Albicans Yeast Cells Research Articles

Elimination of mouse splenic macrophages correlates with increased susceptibility to experimental disseminated candidiasis.

To evaluate the role of macrophages in experimental disseminated candidiasis, mouse splenic macrophages were eliminated by i.v. delivery of liposome-entrapped dichloromethylene diphosphonate (L-Cl2MDP). Splenic tissue sections that were immunoperoxidase-stained with mAbs against marginal zone macrophages (MONTS-4), red pulp macrophages (SK39), and neutrophils (SK208) showed that 3 days after L-Cl2MDP treatment, macrophages but not neutrophils were depleted, and circulating neutrophils responded normally to an irritated peritoneum and showed normal phagocytic ability. That is, in response to thioglycollate in the peritoneum, neutrophils migrated in normal numbers to the peritoneal cavity and expressed the normal activation phenotype of high Mac-1 and low Mel-14 Ag levels. These neutrophils also showed normal ability to ingest Candida albicans yeast cells in both in vitro and in vivo assays. However, the spleens from L-Cl2MDP-treated mice lost their ability to bind yeast, which agrees with our previous findings that yeast cells bind specifically to marginal zone macrophages. When macrophage-depleted were systemically challenged with C. albicans, clearance of viable fungal elements from blood was slower, their kidneys had higher recoverable CFU, and both BALB/cByJ and congenitally thymic-deficient (nude) mouse strains did not survive as long as control mice. Mice given L-Cl2MDP recovered most of their macrophage function by 56 days and became normal in their resistance to C. albicans. These results indicate that macrophages play an important role in host resistance to experimental disseminated candidiasis, but the mechanism does not appear to involve T cell functions.

Mouse sialoadhesin is not responsible for Candida albicans yeast cell binding to splenic marginal zone macrophages

Candida albicans hydrophilic yeast cells specifically adhere to mouse macrophages in the splenic marginal zone and in lymph node subcapsular and medullary sinuses. These macrophages express sialoadhesin that binds erythrocytes, but binding of yeast cells is not mediated by sialoadhesin because (i) erythrocytes did not block yeast cell binding, (ii) yeast cell adherence was unaffected by sialoadhesin-specific monoclonal antibodies, (iii) the purified sialoadhesin did not bind to yeast cells, and (iv) an Fc-sialoadhesin chimera immobilized on plastic did not bind yeast cells. These data give further support for a unique macrophage adhesion system that binds C. albicans.

Adherence of Candida albicans to endothelial cells is inhibited by prostaglandin I2.

The adherence of Candida species yeast cells to bovine and human endothelium was measured. The adherence phenomenon is a saturable event which can be altered by the release of prostaglandin I2 (PGI2 or prostacyclin) by endothelial cells. Release of PGI2 by endothelium has a protective effect, reducing Candida albicans yeast cell adherence to endothelial cells in a dose-dependent manner. Conversely, C. albicans appears to inhibit the release of PGI2 by endothelial cells and thus perhaps is capable of increasing the number of yeast cells which adhere to endothelium. Furthermore, a long-acting analog of PGI2, carbacyclin, reduced the number of yeast cells adhering to human platelet aggregates. Thus, it is possible that the release of PGI2 by endothelium has a protective effect against the hematogenous dissemination of C. albicans and its ability to adhere to vascular endothelium and fibrin-platelet matrices.

Evidence for the presence of a high-affinity laminin receptor-like molecule on the surface of Candida albicans yeast cells.

Two polypeptides of 37 and 67 kDa that bind laminin were detected in cell wall extracts of Candida albicans blastoconidia. The 37-kDa species, found only in yeast cell wall extracts, cross-reacted with a rabbit polyclonal antibody (PAb 4160) directed towards the carboxyl-terminal laminin-binding domain present in the human 67-kDa high-affinity laminin receptor (67LR) and its 37-kDa precursor (37LRP), whereas another antibody (PAb 4056), directed against internal domains of 67LR and 37LRP, recognized a 37-kDa species in wall extracts from both blastoconidia and germinated blastoconidia. Indirect immunofluorescence with PAb 4160 showed a patchy binding pattern only on yeast cells that represented about 10% of the entire blastoconidia population.

