Ultrastructural investigations of the formation of Candida albicans germ tubes and septa.

Germ tubes, blastospores and pseudomycelia of Candida albicans were studied under the electron microscope. Both germ tube and blastospore formation begin with comparable changes in the cell wall of the mother cell. The major part of the germ tube wall consists of the fourth of the five layers forming the C. albicans cell wall. Germ tubes are precursors of mycelia which can ramify. Septa and septal pores can be demonstrated in later phases. Pseudomycelia can be clearly discerned by means of transmission electron microscopy.     

Ethanol-induced germ tube formation in Candida albicans

Ethanol is the first reported compound which can induce germ tube formation in Candida albicans without the addition of any nitrogen-containing nutrients. Conditions controlling induction of germ tubes in C. albicans by ethanol were investigated. Ethanol (17.1 mM) in buffered salts solution containing sodium bicarbonate induced 70 to 80% of yeast phase cells of C. albicans to form germ tubes. Germ tubes could be induced by ethanol (0.08 to 340 mM) at temperatures ranging from 29 to 41 degrees C (optimum 37 degrees C) and at pH values ranging from 3.0 to 8.0 (optimum 5.75). The germ tubes averaged 11 micron in length after 6 h at 37 degrees C. The percentage of cells forming germ tubes decreased as the concentration of cells in the induction solution was increased above 4 X 10(5) cells ml-1. Germ tubes first appeared 45 to 60 min after continuous exposure to ethanol at 37 degrees C and all cells which formed germ tubes did so by 2 h. Germ tube length decreased as the pH was increased but was independent of the concentration of ethanol. Oxygen was required for germ tube formation. In addition to ethanol, 1-propanol, 2-propanol, 1-butanol and acetic acid could induce germ tube formation, whereas methanol could not. These results indicate that the cells must mobilize their endogenous nitrogen and probably carbohydrate reserves in order to initiate formation of germ tubes. The evidence is inconclusive as to whether ethanol itself must be metabolized for germ tube induction to occur, although it is not thought to act by a nonspecific interaction with the cell membrane.     

An investigation of the role of true hypha production in the pathogenesis of experimental oral candidosis

From 427 cases of human oral candidosis 2135 yeast clones were screened for the presence of germ tube-negative C. albicans and variants that formed only pseudohyphae in serum; one strain of each was found. The pathogenic potential of the serum-pseudohyphal and germ tube-negative C. albicans variants was investigated in the oral cavity of the rat; both variants failed to induce palatal candidosis, in contrast to a germ tube-positive C. albicans control strain. The pathogenic potential of C. albicans strains appears to be dependent on the formation of true germ tubes.     

Carbon dioxide induces endotrophic germ tube formation in Candida albicans

Candida albicans formed germ tubes when exposed to air containing 5 to 15% carbon dioxide (CO2). The CO2-mediated germ tube formation occurred optimally at 37 degrees C in a pH range of 5.5 to 6.5. No germ tubes were produced at 25 degrees C, even when the optimal concentration of CO2 (10%) was present in the environment. The requirement of CO2 for germ tube formation could be partially substituted by sodium bicarbonate but not by N2. Carbon dioxide was required to be present throughout the entire course of germ tube emergence suggesting that its role is not limited to an initial triggering of morphogenic change. We suggest that carbon dioxide may be a common effector responsible for the germ tube promoting activity of certain chemical inducers for C. albicans.     

Induction of N-acetyl-D-glucosamine catabolic enzymes and germinative response in Candida albicans

The regulation of N-acetylglucosamine catabolic enzymes was studied in both yeast and germ tube forms of the dimorphic fungus Candida albicans. The induction pattern of these enzymes was the same for yeast cells incubated at 28 degrees C and in cells incubated at 37 degrees C which formed germ tubes. However, the level of activity of these enzymes in germ tube stage is lower as compared to yeast phase cells. A strain of C. albicans that did not form germ tubes was endowed with a pronounced ability for induction of N-acetylglucosamine catabolic enzymes. This result suggests that germ tube formation and N-acetylglucosamine metabolism are mutually exclusive events.     

