Dimorphism in Histoplasma capsulatum: a model for the study of cell differentiation in pathogenic fungi.

Several fungi can assume either a filamentous or a unicellular morphology in response to changes in environmental conditions. This process, known as dimorphism, is a characteristic of several pathogenic fungi, e.g., Histoplasma capsulatum, Blastomyces dermatitidis, and Paracoccidioides brasiliensis, and appears to be directly related to adaptation from a saprobic to a parasitic existence. H. capsulatum is the most extensively studied of the dimorphic fungi, with a parasitic phase consisting of yeast cells and a saprobic mycelial phase. In culture, the transition of H. capsulatum from one phase to the other can be triggered reversibly by shifting the temperature of incubation between 25 degrees C (mycelia) and 37 degrees C (yeast phase). Mycelia are found in soil and never in infected tissue, in contrast to the yeast phase, which is the only form present in patients. The temperature-induced phase transition and the events in establishment of the disease state are very likely to be intimately related. Furthermore, the temperature-induced phase transition implies that each growth phase is an adaptation to two critically different environments. A fundamental question concerning dimorphism is the nature of the signal(s) that responds to temperature shifts. So far, both the responding cell component(s) and the mechanism(s) remain unclear. This review describes the work done in the last several years at the biochemical and molecular levels on the mechanisms involved in the mycelium to yeast phase transition and speculates on possible models of regulation of morphogenesis in dimorphic pathogenic fungi.     

Increasing yeast secretion of heterologous proteins by regulating expression rates and post-secretory loss

Heterologous protein secretion involves the coupled processes of protein synthesis, protein folding, and secretory trafficking. A more complete understanding of how these processes interrelate could help direct optimization of secretion systems. Here we provide a detailed study regarding the dynamics of heterologous protein secretion from yeast in terms of intracellular protein levels, secreted protein levels, and unfolded protein response (UPR). Three different protein expression induction temperatures (20, 30, and 37 degrees C) were investigated as a means to modulate expression rates and thus cellular responses. Inducing at 20 degrees C yielded the slowest initial secretion rate, but the highest absolute level of product. Correspondingly, the level and the rate of both intracellular protein accumulation and unfolded protein response (UPR) activation were also the lowest at 20 degrees C. In addition, secretion ceased after approximately 22 h at 30 and 37 degrees C, respectively, while it was continuous until nutrient depletion at 20 degrees C. Maxima in secretion levels were observed that were a result of the additive effects of secretion cessation and post-secretory protein loss. The post-secretory loss of protein did not appear to result from solution phase proteolysis or aggregation, but required the presence of yeast cells. Refeeding of both yeast nitrogen base and casamino acids successfully prevented the post-secretory loss of protein at both high (37 degrees C) and low (20 degrees C) temperatures, and further increased secretion levels 1.5-fold at 20 degrees C where the secretory pathway was still functioning. Taken together, these findings suggest that there exists an appropriate balance between protein synthesis, processing and secretion rates required for secretion optimization.     

A Highly Thermosensitive and Permeable Mutant of the Marine Yeast Cryptococcus aureus G7a Potentially Useful for Single-Cell Protein Production and its Nutritive Components

The highly thermosensitive and permeable mutants are the mutants from which intracellular contents including proteins can be released when they are incubated both in the low osmolarity water and at the nonpermissive temperature (usually 37 degrees C). After mutagenesis by using nitrosoguanidine, a highly thermosensitive and permeable mutant named Z114 was obtained from the marine yeast Cryptococcus aureus G7a. Of the total protein, 65.3% was released from the mutant cells suspended in distilled water after they were treated at 37 degrees C overnight. However, only 12.3% of the total protein was released from the mutant cells suspended in 1.0 M sorbitol solution after they were treated at 37 degrees C overnight. We found that intracellular density of the mutant treated at 37 degrees C was greatly decreased, and cell volume of the mutant treated at 37 degrees C was increased due to the increased protein release. However, no significant changes in the intracellular density and cell volume of the mutant were observed when its cells suspended in 1.0 M sorbitol solution were treated at 37 degrees C. It was found that no big changes in cell growth, protein content, vitamin C content, nucleic acid content, fatty acids, and amino acid compositions of both the mutant and its wild type were detected. Therefore, the highly thermosensitive and permeable mutant still can be a good candidate as single-cell protein. This means that the method used in this study is a simple and efficient way to release protein from the highly thermosensitive and permeable yeast mutant cells with high protein content.     