Wall formation by Candida albicans yeast cells: synthesis, secretion and incorporation of two types of mannoproteins.

The mannoprotein components solubilized from the walls of Candida albicans blastoconidia following degradation of the glucan network with beta-glucanase (Zymolyase) have higher molecular masses than their probable precursors present in the supernatant of regenerating protoplasts. It therefore appears that the mannoproteins are released from the walls as part of supramolecular complexes. Immunological analysis using both polyclonal and monoclonal antibodies has demonstrated the probable relationship between molecules found in a mixed membrane preparation, those secreted by regenerating protoplasts, and those present in yeast cell walls. Some mannoproteins secreted by protoplasts incubated in the presence of tunicamycin had significantly increased mobility on SDS-PAGE, whereas others were not affected by the treatment. It is therefore possible that two types of mannoproteins are secreted by protoplasts: one carrying N-glycosylated chains (mannan) and one lacking them. All the proteins secreted in the presence of tunicamycin stained with Concanavalin A-peroxidase, demonstrating that they all, including the N-glycosylated ones, carried O-glycosylated sugar residues. Both classes of mannoproteins, secreted independently of each other, were found in the molecular complexes rendered soluble from the wall by Zymolyase digestion. Data obtained with a monoclonal antibody demonstrated the presence of a repeated epitope within one wall protein(s) detectable in a mixed membrane preparation and in the wall complexes released by Zymolyase.

Chemical definition of an epitope/adhesin molecule on Candida albicans.

An antigen (Ag), as defined by reactivity with a monoclonal antibody (mAb 10G), was found on the cell wall surface and on the plasma membrane of Candida albicans yeast cells (Li, R. K., and Cutler, J. E. (1991) J. Gen. Microbiol. 137, 455-464). The 10G Ag from the cell surface location was solubilized by treatment with beta-mercaptoethanol and Zymolyase. Antigenic activity of the extract was lost following periodate oxidation, but was stable to proteolytic enzyme and heat treatments. Mannoproteins in the extract were fractionated by hexadecyltrimethylammonium bromide. A fraction containing the 10G Ag was degraded by mild acid hydrolysis and a tetrahexose eluted from a P-2 size exclusion column was found to be the epitope (10G epitope) part of the 10G Ag. By use of gas-liquid chromatography, mass spectroscopy, and H-1 proton NMR analysis, the 10G epitope was determined to be a linear beta-1,2-linked tetramannose. The 10G Ag, which was immunopurified from the Zymolyase extract, and the 10G epitope were involved in the attachment of C. albicans to mouse spleen marginal zone macrophages. Both the 10G Ag and the purified 10G epitope specifically inhibited C. albicans adherence in an ex vivo binding assay. Latex beads adsorbed with the 10G Ag or the 10G epitope adhered specifically to the splenic marginal zone macrophages. These data show that a beta-1,2-linked mannotetraose part of a C. albicans cell wall mannoprotein is an adhesin site that may play a role in pathogenesis of disseminated candidiasis.

Binding of Candida albicans yeast cells to mouse popliteal lymph node tissue is mediated by macrophages.

