify lovastatin-induced cell death as an apoptosis-like process. Lovastatin did not cause cell death in a leucine auxotroph ofMucorgrown in YNB minimal medium, conditions which support only spherical growth during spore germination. Exogenous dibutyryl-cAMP initiated morphogenesis from hyphal (polar) growth to yeast-like (spherical) growth during spore germination and strongly prevented cell death which resulted from lovastatin treatment. Wortmannin added together with dibutyryl-cAMP showed a synergistic effect in the prevention of fungal cell death. These data suggest that the regulation of lovastatin-induced cell death inMucorrequires a signal transduction pathway(s) involving cAMP whose function is specific to a particular developmental stage.

Amphotericin B toxicity and lethality: a tale of two channels

In spite of its toxic side effects, Amphotericin B (AmB) is the most effective drug in the treatment of visceral Leishmaniasis and systemic fungus infections. However, the toxic side effects of this drug can be substantially reduced when delivered as a complex with liposomes and other lipidic systems. Nonetheless, the mechanisms of AmB antifungal activity and of toxicity to the host are not yet fully understood. The classical pore model of AmB action postulates that a single type of channel permeable to monovalent cations and anions is responsible for the AmB fungicidal and leishmanicidal action, as well as for its toxic side effects. Thus, the loss of membrane cation selectivity, caused by AmB aqueous pores has been proposed as the primary cause of fungal cell death. The hypothesis has been that fungal cell death results from intracellular acidification associated with K + leakage. However, this mechanism of AmB action has been challenged by several investigators on the basis that low AmB concentrations cause K + leakage from sensitive cells which is dissociated from the lethal effects produced by higher AmB concentrations. Our own studies indicate that the formation of aqueous pores by AmB in sterol-containing liposomes is always preceded by the formation of non-aqueous channels. At low AmB concentrations or in the absence of sterols, AmB non-aqueous channels do not evolve to form aqueous pores, nor is the K + leakage they produce lethal to cells. It is only when a ‘critical’ concentration of AmB is reached at the membrane that non-aqueous channels interact with ergosterol or cholesterol to form transmembrane aqueous pores. The pore diameter of the channels formed by AmB is critical for AmB toxicity or lethality because non-aqueous channels are only permeable to urea and monovalent cations, whereas AmB aqueous pores are permeable to monovalent cations and anions (including H + and OH −) and divalent cations such as Ca 2+. In fact, leishmanias are killed rapidly by colloid osmotic lysis due to a net salt influx across the AmB aqueous pores. Fungal cells are protected from osmotic lysis by the presence of a cell wall, but an increased H +/OH − permeability across AmB aqueous pores leads to an elevation of intracellular pH which then results in membrane damage. In host mammalian cells, non-aqueous channels appear to be responsible for some of the toxic but reversible side effects produced by AmB. However, more acute and damaging effects such as those exerted by AmB in kidney tubular cells may be caused by increased salt, Ca 2+and/or H + permeability across aqueous pores. A sustained collapse of pH and Ca 2+ gradients is a mechanism which is also exhibited by molecular inducers of programmed cell death (apoptosis) in eucaryotic cells.

Mechanisms and target sites of damage in killing of Candida albicans hyphae by human polymorphonuclear neutrophils.

Target sites of fungal cell damage were studied to define mechanisms of neutrophil-mediated killing of Candida albicans hyphae. Neutrophils induced hyphal cell wall damage, as evidenced by release of cell wall glycoproteins and confocal microscopic changes. Damage occurred in the presence of neutrophil granule extracts and did not require oxidants. However, oxidation of hyphal surface glycoproteins correlated strongly with parallel increments in fungicidal activity, suggesting that oxidants did contribute to maximal cell wall damage. Neutrophil oxidants also induced hyphal DNA fragmentation, primarily single-strand breakage, as shown by increased electrophoretic migration after nuclease-S1 DNA digestion at single-strand break sites. The onset of damage to hyphal cell walls and DNA preceded detectable neutrophil-mediated fungicidal effects. Likewise, hyphal amino acid and nucleotide turnover as well as ATP initially rose, then declined as lethal effects became detectable. Thus, preceding detectable fungal cell death, neutrophil oxidative and oxygen-independent mechanisms damaged defined targets.

Root Colonization by Bipolaris sorokiniana in Different Cereals and Relations to Lesion Development and Natural Root Cortical Cell Death

AbstractA GUS‐transformed strain of B. sorokiniana was used to study the relationship between fungal growth, lesion development and natural root cortical cell death (RCD) in roots of different cereals. Roots of 10‐day‐old seedlings, grown on filter paper, were inoculated with the fungus and at different time intervals lesion size and GUS‐activity in the roots were determined. A significant, positive correlation was found between GUS‐activity and ergosterol content in barley roots infected with this transformed strain and these results indicate that GUS can be used as a marker to study fungal growth in plant tissue. The fungus had a slower growth rate in resistant barley varieties, i.e., those producing smaller lesions, than in more susceptible varieties.In wheat and triticale, the fungal growth was faster than in barley, despite the smaller and lighter coloured lesions in these species. This may be explained by the fact that wheat and triticale have a faster rate of root cortical senescence than barley. Presumably, dying cortex cells cannot respond to the fungal invasion by producing phenolic compounds that cause browning of the tissue. Among seven investigated cereal species, there was a positive correlation between the degree of RCD and fungal biomass increase in roots after inoculation.