N-acetylglucosamine (GlcNAc) induction of hyphal morphogenesis and transcriptional responses in Candida albicans are not dependent on its metabolism

N-acetylglucosamine (GlcNAc) stimulates important signaling pathways in a wide range of organisms. In the human fungal pathogen Candida albicans, GlcNAc stimulates hyphal cell morphogenesis, virulence genes, and the genes needed to catabolize GlcNAc. Previous studies on the GlcNAc transporter (NGT1) indicated that GlcNAc has to be internalized to induce signaling. Therefore, the role of GlcNAc catabolism was examined by deleting the genes required to phosphorylate, deacetylate, and deaminate GlcNAc to convert it to fructose-6-PO(4) (HXK1, NAG1, and DAC1). As expected, the mutants failed to utilize GlcNAc. Surprisingly, GlcNAc inhibited the growth of the nag1Δ and dac1Δ mutants in the presence of other sugars, suggesting that excess GlcNAc-6-PO(4) is deleterious. Interestingly, both hxk1Δ and an hxk1Δ nag1Δ dac1Δ triple mutant could be efficiently stimulated by GlcNAc to form hyphae. These mutants could also be stimulated to express GlcNAc-regulated genes. Because GlcNAc must be phosphorylated by Hxk1 to be catabolized, and also for it to enter the anabolic pathways that form chitin, N-linked glycosylation, and glycosylphosphatidylinositol anchors, the mutant phenotypes indicate that GlcNAc metabolism is not needed to induce signaling in C. albicans. Thus, these studies in C. albicans reveal a novel role for GlcNAc in cell signaling that may also regulate critical pathways in other organisms.     

N-acetylglucosamine (GlcNAc) Triggers a Rapid,Temperature-Responsive Morphogenetic Program in Thermally Dimorphic Fungi

The monosaccharide N-acetylglucosamine (GlcNAc) is a major component of microbial cell walls and is ubiquitous in the environment. GlcNAc stimulates developmental pathways in the fungal pathogen Candida albicans, which is a commensal organism that colonizes the mammalian gut and causes disease in the setting of host immunodeficiency. Here we investigate GlcNAc signaling in thermally dimorphic human fungal pathogens, a group of fungi that are highly evolutionarily diverged from C. albicans and cause disease even in healthy individuals. These soil organisms grow as polarized, multicellular hyphal filaments that transition into a unicellular, pathogenic yeast form when inhaled by a human host. Temperature is the primary environmental cue that promotes reversible cellular differentiation into either yeast or filaments; however, a shift to a lower temperature in vitro induces filamentous growth in an inefficient and asynchronous manner. We found GlcNAc to be a potent and specific inducer of the yeast-to-filament transition in two thermally dimorphic fungi, Histoplasma capsulatum and Blastomyces dermatitidis. In addition to increasing the rate of filamentous growth, micromolar concentrations of GlcNAc induced a robust morphological transition of H. capsulatum after temperature shift that was independent of GlcNAc catabolism, indicating that fungal cells sense GlcNAc to promote filamentation. Whole-genome expression profiling to identify candidate genes involved in establishing the filamentous growth program uncovered two genes encoding GlcNAc transporters, NGT1 and NGT2, that were necessary for H. capsulatum cells to robustly filament in response to GlcNAc. Unexpectedly, NGT1 and NGT2 were important for efficient H. capsulatum yeast-to-filament conversion in standard glucose medium, suggesting that Ngt1 and Ngt2 monitor endogenous levels of GlcNAc to control multicellular filamentous growth in response to temperature. Overall, our work indicates that GlcNAc functions as a highly conserved cue of morphogenesis in fungi, which further enhances the significance of this ubiquitous sugar in cellular signaling in eukaryotes.     