Recognition of Candida albicans Als3 by the germ tube-specific monoclonal antibody 3D9.3

Monoclonal antibody 3D9.3 (MAb 3D9.3) reacts with the surface of Candida albicans germ tubes and recognizes a protein epitope. We used a two-step chromatography procedure to purify and identify the antigen (3D9) from C. albicans strain 66396 germ tubes. MAb 3D9.3 recognized two intense protein bands at 140 and 180 kDa. A comparative analysis between theoretical and experimental mass spectrum peaks showed that both bands corresponded to Als3. This conclusion was supported by lack of reactivity between MAb 3D9.3 and an als3 Δ /als3 Δ mutant strain, and the fact that an immunoglobulin preparation enriched for Als3 specificity recognized the purified 3D9 antigen. PCR demonstrated that C. albicans strain 66396 has two different-sized ALS3 alleles that correspond to the two purified protein bands. Strain- and species-specificity of the 3D9 epitope were studied with various C. albicans strains and Candida species, such as closely related Candida dubliniensis . The 3D9 epitope was detected only in C. albicans , demonstrating the utility of MAb 3D9.3 for differentiation between C. albicans and C. dubliniensis . Adhesion assays demonstrated that MAb 3D9.3 blocks adhesion of C. albicans germ tubes to human buccal epithelial cells and vascular endothelial cells.     

Structures involved in the binding of human fibrinogen to Candida albicans germ tubes

Recent evidence for the interaction between human fibrinogen and Candida albicans germ tubes have led us to attempt to characterize the structures involved. Using 125I-radiolabeled proteins, fibrinogen purified by affinity chromatography and its plasmin degradation products, the binding sites on the fibrinogen molecule appeared to be located specifically in the D-domain. Conversely to the fibrinogen and the fragment D, radiolabeled fragment E, however, did not interact with cells. The binding was time-dependent, saturable and reversible. Scatchard analysis of the data obtained revealed an average of 6000 binding sites per germ tube with dissociation constant (Kd) of 5.2 X 10(-8) M. No potent competition was observed for a range of different proteins and carbohydrates. Fibrinogen fragment D binding proteins were identified using a dithiothreitol-iodoacetamide extract of the fungus. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, one component of 68 kDa was detected. Thus, the presence of fibrinogen binding proteins specifically localized on the cell wall surface of C. albicans germ tubes may constitute one of the factors involved in the development of candidosis.     

Structures involved in the binding of human fibrinogen to Candida albicans germ tubes

Abstract Recent evidence for the interaction between human fibrinogen and Candida albicans germ tubes have led us to attempt to characterize the structures involved. Using ¹²⁵I-radiolabeled proteins, fibrinogen purified by affinity chromatography and its plasmin degradation products, the binding sites on the fibrinogen molecule appeared to be located specifically in the D-domain. Conversely to the fibrinogen and the fragment D, radiolabeled fragment E, however, did not interact with cell. The binding was time-dependent, saturable and reversible. Scatchard analysis of the data obtained revealed an average of 6000 binding sites per germ tube with dissociation constant ( K _d) of 5.2 × 10⁻⁸ M. No potent competition was observed for a range of different proteins and carbohydrates. Fibrinogen fragment D binding proteins were identified using a dithiothreitol-iodoacetamide extract of the fungus. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, one compotent of 68 kDa was detected. Thus, the presence of fibrinogen binding proteins specifically localized on the cell wall surface of C. albicans germ tubes may constitute one of the factors involved in the development of candidosis.     

Adhesins in Candida albicans.

The adherent properties of Candida albicans blastoconidia and germ tubes have long been appreciated, but little is known about the mechanisms responsible for adherence. Recently, three genes, ALA1, ALS1 and HWP1, encoding proteins with adherent properties and motifs consistent with linkage to the beta-1, 6-glucan of C. albicans cell walls have provided insight into the topology of protein adhesins. Hwp1, a developmentally regulated adhesin of germ tubes and hyphae, attaches to buccal epithelial cells by an unconventional, transglutaminase-mediated mechanism of adhesion.     

Aggregation and disaggregation of Candida albicans germ-tubes

Abstract The spontaneous aggregation of Candida albicans germ tubes, which occurs in medium 199, has been studied. The disaggregating activity of various substances, such as sugars, enzymes and sulphydryl compounds has been tested by addition of these substances to the culture medium. Glutathione and dithiothreitol were the most effective in inhibiting aggregation without disturbing germ tube formation.
In a buffer solution, pH 7, the previously aggregated tubes were irreversibly dispersed by pronase. In the presence of glutathione and dithiothreitol, the process was reversible, since after washing with distilled water, germ tubes aggregated immediately.     