Effects of temperature acclimation on Neurospora phospholipids. Fatty acid desaturation appears to be a key element in modifying phospholipid fluid properties

Experiments were conducted on the effect of growth temperature on phospholipids of Neurospora. Strains grown at high (37 degrees C) and low (15 degrees C) temperatures show large differences in the proportions of phospholipid fatty acid alpha-linolenate (18 : 3) which can vary by 10-fold over this temperature range. Changes in the phospholipid base composition are less dramatic; the most significant is an increase in phosphatidylethanolamines at low temperatures accompanied by a concomitant decrease in phosphatidylcholine. It appears that phospholipid fatty acid desaturation is closely regulated with respect to growth temperature. Over the 37 to 15 degrees C growth temperature range there appear to be at least two desaturase systems in Neurospora which are under different controls. Production of 18 : 1 and 18 : 2 species appears to occur at high levels over the entire temperature range, whereas the production of 18 : 3 seems to be inversely related to growth temperature. Shifting 37 degrees C-acclimated cultures to 15 degrees C produces a growth lag period of approximately 3 h, during which the level of 18 : 3 increases markedly. Differential scanning calorimetry of phospholipids from 37 degrees C cells shows a phase transition at -22 degrees C while lipids from 15 degrees C cultures exhibit a phase transition with reduced enthalpy at about -41 degrees C. The data are consistent with the idea that phospholipid composition in Neurospora is under strict control and suggest that membrane fluidity is regulated with respect to growth temperature through changes in membrane lipid composition.     

Beta-adrenergic modulation of cardiac ion channels. Differential temperature sensitivity of potassium and calcium currents

beta-Adrenergic stimulation of ventricular heart cells results in the enhancement of two important ion currents that regulate the plateau phase of the action potential: the delayed rectifier potassium channel current (IK) and L-type calcium channel current (ICa). The temperature dependence of beta-adrenergic modulation of these two currents was examined in patch-clamped guinea pig ventricular myocytes at various steps in the beta-receptor/cyclic AMP-dependent protein kinase pathway. External applications of isoproterenol and forskolin were used to activate the beta-receptor and the enzyme adenylate cyclase, respectively. Internal dialysis of cyclic 3',5'-adenosine monophosphate (cAMP) or the catalytic subunit of cAMP-dependent protein kinase (CS), as well as the external addition of 8-chlorphenylthio cAMP (CPT-cAMP) was applied to increase intracellular levels of cAMP and CS. Isoproterenol-mediated increases in IK, but not ICa, were found to be very temperature dependent over the range of 20-37 degrees C. At room temperature (20-22 degrees C) isoproterenol produced a large (threefold) enhancement of ICa but had no effect on IK. In contrast, at warmer temperatures (30-37 degrees C) both currents increased in the presence of this agonist and the kinetics of IK were slowed at -30 mV. A similar temperature sensitivity also existed after exposure to forskolin, CPT-cAMP, cAMP, and CS, suggesting that this temperature sensitivity of IK may arise at the channel protein level. Modulation of IK during each of these interventions was accompanied by a slowing in IK kinetics. Thus, regulation of cardiac potassium channels but not calcium channels involves a temperature-dependent step that occurs after activation of the catalytic subunit of cAMP-dependent protein kinase.     

Ultrastructural study of the yeast-mycelium transition in Histoplasma capsulatum. II. Changes at 34 degrees C

The ultrastructural changes which occur during the mycelium to yeast transition in Histoplasma capsulatum induced by a temperature shift from 25 degrees C to 34 degrees C are described and compared to those observed after a temperature shift from 25 degrees C to 37 degrees C. 24 hours after the temperature shift to 34 degrees C only 8% of the cells are lysed. However, many mitochondria have lost their characteristic elongated form and have become rounded. Vesicular cristae which are no longer oriented parallel to the long axis of the mitochondria are also observed. In contrast a temperature shift from 25 degrees C to 37 degrees C induces lysis of 70% of the cells; mitochondria are rarely observed in the remaining cells. These ultrastructural changes can be correlated with the uncoupling of oxidative phosphorylation and the production of heat shock proteins.     

Changes in cyclic nucleotide levels and dimorphic transition in Candida albicans.

