Vacuoles and fungal biology

Fungal vacuoles have long been recognised as versatile organelles, involved in many aspects of protein turnover, cellular homeostasis, membrane trafficking, signalling and nutrition. Recent research has also revealed an expanding repertoire of physiological functions for fungal vacuoles that are vital for fungal growth, differentiation, symbiosis and pathogenesis. Vacuole-mediated long-distance nutrient transporting systems have been shown to facilitate mycelial foraging and long-distance communication in saprophytes and mycorrhizal fungi. Some hyphae of plant and human fungal pathogens can grow under severely nutrient-limited conditions by expanding the vacuolar space rather than synthesising new cytoplasm and organelles. Autophagy has been recognised as a crucial process in plant pathogens for the initiation of appressorium formation. These studies demonstrate the importance of fungal vacuoles as organelles that are essential for many of the attributes that define the activities and roles of fungi in their natural environments.     

Transformation of steroids by fungal protoplasts

Summary Protoplasts of Cunninghamella elegans transformed cortexolone to the same products as did the mycelium. Transformation of the steroid by non-induced mycelium and by protoplasts released from it was almost completely inhibited by cycloheximide. However, hydroxylation of cortexolone was not affected by this antibiotic if mycelium grown in the presence of an enzyme inducer or protoplasts obtained from the induced mycelium were used. The transformation rate of protoplasts, on the basis of dry weight or protein units, was about four times higher than that of the mycelium, indicating that the mycelial cell wall was a serious rate-limiting factor in steroid bioconversion.     

Conditions for induced fusion of fungal protoplasts in polyethylene glycol solutions

Solutions containing polyethylene glycol MW 6000 (PEG) induced fusion of protoplasts of Penicillium chrysogenum. Balanced heterokaryons were formed by fusion of nutritionally complementing protoplasts. Heterokaryotic fusion products were obtained up to a frequency of 4% of the number of protoplasts, surviving the fusion treatment. Investigation of the conditions, necessary to achieve this high fusion frequency, showed that supplementing the PEG solution with Ca⁺⁺ and adjustment to high pH gave the best results. Mechanisms of fusion of fungal protoplasts by PEG, calcium and alkaline pH are discussed in view of the obtained results.     

Conditions for induced fusion of fungal protoplasts in polyethylene glycol solutions.

Solutions containing polyethylene glycol MW 6000 (PEG) induced fusion of protoplasts of Penicillium chrysogenum. Balanced heterokaryons were formed by fusion of nutritionally complementing protoplasts. Heterokaryotic fusion products were obtained up to a frequency of 4% of the number of protoplasts, surviving the fusion treatment. Investigation of the conditions, necessary to achieve this high fusion frequency, showed that supplementing the PEG solution with Ca++ and adjustment to high pH gave the best results. Mechanisms of fusion of fungal protoplasts by PEG, calcium and alkaline pH are discussed in view of the obtained results.     

Vacuoles and prevacuolar compartments

Plant vacuoles are complex and dynamic organelles. Important advances have been made in our understanding of the transporters present in the tonoplast and of the molecular interactions that allow targeting to vacuoles. Despite these advances, markers that permit vacuoles to be defined unambiguously have not yet been identified.     

Parsley protoplasts retain differential responsiveness to u.v. light and fungal elicitor

The differential response of cultured parsley cells to u.v. irradiation and elicitor treatment is a paradigm for analysis of specific plant defense responses. We demonstrate that freshly isolated parsley protoplasts, in the absence of detectable cell wall, maintain fully the ability to be activated by these important environmental factors. Stimulated protoplasts synthesize typical qualitative patterns and amounts of potentially protective flavonoid glycosides and coumarin phytoalexins following either u.v. irradiation or treatment with fungal elicitor, respectively. Induced accumulation of mRNAs and enzymes of the phenylpropanoid biosynthetic pathways is nearly identical in protoplasts and cells. Stimulation of protoplasts with elicitor requires only a short period of contact, which is not sufficient for cell wall regeneration. Importantly, there is no activation of these pathways during protoplast preparation. These results establish that parsley protoplasts respond appropriately to two physically distinct stimuli and might serve as an especially suitable system for the analysis of signal transduction and gene activation.     

Polyethylene glycol-induced internalization of bacteria into fungal protoplasts: electron microscopic study and optimization of experimental conditions

We studied the mechanism of internalization of Escherichia coli into Saccharomyces cerevisiae induced by polyethylene glycol (PEG) and optimized the experimental conditions. Transmission electron microscope studies revealed that the principal factor involved in the internalization was the degree of cell aggregation attained. Internalization occurred mainly by an endocytosis-like mechanism and took place during the elimination of PEG. The optimum conditions were to treat a mixed pellet of both microorganisms with 15% PEG and then gradually dilute the polymer. The same conditions were applied to E. coli and Aspergillus nidulans, with similar results.     