Cytoplasmic alkalinization during germ tube formation in Candida albicans

Weak acids were used to measure the internal pH of yeast cells of Candida albicans that had been induced to form buds or germ tubes. Under conditions that supported germ tube formation the internal pH rose from around 6.8 to over 8.0 after 30 min in two different induction media. Internal pH measured by 31P NMR confirmed this pattern and also showed that the internal pH fell to around 7.0 prior to the outgrowth of germ tubes. Conditions which led to budding induced less cytoplasmic alkalinization. This alkalinization was brought about when cells were inoculated into media of neutral pH and at an increased temperature. Increasing the temperature of the medium augmented the alkalinization of the cytoplasm induced by raising the external pH. Strains of C. albicans defective in the ability to produce germ tubes did not show this dramatic cytoplasmic alkalinization under conditions which normally supported filamentous growth. The raising of internal pH may be due to the activation of the plasma membrane proton-pumping ATPase since diethylstilboestrol inhibited the cytoplasmic alkalinization and germ tube formation without causing irreversible loss of cell viability. The results show that the induction of the dimorphic transition in this organism is accompanied by a steep rise in internal pH. It is not known whether these changes are the cause or consequence of morphogenesis.     

Effects of culture density on the kinetics of germ tube formation in Candida albicans

The relationship between culture density or phase of growth at 24.5 degrees C and the ability of Candida albicans to form germ tubes when shifted to 37 degrees C was investigated. Evidence is presented demonstrating germ tube production from liquid synthetic medium cultures at all phases of growth. Previous studies reported that only cells from stationary phase cultures were competent to form germ tubes. Comparisons between exponential and stationary phase cultures indicate more rapid and more synchronous germ tube production from cells growing in the exponential phase.     

The requirements for bicarbonate and metabolism of the inducer during germ tube formation by Candida albicans

Factors affecting germ tube formation in Candida albicans at suboptimal temperatures were investigated. Candida albicans formed germ tubes between 22 and 30 degrees C in solution when incubated without shaking, in the presence of bicarbonate (2 mg mL-1). Other conditions depended on the inducer used. Proline could induce germ tube formation optimally only when its concentration was between 200 and 400 mM. A concentration of 0.05 mM N-acetylglucosamine was sufficient to induce germ tube formation. N-Acetylglucosamine could induce germ tube formation at 30 but not at 25 degrees C. N-Acetylglucosamine induced germ tube formation was most reproducible when the cells were first starved by incubation in water for 16-24 h at 20 degrees C. Germ tubes induced by proline could be formed at pH values between 3.8 and 9.0 at 30 degrees C, but only between 7.0 and 7.5 at 25 degrees C. The addition of 0.05 to 5 mM glucose to a 5 mM proline induction solution allowed germ tube formation at 30 but not at 25 degrees C. Glucose (400 mM) did not suppress germ tube formation at 30 degrees C but only 5 mM was sufficient to cause a 65% suppression at 25 degrees C. The results show the importance of CO2 and (or) bicarbonate to the induction of germ tube formation and are consistent with the metabolism of the inducer.     

Influence of applied electrical fields on yeast and hyphal growth of Candida albicans

Yeast cells of Candida albicans which had been attached to polylysine-coated microscope slides were induced to form buds or germ tubes in the presence of external electrical fields. The sites of budding and germ tube formation and the growth of germ tubes and hyphal branches were polarized preferentially towards the cathode. Buds were not converted to pseudohyphae or germ tubes by the field and the field had no effect on the positioning of nuclei or septa in the yeast cell or germ tube. Buds were less polarized than germ tubes at any given applied voltage. The polarization of buds reached a peak at an electrical field of 12 mV per cell. Polarization of germ tubes was biphasic, increasing rapidly with increasing field strengths up to 5 mV per cell, and then more slowly in stronger fields. An electrical field was only required for a fraction of the time taken for germ tubes to start to form, so cells retained a memory of experiencing an electrical field which influenced the selection of sites of evagination. Increasing the electrical field delayed the time of germ tube evagination and inhibited the rate of germ tube extension. Unlike previous findings with other filamentous fungi, germ tubes grew unidirectionally towards the cathode for extended periods and did not deviate to a perpendicular orientation. This result suggests that the septal pore of the filamentous form may have high electrical resistance and would act as an effective barrier to solute transport between intercalary compartments.     