The relationship between the levels of cyclic nucleotides and dimorphic transition in Candida albicans was examined. The results showed that cells of this pathogenic fungus contained both cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine 3',5'-monophosphate (cGMP), the concentration of the latter being about one-tenth that of the former in stationary-phase cells of the yeast form. Our results further indicated that germ tube formation induced by incubation at 40 degrees C followed a rise in cAMP concentration in the cell with no accompanying change in cGMP content. Cysteine, which suppressed germination, also reversed the increase in intracellular cAMP concentration. Dibutyryl cAMP (1 MM) significantly promoted germination in proline medium at temperatures of 32 to 34 degrees C. These results suggested that cAMP was one of the controlling factors in the morphological transition in Candida albicans.     

gamma-Glutamyltranspeptidase from Escherichia coli K-12: formation and localization.

Escherichia coli cells showed maximum activity of gamma-glutamyltranspeptidase (EC 2.3.2.2) when they were grown at 20 degrees C, 14% of maximum activity at 37 degrees C, and none at 43 degrees C. The enzyme activity of intact cells grown at 20 degrees C was stably maintained after the temperature was changed to 45 degrees C. The activity increased during the exponential phase, and maximum activity was found at stationary phase. Its intracellular localization in the periplasmic space was confirmed.     

Efficiency of acetylcholine receptor subunit assembly and its regulation by cAMP

Assembly of nicotinic acetylcholine receptor (AChR) subunits was investigated using mouse fibroblast cell lines stably expressing either Torpedo (All-11) or mouse (AM-4) alpha, beta, gamma, and delta AChR subunits. Both cell lines produce fully functional cell surface AChRs. We find that two independent treatments, lower temperature and increased intracellular cAMP can increase AChR expression by increasing the efficiency of subunit assembly. Previously, we showed that the rate of degradation of individual subunits was decreased as the temperature was lowered and that Torpedo AChR expression was acutely temperature sensitive, requiring temperatures lower than 37 degrees C. We find that Torpedo AChR assembly efficiency increases 56-fold as the temperature is decreased from 37 to 20 degrees C. To determine how much of this is a temperature effect on degradation, mouse AChR assembly efficiencies were determined and found to be only approximately fourfold more efficient at 20 than at 37 degrees C. With reduced temperatures, we can achieve assembly efficiencies of Torpedo AChR in fibroblasts of 20-35%. Mouse AChR in muscle cells is also approximately 30% and we obtain approximately 30% assembly efficiency of mouse AChR in fibroblasts (with reduced temperatures, this value approaches 100%). Forskolin, an agent which increases intracellular cAMP levels, increased subunit assembly efficiencies twofold with a corresponding increase in cell surface AChR. Pulse-chase experiments and immunofluorescence microscopy indicate that oligomer assembly occurs in the ER and that AChR oligomers remain in the ER until released to the cell surface. Once released, AChRs move rapidly through the Golgi membrane to the plasma membrane. Forskolin does not alter the intracellular distribution of AChR. Our results indicate that cell surface expression of AChR can be regulated at the level of subunit assembly and suggest a mechanism for the cAMP-induced increase in AChR expression.     

Adaptation of the yeast Yarrowia lipolytica to heat shock

The adaptive response of the yeast Yarrowia lipolytica to heat shock has been studied. Experiments showed that, after 10 min of incubation at 45 degrees C, the survival rate of Yarrowia lipolytica cells was less than 0.1%. Stationary-phase yeast cells were found to be more thermotolerant than exponential-phase cells. The 60-min preincubation of cells at 37 degrees C or pretreatment with low concentrations of H2O2 (0.5 mM) and menadione (0.05 mM) made them more tolerant to heat and to oxidative stress (120 mM hydrogen peroxide). The pH dependence of yeast thermotolerance has also been studied. The adaptation of yeast cells to heat shock and oxidative stress was found to be associated with a decrease in the intracellular level of cAMP and an increase in the activity of antioxidant enzymes (catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase).     