We previously reported that Candida albicans yeast cells adhere to the macrophage-rich medullary and subcapsular sinus areas of mouse lymph node tissue. To determine whether the yeast cell-lymph node interaction is mediated by macrophages, the effect of specific elimination of macrophages on yeast cell binding was studied, and yeast cell adherence was correlated with the ingestion of India ink by lymph node cells. Macrophage elimination was done by use of liposome-containing dichloromethylene diphosphonate (L-Cl2MDP). Mice were injected in the hind footpads with the L-Cl2MDP preparation, popliteal lymph nodes were removed 5 days later, and yeast cell adherence was determined by an ex vivo binding assay. As controls, lymph nodes from mice that received footpad injections of either phosphate-buffered saline (PBS) alone or liposome-containing PBS were used. Use of macrophage- and neutrophil-specific monoclonal antibodies in tissue immunostaining showed that the L-Cl2MDP treatment eliminated macrophages but not neutrophils from the medullary and subcapsular sinus areas of the popliteal lymph nodes. A striking reduction of yeast cell adherence occurred with lymph nodes from L-Cl2MDP-treated mice compared with lymph nodes from control animals. The lymph node-yeast cell binding patterns of L-Cl2MDP-treated and control mice were the same regardless of mouse strain, sex, or T-cell competency. Results of India ink experiments, in which India ink was injected into footpads of mice and was rapidly taken up by popliteal lymph node macrophages, showed a strong correlation between yeast adherence and India ink staining of cells. In addition, the interaction of yeast cells with lymph node tissue from normal mice was not significantly affected by the addition of two extracellular matrix proteins, fibronectin and laminin, during the ex vivo adherence assay. These data indicate that medullary and subcapsular sinus lymph node macrophages express an adhesion system similar to that described for mouse splenic marginal zone macrophages.

Adherence of Candida albicans to immobilized extracellular matrix proteins is mediated by calcium-dependent surface glycoproteins

The adherence of Candida albicans to the extracellular matrix proteins type I collagen and fibronectin (Fn) is dependent upon the presence of extracellular calcium. In the absence of calcium, 14±3% of yeast cells added to type I collagen adhered, whereas 62±3% of yeast cells adhered in the presence of 2 mM calcium. EDTA and EGTA reduced C. albicans adherence in a dose-dependent manner. Calcium, and to a lesser extent, cesium and magnesium, enhanced yeast cell adherence. The fluorescent probe, terbium (Tb +3), bound to the surface of yeast cells in a dose-dependent manner demonstrating the presence of cationic metal-binding sites on the yeast cell surface. When extracts of C. albicans yeast cells were applied to columns of Fn or gelatin coupled to agarose, two surface proteins of C. albicans were specifically eluted with 10 mM EDTA, 2% α-methylmannopyranoside or an Arg-Gly-Asp-containing peptide. The fungal proteins had relative molecular masses of 60 000 and 105 000 in the unreduced state. The proteins were present in the cell membrane as well as the cell wall and were demonstrated to be glycoproteins by their ability to bind concanavalin A. Immunoblot analysis of yeast extracts demonstrated that anti-integrin antibodies to the human fibronectin, vitronectin and complement receptor cross-reacted with the Candida 60 kDa glycoprotein. Thus, calcium-dependent fungal cell wall glycoproteins likely related to integrins may be receptors responsible for yeast cell adherence to host tissue such as the extracellular matrix.

Inhibition of pseudohyphal growth as a neutrophil-mediated host defense mechanism against experimental deep Candida albicans infections in mice.

Neutrophils have recently been found to have microbistatic activity in addition to their well-described microbicidal mechanisms; therefore, a murine model of deep candidiasis was used to evaluate the possibility that both antimicrobial activities might participate in the host defense against this type of infection. In nonleukopenic animals, subcutaneous injection of Candida albicans yeast cells produced local abscesses and early metastatic spread of the infection to the kidneys. Although completely isolated within a dense neutrophilic infiltrate, a significant proportion of the fungal cells in the subcutaneous abscesses of these animals remained viable for at least 6 to 10 days after inoculation; the infections at this site were observed to resolve after spontaneous rupture of the abscesses. Growth of the organisms appeared to be suppressed at later time points in these animals, as evidenced by the markedly reduced proportions of multicelled pseudohyphae (representing organisms that had undergone cell division) observed in their abscess exudates as compared with those in samples from leukopenic animals. In addition, at 10 days after inoculation, very few multicelled pseudohyphae were observed histologically in the subcutaneous infections of nonleukopenic animals, whereas masses of these forms were found in leukopenic ones. Fluids from the abscesses of the nonleukopenic animals appeared to contain growth-inhibiting activity for C. albicans, in that the organisms could not grow in them unless additional zinc were to be added to the medium. This type of zinc-reversibility is a characteristic of neutrophil microbistatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Yeast-specific DNA probes and their application for the detection of Candida albicans.