Phosphatidylserine is involved in the ferrichrome-induced plasma membrane trafficking of Arn1 in Saccharomyces cerevisiae

Arn1 is an integral membrane protein that mediates the uptake of ferrichrome, an important nutritional source of iron, in Saccharomyces cerevisiae. In the absence of ferrichrome, Arn1p is sorted directly from the trans-Golgi network to the vacuolar lumen for degradation. In the presence of low levels of ferrichrome, the siderophore binds to a receptor domain on Arn1, triggering the redistribution of Arn1 to the plasma membrane. When extracellular ferrichrome levels are high, Arn1 cycles between the plasma membrane and intracellular vesicles. To further understand the mechanisms of trafficking of Arn1p, we screened 4580 viable yeast deletion mutants for mislocalization of Arn1-GFP using synthetic genetic array technology. We identified over 100 genes required for trans-Golgi network-to-vacuole trafficking of Arn1-GFP and only two genes, SER1 and SER2, required for the ferrichrome-induced plasma membrane trafficking of Arn1-GFP. SER1 and SER2 encode two enzymes of the major serine biosynthetic pathway, and the Arn1 trafficking defect in the ser1Δ strain was corrected with supplemental serine or glycine. Plasma membrane trafficking of Hxt3, a structurally related glucose transporter, was unaffected by SER1 deletion. Serine is required for the synthesis of multiple cellular components, including purines, sphingolipids, and phospholipids, but of these only phosphatidylserine corrected the Arn1 trafficking defects of the ser1Δ strain. Strains with defects in phospholipid synthesis also exhibited alterations in Arn1p trafficking, indicating that the intracellular trafficking of some transporters is dependent on the phospholipid composition of the cellular membranes.     

The Plasma Membrane Proton Pump PMA-1 Is Incorporated into Distal Parts of the Hyphae Independently of the Spitzenk?rper in Neurospora crassa

Most models for fungal growth have proposed a directional traffic of secretory vesicles to the hyphal apex, where they temporarily aggregate at the Spitzenkörper before they fuse with the plasma membrane (PM). The PM H⁺-translocating ATPase (PMA-1) is delivered via the classical secretory pathway (endoplasmic reticulum [ER] to Golgi) to the cell surface, where it pumps H⁺ out of the cell, generating a large electrochemical gradient that supplies energy to H⁺-coupled nutrient uptake systems. To characterize the traffic and delivery of PMA-1 during hyphal elongation, we have analyzed by laser scanning confocal microscopy (LSCM) strains of Neurospora crassa expressing green fluorescent protein (GFP)-tagged versions of the protein. In conidia, PMA-1-GFP was evenly distributed at the PM. During germination and germ tube elongation, PMA-1-GFP was found all around the conidial PM and extended to the germ tube PM, but fluorescence was less intense or almost absent at the tip. Together, the data indicate that the electrochemical gradient driving apical nutrient uptake is generated from early developmental stages. In mature hyphae, PMA-1-GFP localized at the PM at distal regions (>120 μm) and in completely developed septa, but not at the tip, indicative of a distinct secretory route independent of the Spitzenkörper occurring behind the apex.     

Ctr2 is partially localized to the plasma membrane and stimulates copper uptake in COS-7 cells

Ctr1 (copper transporter 1) mediates high-affinity copper uptake. Ctr2 (copper transporter 2) shares sequence similarity with Ctr1, yet its function in mammalian cells is poorly understood. In African green monkey kidney COS-7 cells and rat tissues, Ctr2 migrated as a predominant band of approximately 70 kDa and was most abundantly expressed in placenta and heart. A transiently expressed hCtr2-GFP (human Ctr2-green fluorescent protein) fusion protein and the endogenous Ctr2 in COS-7 cells were mainly localized to the outer membrane of cytoplasmic vesicles, but were also detected at the plasma membrane. Biotinylation of Ctr2 with the membrane-impermeant reagent sulfo-NHS-SS-biotin [sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate] confirmed localization at the cell surface. Cells expressing hCtr2-GFP hyperaccumulated copper when incubated in medium supplemented with 10 microM CuSO(4), whereas cells depleted of endogenous Ctr2 by siRNAs (small interfering RNAs) accumulated lower levels of copper. hCtr2-GFP expression did not affect copper efflux, suggesting that hCtr2-GFP increased cellular copper concentrations by promoting uptake at the cell surface. Kinetic analyses showed that hCtr2-GFP stimulated saturable copper uptake with a K(m) of 11.0+/-2.5 microM and a K(0.5) of 6.9+/-0.7 microM when data were fitted to a rectangular hyperbola or Hill equation respectively. Competition experiments revealed that silver completely inhibited hCtr2-GFP-dependent copper uptake, whereas zinc, iron and manganese had no effect on uptake. Furthermore, increased copper concentrations in hCtr2-GFP-expressing cells were inversely correlated with copper chaperone for Cu/Zn superoxide dismutase protein expression. Collectively, these results suggest that Ctr2 promotes copper uptake at the plasma membrane and plays a role in regulating copper levels in COS-7 cells.     