Morphological studies of N-acetylglucosamine induced germ tube formation by Candida albicans

In N-acetylglucosamine induced germ tube formation by Candida albicans, multiple (up to five) protuberances appeared within 90 min at 37 degrees C on each yeast cell. The protuberances were extensions of the cytosol and contained vesiclelike structures. Usually only one protuberance subsequently developed into a germ tube. The germ tubes emanated from all aspects of the cell surface but seldom from the budding (long axis) poles. Pseudohyphae, which originated from the budding pole, exhibited a marked constriction at the site of emergence and were 0.6-2.5 microns in diameter compared with a diameter of 0.6-0.8 micron for germ tubes. The presence of septa confirmed that germ tubes are precursors of septate mycelia. Ultrathin-section transmission electron microscopy of aldehyde plus osmium fixed cells revealed electron-lucent walls with a thin electron-dense outer layer. A fibrillar border was also routinely associated with germ tubes. Poststaining with potassium permanganate revealed, in addition, a previously invisible fuzzy layer on the outer region of the cell wall which extended over bud scars and germ tubes and which coalesced at sites of contact between cells.     

Adherence of germ tubes of Candida albicans to tissues from immunocompromised mice

Abstract The influence of immune status of the host on binding of germ tubes of Candida albicans to murine tissue sections in an ex vivo assay was examined. Generally, germ tubes appeared randomly adhered to the tissues examined and binding was unaffected by immunodeficiency induced by treatment with cyclophosphamide and cortisone acetate. Adherence was somewhat reduced in spleen and kidney sections or increased in liver sections and unchanged in lymph node sections from treated mice compared to sections from control animals. Scanning electron micrographs showed organisms appeared to be loosely or tightly bound to the surface or partially embedded in spleen sections from both control and treated mice. These observations suggested that qualitative and quantitative difference in adhesion of germ tubes to various tissues may contribute little to the susceptibility of the immunodeficient animal to candidal infection.     

Quantification of thigmotropism (contact sensing) of Candida albicans and Candida tropicalis

To quantify the thigmotropism, we adapted the our previous method using a chemotaxifilter system in combination with a bioluminescent adenosine triphosphate (ATP) assay based on firefly luciferase-luciferin system and analyzed the relationship between the ability of germ tube formation and thigmotropism of C. albicans and C. tropicalis. Both the ability to form germ tube and the amount of hyphae exhibiting thigmotropism varied depending upon both the species and strains of Candida. C. albicans formed more germ tubes than C. tropicalis. A good correlation was observed between the ability to form a germ tube and the capacity for thigmotropism, and the results gave a level of significance (p<0.05). Further, SEM observation revealed that relatively long hyphae of C. tropicalis with penetrated through the pores of filter membrane. This phenomenon may be of importance in the development of pathogenesis of C. tropicalis as well as C. albicans. This revised version was published online in August 2006 with corrections to the Cover Date.     

Germ tube formation from zonal rotor fractions of Candida albicans.

Homogenous cell populations of increasing cell volume may have been isolated from exponential and stationary culture of Candida albicans by centrifugation on a sucrose gradient. Observations of the yeast-mycelial transition using these populations showed the following. (i) No fraction from early logarithmic phase cells was unable to undergo morphological transition. (ii) The time of initiation of germ tube production was correlated with cell size in stationary-phase cultures. (iii) The rate of appearance of germ tubes was nearly identical in all fractions measured. (iv) Addition of N-acetyl-D-glucosamine to homogeneous cell populations decreased the time of initial appearance of germ tubes but did not affect the rate of appearance after initiation.     

Dynamic changes of the cell wall surface of Candida albicans associated with germination and adherence

The distribution of mannoproteins at the cell wall surface of Candida albicans was analyzed during the process of germination in conditions favoring adherence of germ tubes to a plastic matrix. Three cytochemical methods allowing the detection of concanavalin A binding sites, anionic sites and the enzyme acid phosphatase, respectively were used. All three methods gave similar results, indicating a spatial and temporal reorganization of some cell wall mannoproteins: a strong labeling was observed on blastoconidia; in contrast, as soon as the emergence of germ tubes took place, these reactions decreased dramatically at the surface of mother cells, whereas the germ tube surface was strongly stained. Some new components with multiple biological activities were detected at the germ-tube surface. Indeed, among mannoproteins responsible for an enhanced adhesion to plastic surfaces, two components with molecular weights of 68 and 60 to 62 kDa were shown to interact with laminin, fibrinogen, and C3d. This study therefore indicates that germination, and then adherence of germ tubes, imply a degradation of surface mannoproteins, and a simultaneous presentation of new molecules which can interact with their nonbiological materials or host proteins.