Vasopressin V2-receptor mobile fraction and ligand-dependent adenylate cyclase activity are directly correlated in LLC-PK1 renal epithelial cells

The role of hormone receptor lateral mobility in signal transduction was studied using a cellular system in which the receptor mobile fraction could be reversibly modulated to largely varying extents. The G-protein-coupled vasopressin V2-type receptor was labeled in LLC-PK1 renal epithelial cells using a fluorescent analogue of vasopressin, and receptor lateral mobility measured using fluorescence microphotolysis (fluorescence photobleaching recovery). The receptor mobile fraction (f) was approximately 0.9 at 37 degrees C and less than 0.1 at 10 degrees C, in accordance with previous studies. When cells were incubated for 1 h at 4 degrees C without hormone, and then warmed up to 37 degrees C and labeled with the vasopressin analogue, f increased from approximately 0.4 to 0.8 over approximately 1 h. The apparent lateral diffusion coefficient was not markedly affected by temperature pretreatment. Studies with radiolabeled vasopressin indicated that temperature pretreatment influenced neither receptor number nor binding/internalization kinetics. F-actin staining revealed that temperature change resulted in reversible changes of cytoskeletal structure. The maximal rate of in vivo cAMP production at 37 degrees C in response to vasopressin, but not to forskolin (receptor-independent agonist), was also markedly influenced by preincubation of cells at 4 degrees C, thus paralleling the effects of temperature preincubation on f. A linear correlation between f and maximal cAMP production was observed, suggesting that the receptor mobile fraction is a key parameter in hormone signal transduction in vivo. We conclude that mobile receptors are required to activate G-proteins, and discuss the implications of this for signal transduction mechanisms.     

Oscillations of cAMP with the cardiac cycle

Oscillations of cAMP with the cardiac cycle were demonstrated in the rat heart using a stimulator-triggered rapid freeze-clamp to decrease the temperature of the heart from 37 degrees C to -80 degrees C in 5 msec (20,000 degrees/sec) at a predetermined phase of the cardiac cycle. The nucleotide, cAMP, oscillated 60% with the cardiac cycle during normal working conditions, the higher cAMP value occurring during systole.     

Recognition of different pools of phosphatidylglycerol in intact cells and isolated membranes of Acholeplasma laidlawii by phospholipase A2.

Phospholipase A2 (EC 3.1.1.4) from pig pancreas hydrolyzes phosphatidylglycerol in intact cells and isolated membranes of Acholeplasma laidlawii. Complete degradation of phosphatidylglycerol in intact cells at 37 degrees C does not result in lysis as shown by the retention of intracellular K+ ions and the cytoplasmic glucose-6-phosphatase, as well as the inability to detect activity of membrane-bound intracellular NADH-oxidase. A. laidlawii was grown on linoleic acid. Phospholipase A2 treatment of these cells at 5 degrees C, at which temperature the lipids are still in the liquid-crystalline state, results in a rapid breakdown of 50% of the phosphatidylglycerol. The residual phosphatidylglycerol can be hydrolyzed only at elevated temperatures and at much smaller rates, depending strongly on the incubation temperature. When membranes isolated from these cells are incubated at 5 degrees C, 70% of the phosphatidylglycerol is hydrolyzed immediately. The hydrolysis of the residual 30% is again strongly temperature dependent. Cells were grown on palmitate, elaidate, or oleate to investigate possible effects of the lipid phase transition on the accessibility of phosphatidylglycerol for phospholipase A2. Under conditions in which all the lipid is in the solid state, no hydrolysis occurs. When solid and liquid-crystalline lipid phases coexist, a limited hydrolysis of phosphatidylglycerol can be observed. The results demonstrate the disposition of phosphatidylglycerol in three different pools in the membrane of A. laidlawii. Phospholipase A2 has been used to discriminate between these pools and to estimate the amount of phosphatidylglycerol which is present in the liquid-crystalline phase. The present data, however, do not allow a definite localization of the phosphatidylglycerol pools.     

Calorimetric and spectroscopic studies of lipid thermotropic phase behavior in liver inner mitochondrial membranes from a mammalian hibernator

Arrhenius plots of various enzyme and transport systems associated with the liver mitochondrial inner membranes of ground squirrels exhibit changes in slope at temperatures of 20-25 degrees C in nonhibernating but not in hibernating animals. It has been proposed that the Arrhenius breaks observed in nonhibernating animals are the result of a gel to liquid-crystalline phase transition of the mitochondrial membrane lipids, which also occurs at 20-25 degrees C, and that the absence of such breaks in hibernating animals is due to a major depression of this lipid phase transition to temperatures below 4 degrees C. In order to test this hypothesis, we have examined the thermotropic phase behavior of liver inner mitochondrial membranes from hibernating and nonhibernating Richardson's ground squirrels, Spermophilus richardsonii, by differential scanning calorimetry and by 19F nuclear magnetic resonance and fluorescence polarization spectroscopy. Each of these techniques indicates that no lipid phase transition occurs in the membranes of either hibernating or nonhibernating ground squirrels within the physiological temperature range of this animal (4-37 degrees C). Moreover, differential scanning calorimetric measurements indicate that only a small depression of the lipid gel to liquid-crystalline phase transition, which is centered at about -5 degrees C in nonhibernating animals and at about -9 degrees C in hibernators, occurs. We thus conclude that the Arrhenius plot breaks observed in some membrane-associated enzymatic and transport activities of nonhibernating animals are not the result of a lipid phase transition and that a major shift in the gel to liquid-crystalline lipid phase transition temperature is not responsible for seasonal changes in the thermal behavior of these inner mitochondrial membrane proteins.     