Response of carrot protoplasts and protoplast-derived aggregates to selection using a fungal culture filtrate of Alternaria radicina

Protoplasts isolated from three accessions of cultivated carrot and 5-day-old protoplast-derived aggregates were subjected to selection to identify somaclonal variants with enhanced tolerance to the fungal disease black rot incited by Alternaria radicina. Different concentrations [1, 2, 3.5, 5, 10, 20, 35 and 50 % (v/v)] of a fungal culture filtrate (FCF) from 2-week-old liquid cultures of A. radicina were used. Protoplasts and aggregates were subjected to short-term selection for a period of 10 days. All FCF concentrations added to the cultures on the day of isolation decreased protoplast survival frequency and plating efficiency, while FCF applied 5 days later inhibited cell divisions in 5–50 % concentrations. The responses of protoplasts to the treatment were genotype dependent. Most R0 plants were regenerated in all accessions from cell lines grown with 1 % FCF, while only a few plants were produced from 2 to 3.5 % FCF-treated cultures of ‘Dolanka’ and the breeding line ‘9304B’, respectively. Nineteen-percent of putative stress-tolerant regenerants were tetraploids, while only 5 % tetraploids were observed in the control. The incidence of unique random amplified polymorphic DNA fragments indicating possible chromosomal rearrangements was low and did not differ among regenerants after selection and those derived from the control. Mobilization of miniature inverted repeat transposable elements was not observed. Some R0 individuals regenerated both from FCF-treated and untreated cultures showed lower susceptibility to A. radicina in a laboratory assay in comparison to control plants grown from seed. Regenerants from FCF-treated cultures showed lower frequency of flowering plants and a higher rate of male sterility. Pollen viability of the putative stress-tolerant regenerants varied over a wide range (6–98 %), independently of in vitro selection conditions. Our data suggest that A. radicina FCF may be feasible for the in vitro selection to generate plants with superior phenotypic performance against A. radicina.     

High-affinity binding of fungal beta-glucan fragments to soybean (Glycine max L.) microsomal fractions and protoplasts

We have recently reported the existence of binding sites in soybean membranes for a beta-glucan fraction derived from the fungal pathogen Phytophthora megasperma f. sp. glycinea, which may play a role in the elicitor-mediated phytoalexin response of this plant [Schmidt, W. E. & Ebel, J. (1987) Proc. Natl Acad. Sci. USA 84, 4117-4121]. The specificity of beta-glucan binding to soybean membranes has now been investigated using a variety of competing polyglucans and oligoglucans of fungal origin. P. megasperma beta-glucan binding showed high apparent affinity for branched glucans with degrees of polymerization greater than 12. Binding affinity showed good correlation with elicitor activity as measured in a soybean cotyledon bioassay. Modification of the glucans at the reducing end with phenylalkylamine reagents had no effect on binding affinity. This characteristic was used to synthesize an oligoglucosyl tyramine derivative suitable for radioiodination. The 125I-glucan (15-30 Ci/mmol) provided higher sensitivity and lower detection limits for the binding assays while behaving in a manner identical to the [3H]glucan used previously. More accurate determinations of the Kd value for glucan binding indicated a higher affinity than previously shown (37 nM versus 200 nM). The 125I-glucan was used to provide the first reported evidence of specific binding of a fungal beta-glucan fraction in vivo to soybean protoplasts. The binding affinity to protoplasts proved identical to that found in microsomal fractions.     

The Isolation of Vacuoles from Candida Utilis

Vacuoles containing S-adenosylmethionine have been isolated from the yeast, Candida utilis, by the osmotic lysis of its spheroplasts and differential centrifugation. Culture in the presence of L-methionine and harvest in the stationary phase contributed to the ease of isolation and stability of the vacuoles. The vacuoles showed retentiveness for the sulfonium compound and responded to the vital-staining reaction with Neutral Red. Ultraviolet micrography indicated the homogeneity of the vacuolar preparation.     

Maturation of Autophagic Vacuoles in Mammalian Cells

The autophagic process was first described in mammalian cells several decades ago. After their formation as double-membraned vacuoles containing cytoplasmic material, autophagic vacuoles or autophagosomes undergo a stepwise maturation including fusion with both endosomal and lysosomal vesicles. However, the molecular mechanisms regulating these fusion steps have begun to emerge only recently. The list of newly discovered molecules that regulate the maturation of autophagosomes to degradative autolysosomes includes the AAA ATPase SKD1, the small GTP binding protein Rab7, and possibly also the Alzheimer-linked presenilin 1. This review combines previous data on the endo/lysosomal fusion steps during autophagic vacuole maturation with recent findings on the molecules regulating these fusion steps. Interestingly, autophagic vacuole maturation appears to be blocked in certain human diseases including neuronal ceroid lipofuscinosis and Danon disease. This suggests that autophagy has important housekeeping or protective functions, because a block in autophagic maturation causes a disease.     

Transport Processes in Vacuoles of Higher Plants

The vacuole is the largest compartment of a mature plant cell and serves as an internal reservoir of metabolites and nutrients. In the last years transport of solutes across the tonoplast has been intensively investigated. It was shown that two different proton pumps reside in the tonoplast. These pumps generate an electrochemical gradient which can be used as an energy-source to accumulate solutes. Cation uptake is driven by an H⁺ antiport mechanism. Anions are accumulated in response to the inside positive membrane potential. In addition, the existence of ion channels was shown using the patch clamp technique. The aim of this review is to compare and to discuss the present state of our knowledge of solute transport across the tonoplast.     