Two DNA fragments cloned from the genome of Candida albicans ATCC 10261 may be useful in the rapid diagnosis of disseminated candidosis. One sequence (probe EOB1) was specific for C. albicans (positive hybridisation with 45 strains tested). The second sequence (probe EOB2) detected C. albicans, as well as five other pathogenic Candida spp. and Saccharomyces cerevisiae, but did not react with human or bacterial DNA. Both probes were repetitive sequences in the genome of C. albicans. Probe EOB1 was used to detect, without DNA amplification, 500 C. albicans yeast cells in 1 ml of human blood.

Gamma interferon modifies CD4+ subset expression in murine candidiasis.

A single injection of monoclonal antibody to gamma interferon administered in conjunction with a live Candida albicans yeast cell vaccine resulted in the detection of nonprotective Th2 rather than protective Th1 responses and altered the early expression of interleukin 4 and gamma interferon mRNA in CD4+ cells.

Evidence that Candida albicans binds via a unique adhesion system on phagocytic cells in the marginal zone of the mouse spleen.

We recently demonstrated by using an ex vivo adhesion assay that Candida albicans yeast cells exhibit a unique binding affinity for the marginal zone of the spleen. This binding event provides a working model for studying mechanisms of organ dissemination of the fungus from the blood. By using the ex vivo assays reported here, we showed by bright-field and electron microscopic techniques that mouse spleen marginal zone cells capable of ingesting India ink particles are also involved in yeast cell attachment. During splenic clearance of yeast cells from the circulation in vivo, C. albicans is also associated exclusively with marginal zone cells capable of ingesting India ink. The ability to ingest the ink particles is not necessarily related to yeast cell adherence, because the fungal cells did not bind to phagocytic cells in the splenic red pulp. In fact, the marginal zone phagocytic cells appear to have a unique binding system, because yeast cells also did not bind to phagocytes in other tissues, such as the thymus and peritoneum, or to seven different myeloid cell lines. In addition, antibodies to a number of well-characterized murine adhesion molecules, such as leukocyte integrins, LECAM-1, and CD44, had no effect on binding. On the basis of these results, we propose that splenic marginal zone phagocytes express a novel adhesion system that involves either a unique adhesion molecule or previously described adhesion molecules with unique binding activities.

Mechanisms of aggregation accompanying morphogenesis in Candida albicans.

Cultures of Candida albicans yeast cells do not normally aggregate, but extensive aggregation accompanies the induction of mycelial growth, indicating the occurrence of cell surface changes during the yeast to mycelial transition. Aggregation correlated with the formation of germ tubes as did changes in surface charge determined by attachment to ion exchange sepharose beads. Yeast cells of all strains examined were negatively charged and attachment to positively charged (DEAE) sepharose beads increased following germ tube formation. If Mg2+ was present during germ tube formation, a high degree of clumping occurred that could only be dispersed by treatment with protein-disrupting agents. Trypsin, chymotrypsin, SDS, urea, guanidine HCl and dithiothreitol but not EDTA or EGTA caused irreversible dispersal of aggregates, although germ tube aggregates dispersed by treatment with buffers at high pH reaggregated if neutralized or if calcium ions were added. Germ tube cultures produced in divalent cation-deprived medium formed aggregates that were readily dispersed by washing. However, the addition of Mg2+ or other divalent cations (Ca2+, Zn2+, Cu2+, Fe2+) caused immediate aggregation of these cultures. These results suggest that divalent cation crossbridging between opposing anionic sites and protein interactions act synergistically to promote aggregation of C. albicans germ tube cells.