Domains and anomalous adsorption isotherms of dipalmitoylphosphatidylcholine membranes and lipophilic ions: pentachlorophenolate, tetraphenylborate, and dipicrylamine.

Dipalmitoylphosphatidylcholine (DPPC) vesicles acquire negative surface charge on adsorption of negatively charged pentachlorophenolate (PCP-), and lipophilic ions tetraphenylborate (TPhB-), and dipicrylamine (DPA-). We have obtained (a) zeta-potential isotherms from the measurements of electrophoretic mobility of DPPC vesicles as a function of concentration of the adsorbing ions at different temperatures (25-42 degrees C), and (b) studied the effect of PCP- on gel-to-fluid phase transition by measuring the temperature dependence of zeta-potential at different PCP- concentrations. The zeta-potential isotherms of PCP- at 25, 32, and 34 degrees C correspond to adsorption to membrane in its gel phase. At 42 degrees C the zeta-potential isotherm corresponds to membrane in its fluid phase. These isotherms are well described by a Langmuir-Stern-Grahame adsorption model proposed by McLaughlin and Harary (1977. Biochemistry. 15:1941-1948). The zeta-potential isotherm at 37 degrees C does not follow the single-phase adsorption model. We have also observed anomalous adsorption isotherms for lipophilic ions TPhB- and DPA- at temperatures as low as 25 degrees C. These isotherms demonstrate a gel-to-fluid phase transition driven by ion adsorption to DPPC membrane during which the membrane changes from weakly to a strongly adsorbing state. The anomalous isotherm of PCP- and the temperature dependence of zeta-potential can be described by a two-phase model based on the combination of (a) Langmuir-Stern-Grahame model for each phase, (b) the coexistence of gel and fluid domains, and (c) depression of gel-to-fluid phase transition temperature by PCP-. Within the anomalous region the magnitude of zeta-potential rapidly increases concentration of adsorbing species, which was characterized in terms of a Esin-Markov coefficient. This effect can be exploited in membrane-based devices. Comments are also made on the possible effect of PCP, as an uncoupler, in energy transducing membranes.     

Regulation of dimorphism in Histoplasma capsulatum by cyclic adenosine 3',5'-monophosphate.

During temperature-induced transition of the dimorphic pathogenic fungus Histoplasma capsulatum from the single yeast cell form to the multicellular mycelial form, there was an increase in intracellular cyclic adenosine 3',5'-monophosphate (cAMP) levels as well as a striking accumulation of cAMP in the medium. cAMP levels also changed during the reverse mycelium-to-yeast transition.     

The relation of endogenous adenosine cyclic 3':5'-monophosphate to the antagonistic effects of adenosine and colchicine on cell shape.

Adenosine and colchicine have antagonistic effects on cell shape. When Chinese Hamster lung fibroblasts (CHE36-6) or SV40 transformed 3T3 (SV3T3) cells are incubated with colchicine (1 muM) for one hour at 37 degrees C, they round up into spheres with short spikes. Cells treated with adenosine (1 muM-minus 4 mM) for one hour become refractile and develop spindly processes. However, when the two compounds are added simultaneously, the characteristic responses to either drug are abolished and the cells appear normal. The counteraction is specific for adenine and its derivatives, adenosine being the most effective of the compounds we tested. Accumulation of colchicine or adenosine is not altered significantly by the presence of the other drug, ruling out decreases in uptake as the basis of the mutual antagonism. The morphological changes can be observed under conditions where there are no changes in intracellular cAMP levels (such as incubation with low concentrations of adenosine or cordycepin, an adenosine analog that cannot be directly converted to cAMP). Colchicine does not alter cAMP content of control or adenosine-treated cells. These data show that adenine compounds have potent effects on cell shape, and the antagonistic effects of adenosine and colchicine on cell shape are not mediated through changes in intracellular cAMP levels.