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe pneumonia "Legionnaires'' disease"¹. In protozoan and mammalian phagocytes, L. pneumophila employs a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates as many as 275 "effector" proteins into host cells. The effectors manipulate host proteins as well as lipids and communicate with secretory, endosomal and mitochondrial organelles^2-4.The formation of LCVs represents a complex, robust and redundant process, which is difficult to grasp in a reductionist manner. An integrative approach is required to comprehensively understand LCV formation, including a global analysis of pathogen-host factor interactions and their temporal and spatial dynamics. As a first step towards this goal, intact LCVs are purified and analyzed by proteomics and lipidomics.The composition and formation of pathogen-containing vacuoles has been investigated by proteomic analysis using liquid chromatography or 2-D gel electrophoresis coupled to mass-spectrometry. Vacuoles isolated from either the social soil amoeba Dictyostelium discoideum or mammalian phagocytes harboured Leishmania⁵, Listeria⁶, Mycobacterium⁷, Rhodococcus⁸, Salmonella⁹ or Legionella spp.¹⁰. However, the purification protocols employed in these studies are time-consuming and tedious, as they require e.g. electron microscopy to analyse LCV morphology, integrity and purity. Additionally, these protocols do not exploit specific features of the pathogen vacuole for enrichment.The method presented here overcomes these limitations by employing D. discoideum producing a fluorescent LCV marker and by targeting the bacterial effector protein SidC, which selectively anchors to the LCV membrane by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P)^3,11 . LCVs are enriched in a first step by immuno-magnetic separation using an affinity-purified primary antibody against SidC and a secondary antibody coupled to magnetic beads, followed in a second step by a classical Histodenz density gradient centrifugation^12,13 (Fig. 1).A proteome study of isolated LCVs from D. discoideum revealed more than 560 host cell proteins, including proteins associated with phagocytic vesicles, mitochondria, ER and Golgi, as well as several GTPases, which have not been implicated in LCV formation before¹³. LCVs enriched and purified with the protocol outlined here can be further analyzed by microscopy (immunofluorescence, electron microscopy), biochemical methods (Western blot) and proteomic or lipidomic approaches.     

Polyethylene glycol-induced internalization of bacteria into fungal protoplasts: electron microscopic study and optimization of experimental conditions.

We studied the mechanism of internalization of Escherichia coli into Saccharomyces cerevisiae induced by polyethylene glycol (PEG) and optimized the experimental conditions. Transmission electron microscope studies revealed that the principal factor involved in the internalization was the degree of cell aggregation attained. Internalization occurred mainly by an endocytosis-like mechanism and took place during the elimination of PEG. The optimum conditions were to treat a mixed pellet of both microorganisms with 15% PEG and then gradually dilute the polymer. The same conditions were applied to E. coli and Aspergillus nidulans, with similar results.     

Comparative Study of the Structure of Gas Vacuoles

The fine structure of gas vacuoles was examined in two blue-green algae, two green bacteria, three purple sulfur bacteria, and two halobacteria. The gas vacuole is a compound organelle, composed of a variable number of gas vesicles. These are closed, cylindrical, gas-containing structures with conical ends, about 80 to 100 nm in width and of variable length, ranging from 0.2 to over 1.0 mum. The wall of the gas vesicle is a non-unit membrane 2 to 3 nm in thickness, bearing very regular striations with a periodicity of 4 nm, oriented more or less at right angles to the long axis of the cylinder. This fine structure could be clearly resolved in isolated gas vesicles prepared from a blue-green alga and from Halobacterium halobium, and its presence in the gas vesicles of the green bacterium Pelodictyon clathratiforme was inferred from thin sections. The gas vacuole thus appears to be a homologous organelle in all of these procaryotic groups. Minor differences with respect to the length and arrangement of the gas vesicles were observed. In blue-green algae and green bacteria, the vesicles are relatively long and tend to be arrayed in parallel bundles; in purple sulfur bacteria and Halobacterium, they are shorter and more irregularly distributed in the cell.     

Lipidic Vacuoles in Plasmodium knowlesi Erythrocytic Schizonts

ABSTRACT. Electron microscopy of schizont development in erythrocytic Plasmodium knowlesi has revealed that spheroidal vacuoles 250 nm in diameter with semi-dense contents appear at the periphery of the parasite prior to the budding of merozoites. When treated with non-polar solvents, their contents are completely extracted, and after fixation in tannic-glutaraldehyde they contain regular lamellae with a periodicity of 5.5 nm. Both of these reactions are typical of lipids. Some of these structures are associated with phagosomal vacuoles which may contribute to their lamellae. They disappear at the onset of merozoite formation, but membranous whorls of various sizes continue to be associated with the schizont surface during budding of merozoites. It is suggested that the lipidic vacuoles are a source of preformed lipid which can be utilized rapidly during the generation of merozoites.