Identification of a vacuolar sucrose transporter in barley and Arabidopsis mesophyll cells by a tonoplast proteomic approach

The vacuole is the main cellular storage pool, where sucrose (Suc) accumulates to high concentrations. While a limited number of vacuolar membrane proteins, such as V-type H(+)-ATPases and H(+)-pyrophosphatases, are well characterized, the majority of vacuolar transporters are still unidentified, among them the transporter(s) responsible for vacuolar Suc uptake and release. In search of novel tonoplast transporters, we used a proteomic approach, analyzing the tonoplast fraction of highly purified mesophyll vacuoles of the crop plant barley (Hordeum vulgare). We identified 101 proteins, including 88 vacuolar and putative vacuolar proteins. The Suc transporter (SUT) HvSUT2 was discovered among the 40 vacuolar proteins, which were previously not reported in Arabidopsis (Arabidopsis thaliana) vacuolar proteomic studies. To confirm the tonoplast localization of this Suc transporter, we constructed and expressed green fluorescent protein (GFP) fusion proteins with HvSUT2 and its closest Arabidopsis homolog, AtSUT4. Transient expression of HvSUT2-GFP and AtSUT4-GFP in Arabidopsis leaves and onion (Allium cepa) epidermal cells resulted in green fluorescence at the tonoplast, indicating that these Suc transporters are indeed located at the vacuolar membrane. Using a microcapillary, we selected mesophyll protoplasts from a leaf protoplast preparation and demonstrated unequivocally that, in contrast to the companion cell-specific AtSUC2, HvSUT2 and AtSUT4 are expressed in mesophyll protoplasts, suggesting that HvSUT2 and AtSUT4 are involved in transport and vacuolar storage of photosynthetically derived Suc.     

A Protein Inhibiting Pancreatic Lipase Activity in Soybean Seeds

D-Glucosamine (GlcN) inhibited hyphal growth of Pyricularia oryzae P₂ although it did not inhibit germination of conidia. The degree of inhibition varied according to the sugar, being the highest in the case of sucrose and the lowest in the case of glucose among the sugars tested. Uptake of GlcN was severely inhibited by glucose whereas uptake of glucose was not inhibited by GlcN. Glucose was the most effective among the sugars tested in the inhibition of GlcN uptake. GlcN caused morphological changes in both colonies and hyphae. The most characteristic change was a swelling of hyphae. Analysis of cell walls indicated that the cell wall of swollen hyphae contained more than twice as much N-acetylglucosamine and smaller amounts of other sugars as those found in normal hyphae.     

Composition and Ultrastructure of Streptomyces venezuelae

Streptomyces venezuelae is a filamentous bacterium with branching vegetative hyphae embedded in the substrate and aerial hyphae bearing spores. The exterior of the spore is inlaid with myriads of tiny rods which can be removed with xylene. The spore wall is approximately 30 nanometers thick. Occasionally, it can be seen that the plasma membrane and the membranous bodies within a spore are connected. The spore's germ plasm is not separated from the cytoplasm by a nuclear envelope. The cell walls of the vegetative hyphae, which are about 15 nanometers thick, are structurally and chemically similar to those of gram-positive bacteria. The numerous internal membranous bodies, some of which arise from the plasma membrane of the vegetative hypha, may be vesicular, whirled, or convoluted. Membranous bodies are usually prominent at the hyphal apices and are associated with septum formation. The germ plasm is an elongate, contorted, centrally placed area of lower electron density than the hyphal cytoplasm. The spores differ from the vegetative hyphae, not only in fine structure, but also in the arginine and leucine contents of their total cellular proteins.     

Hyphal death during colony development in Streptomyces antibioticus: morphological evidence for the existence of a process of cell deletion in a multicellular prokaryote

During the life cycle of the streptomycetes, large numbers of hyphae die; the surviving ones undergo cellular differentiation and appear as chains of spores in the mature colony. Here we report that the hyphae of Streptomyces antibioticus die through an orderly process of internal cell dismantling that permits the doomed hyphae to be eliminated with minimum disruption of the colony architecture. Morphological and biochemical approaches revealed progressive disorganization of the nucleoid substructure, followed by degradation of DNA and cytoplasmic constituents with transient maintenance of plasma membrane integrity. Then the hyphae collapsed and appeared empty of cellular contents but retained an apparently intact cell wall. In addition, hyphal death occurred at specific regions and times during colony development. Analysis of DNA degradation carried out by gel electrophoresis and studies on the presence of dying hyphae within the mycelium carried out by electron microscopy revealed two rounds of hyphal death: in the substrate mycelium during emergence of the aerial hyphae, and in the aerial mycelium during formation of the spores. This suggests that hyphal death in S. antibioticus is somehow included in the developmental program of the organism.     