Effect of an arginine-glycine-aspartic acid-containing peptide on hematogenous candidal infections in rabbits

The adherence of Candida albicans yeast cells to the subendothelial extracellular matrix, fibronectin, laminin, and type I and IV collagen was tested. Fibronectin (10(-7) M) and a peptide, PepTite-2000 (Telios Pharmaceuticals Inc., San Diego, Calif.), containing the sequence arginine-glycine-aspartic acid (RGD) inhibited Candida adherence to these targets by greater than 90%. When C. albicans was perfused over ex vivo rabbit aortic endothelium, there was no significant difference in the amount of adherence in the presence or absence of the RGD-containing peptide. However, the RGD-containing peptide reduced the number of Candida organisms present in liver, brain, heart, and kidneys (P less than 0.05) of rabbits 4 h after intravenous inoculation of 5 x 10(7) C. albicans yeast cells. The peptide also reduced the number of macroscopic Candida abscesses in the kidneys of rabbits 72 h after intravenous inoculation of 10(7) C. albicans yeast cells (P less than 0.05). Inhibition of Candida adherence in vitro and in vivo may occur because the peptide blocks a fungal receptor that is necessary for adherence.

The Postantifungal Effect: Synergistic Interactions of Cispentacin and Pradimicin (BMY-28864) with Flucytosine against Candida albicans.

A turbidometric method was used to evaluate Candida albicans yeast cell growth and to quantitate the in vitro postantifungal effect (PAFE) after exposure to various concentrations of cispentacin and pradimicin (BMY-28864), alone and in combination with flucytosine, for 2hr at 30°C and 37°C. Initial MIC and FIC (fractional inhibitory concentration) assays were made to determine approximate drug concentrations to use in the PAFE studies. The PAFE was calculated by the difference in time (hr) required for growth of the control and test cultures to reach the 0.5 absorbance level following removal of the drug by dilution. A synergistic PAFE was evidenced with both cispentacin and pradimicin (BMY-28864) when combined with flucytosine. Optimal PAFEs ranging from 5.7 to 18.5hr were induced by concentrations of cispentacin (0.078 to 0.313μ/ml) and flucytosine (0.049-0.098μg/ml) and from 3.3 to 11.7hr with pradimicin (BMY-28864) (0.078 to 0.313μg/ml) and flucytosine (0.049-0.098μg/ml). The PAFEs persisted for 1.9 to 4.8hr (cispentacin+flucytosine) and for 0.3 to 2.9hr (pradimicin-BMY-28864+flucytosine) longer than those achieved with either agent alone.

Turbidometric characterization of the postantifungal effect: comparative studies with amphotericin B, 5‐fluorocytosine and miconazole on Candida albicans

The phenomenon of persistent suppression of Candida albicans yeast cell growth after short drug exposures (postantifungal effect, PAFE) was determined by performing comparative studies using different concentrations of 5-fluorocytosine (5-FC), amphotericin B (AMPH) and miconazole (MCZ). An in vitro turbidometric method was used to measure cell growth and to quantitate the PAFE after removal of the drug by dilution following exposure to C. albicans yeast cells for 0.5 h, 1 h or 2 h. The PAFE was determined by the difference in time (h) required for growth of the control and test cultures to increase to the 0.5 absorbance level following removal of the antifungal agent. A PAFE was demonstrated with each agent and generally the length of the PAFE was dependent upon the concentration of the drug and the time of exposure. An exposure time of 0.5 h resulted in PAFEs ranging from 0.6 to 16.7 h with 5-FC, 0 to 16.5 h with AMPH and 0.1 to 14.1 h with MCZ. In most instances exposure of the cells to each drug for 1 or 2 h resulted in slightly longer PAFEs, respectively. Longer PAFEs were induced with lower concentrations of 5-FC as compared to AMPH and MCZ. The data from such PAFE assays may be useful for determining in vivo treatment regimens, since longer PAFEs may allow for intermittent dosing instead of continuous drug administration.