Induction of germ tube formation by N-acetyl-D-glucosamine in Candida albicans: uptake of inducer and germinative response

A number of strains of Candida albicans were tested for germ tube formation after induction by N-acetyl-D-glucosamine (GlcNAc) and other simple (proline, glucose plus glutamine) or complex (serum) compounds. A proportion of strains (high responders) were induced to form germ tubes evolving to true hyphae by GlcNAc alone or by proline or glucose plus glutamine mixture. The majority of strains were low responders because they could be induced only by serum or GlcNAc-serum medium. Two strains were found to be nonresponders: they grew as pseudohyphae in serum. Despite minor quantitative differences, all strains efficiently utilized GlcNAc for growth under the yeast form at 28 degrees C. They also had comparable active, inducible, and constitutive uptake systems for GlcNAc. During germ tube formation in GlcNAc, the inducible uptake system was modulated, as expected from induction and decay of GlcNAc kinase. Uranyl acetate, at a concentration of 0.01 mM, inhibited both GlcNAc uptake and germ tube formation and was reversed by phosphates. Germinating and nongerminating cells differed in the rapidity and extent of GlcNAc incorporation into acid-insoluble and alkali-acid-insoluble cell fractions. During germ tube formation induced by proline, GlcNAc was almost totally incorporated into the acid-insoluble fraction after 60 min. Moreover, hyphal development on induction by either GlcNAc or proline was characterized by an apparent "uncoupling" between protein and polysaccharide metabolism, the ratio between the two main cellular constituents falling from more than 1 to less than 0.5 after 270 min of development. The data suggest that utilization of the inducer for wall synthesis is a determinant of germ tube formation C. albicans but that the nature and extent of inducer uptake is not a key event for this phenomenon to occur.     

Functional characterization of myosin I tail regions in Candida albicans

The molecular motor myosin I is required for hyphal growth in the pathogenic yeast Candida albicans. Specific myosin I functions were investigated by a deletion analysis of five neck and tail regions. Hyphal formation requires both the TH1 region and the IQ motifs. The TH2 region is important for optimal hyphal growth. All of the regions, except for the SH3 and acidic (A) regions that were examined individually, were required for the localization of myosin I at the hyphal tip. Similarly, all of the domains were required for the association of myosin I with pelletable actin-bound complexes. Moreover, the hyphal tip localization of cortical actin patches, identified by both rhodamine-phalloidin staining and Arp3-green fluorescent protein signals, was dependent on myosin I. Double deletion of the A and SH3 domains depolarized the distribution of the cortical actin patches without affecting the ability of the mutant to form hyphae, suggesting that myosin I has distinct functions in these processes. Among the six myosin I tail domain mutants, the ability to form hyphae was strictly correlated with endocytosis. We propose that the uptake of cell wall remodeling enzymes and excess plasma membrane is critical for hyphal formation.     

Identification of functional domains of Mid1, a stretch-activated channel component, necessary for localization to the plasma membrane and Ca2+ permeation

The Saccharomyces cerevisiae MID1 gene product (Mid1) is a stretch-activated Ca(2+)-permeable channel component required for Ca2+ influx and the maintenance of viability of cells exposed to the mating pheromone, alpha-factor. It is composed of 548-amino-acid (aa) residues with four hydrophobic segments, H1 (aa 2-22), H2 (aa 92-111), H3 (aa 337-356) and H4 (aa 366-388). It also has 16 putative N-glycosylation sites. In this study, sequentially truncated Mid1 proteins conjugated with GFP were expressed in S. cerevisiae cells. The truncated protein containing the region from H1 to H3 (Mid1(1-360)-GFP) localized normally in the plasma and endoplasmic reticulum (ER) membranes and complemented the low viability and Ca(2+)-uptake activity of the mid1 mutant, whereas Mid1(1-133)-GFP containing the region from H1 to H2 did not. Mid1(Delta3-22)-GFP lacking the H1 region failed to localize in the plasma membrane. Membrane fractionation showed that Mid1(1-22)-GFP containing only H1 localized in the plasma membrane in the presence of alpha-factor, suggesting that H1 is a signal sequence responsible for the alpha-factor-induced Mid1 delivery to the plasma membrane. The region from H1 to H3 is required for the localization of Mid1 in the plasma and ER membranes. Finally, trafficking of Mid1-GFP to the plasma membrane was dependent on the N-glycosylation of Mid1 and the transporter protein Sec12.     

Restricted sugar uptake by sugar-induced internalization of the yeast lactose/galactose permease Lac12

Kluyveromyces lactis Lac12 permease mediates lactose and low-affinity galactose transports. In this study we investigated the effects of carbon sources on internalization of Lac12 using a LAC12-GFP fusion construct. When galactose- or lactose-grown cells are shifted to a fresh sugar medium, Lac12-GFP is removed from the plasma membrane and is localized intracellularly. Surprisingly, either galactose or lactose in the new media caused the internalization, and cells responded differently to these two sugars. Our results reveal that this process is dependent on sugar species and also sugar concentration. Lac12-GFP internalization causes reduction of [C(14) ]lactose uptake rates and also occurs in a Klsnf1 mutant strain; it is therefore independent of KlSnf1 activity. We suggest that glucose-6-phosphate is the intracellular signal, as internalization was induced by 2-deoxyglucose, and inhibition of phosphoglucomutase by lithium prevented galactose- but not lactose- or glucose-induced internalization. Lac12-GFP internalization was not triggered by 6-deoxyglucose, and was irreversible in the absence of protein synthesis.     

The Arabidopsis concentration-dependent influx/efflux transporter ABCB4 regulates cellular auxin levels in the root epidermis

Arabidopsis ATP-binding cassette B4 (ABCB4) is a root-localised auxin efflux transporter with reported auxin uptake activity in low auxin concentrations. Results reported here demonstrate that ABCB4 is a substrate-activated regulator of cellular auxin levels. The contribution of ABCB4 to shootward auxin movement at the root apex increases with auxin concentration, but in root hair elongation assays ABCB4-mediated uptake is evident at low concentrations as well. Uptake kinetics of ABCB4 heterologously expressed in Schizosaccharomyces pombe differed from the saturation kinetics of AUX1 as uptake converted to efflux at threshold indole-3-acetic acid (IAA) concentrations. The concentration dependence of ABCB4 appears to be a direct effect on transporter activity, as ABCB4 expression and ABCB4 plasma membrane (PM) localisation at the root apex are relatively insensitive to changes in auxin concentration. However, PM localization of ABCB4 decreases with 1-naphthylphthalamic acid (NPA) treatment. Unlike other plant ABCBs studied to date, and consistent with decreased detergent solubility, ABCB4(pro) :ABCB4-GFP is partially internalised in all cell types by 0.05% DMSO, but not 0.1% ethanol. In trichoblasts, ABCB4(pro) :ABCB4-GFP PM signals are reduced by >200 nm IAA and 2,4-dichlorophenoxyacetic acid (2,4-D). In heterologous systems and in planta, ABCB4 transports benzoic acid with weak affinity, but not the oxidative catabolism products 2-oxindole-3-acetic-acid and 2-oxindole-3-acetyl-β-D-glucose. ABCB4 mediates uptake, but not efflux, of the synthetic auxin 2,4-D in cells lacking AUX1 activity. Results presented here suggest that 2,4-D is a non-competitive inhibitor of IAA transport by ABCB4 and indicate that ABCB4 is a target of 2,4-D herbicidal activity.     

Cytological analysis of mycelial incompatibility in Helicobasidium mompa

When the mycelia of Helicobasidium mompa encounter mycelia with a different genetic background, distinct demarcation lines form. The hyphae of H. mompa induce heterogenic incompatibility accompanied by active programmed cell death (PCD) process. In this study, we observed hyphal interaction between compatible and incompatible H. mompa pairs by means of light and electron microscopy. PCD started with one of the two approaching hyphae. Heterochromatin condensation and genomic DNA laddering were not observed. Moreover, cell damage began with the tonoplast and continued with the plasma membrane and nuclear membrane, suggesting that the PCD observed in heterogenic incompatibility of H. mompa is a vacuole-mediated process.