Production of granulocyte-macrophage colony-stimulating factor by large granular lymphocytes stimulated with Candida albicans: role in activation of human neutrophil function

In the present study, culture supernatants from larger granular lymphocytes (LGL) that were activated with Candida albicans antigens were shown to stimulate the ability of neutrophils to inhibit fungal growth. Identification of the activation factors showed that granulocyte-macrophage colony-stimulating factor (GM-CSF), a hematopoietic growth factor, was involved. Human peripheral blood mononuclear cells were fractionated by Percoll density centrifugation and each subpopulation of cells was stimulated with C albicans yeast cells. GM-CSF was produced in those fractions enriched for LGL, but not T lymphocytes or adherent monocytes. Additionally, the phenotype of the GM-CSF-producing cell was found to be CD2+, CD16+, HLA-DR+, and negative for CD4, CD8, and CD15. Kinetic studies demonstrated that GM- CSF appeared in the supernatants within 2 days of culture and continued to be produced up to 7 days. Optimal stimulation of LGL was seen at a ratio of 3 heat-killed C albicans yeast cells per LGL. Thus, LGL play an important role in host defense against this opportunistic pathogen by producing cytokines, including GM-CSF, which in turn activates the fungicidal activity of neutrophils.

Production of Granulocyte-Macrophage Colony-Stimulating Factor by Large Granular Lymphocytes Stimulated With Candida Albicans: Role in Activation of Human Neutrophil Function

In the present study, culture supernatants from larger granular lymphocytes (LGL) that were activated with Candida albicans antigens were shown to stimulate the ability of neutrophils to inhibit fungal growth. Identification of the activation factors showed that granulocyte-macrophage colony-stimulating factor (GM-CSF), a hematopoietic growth factor, was involved. Human peripheral blood mononuclear cells were fractionated by Percoll density centrifugation and each subpopulation of cells was stimulated with C albicans yeast cells. GM-CSF was produced in those fractions enriched for LGL, but not T lymphocytes or adherent monocytes. Additionally, the phenotype of the GM-CSF–producing cell was found to be CD2+, CD16+, HLA-DR+, and negative for CD4, CD8, and CD15. Kinetic studies demonstrated that GM-CSF appeared in the supernatants within 2 days of culture and continued to be produced up to 7 days. Optimal stimulation of LGL was seen at a ratio of 3 heat-killed C albicans yeast cells per LGL. Thus, LGL play an important role in host defense against this opportunistic pathogen by producing cytokines, including GM-CSF, which in turn activates the fungicidal activity of neutrophils.

Differential adherence of hydrophobic and hydrophilic Candida albicans yeast cells to mouse tissues

Using an ex vivo binding assay, we previously demonstrated that yeast cells grown at 37 degrees C display binding specificity in mouse spleen, lymph node, and kidney tissues. In spleen and lymph node tissues, binding was predominantly in regions rich in macrophages. Here, we tested the possibility that hydrophobic and hydrophilic cells bind differentially to host tissues. Hydrophobic and hydrophilic yeast cells of four Candida albicans strains were incubated for 15 min at 4 degrees C with cryostat sections of organs that had been rapidly frozen after removal from BALB/cByJ mice. Unattached cells were removed by washing, and the sections were examined. Hydrophobic cells bound diffusely and abundantly to all tissues, while hydrophilic cell attachment was restricted to specific sites. For example, hydrophobic cells bound to the white and red pulp and the marginal zones in spleens, whereas hydrophilic cells attached primarily to the marginal zones. Hydrophobic yeast cells attached throughout lymph node tissue including paracortical areas, but hydrophilic cell attachment occurred primarily at the subcapsular and trabecular sinuses, EDTA inhibited the adherence of hydrophilic cells but not hydrophobic cells to mouse tissues. Hydrophobic C. albicans strains displaying similar levels of hydrophobicity differed quantitatively in their levels of attachment to kidney and spleen tissues, confirming our earlier observation that surface hydrophobicity is not the sole determinant in adherence to host cells. Other studies have shown that hydrophobic and hydrophilic cells display different virulence characteristics related to their surface properties and that hydrophobic cells are more virulent than hydrophilic cells. Taken together, the present results suggest that the enhanced virulence of hydrophobic cells over hydrophilic cells may be due, in part, to the potential of hydrophobic cells to bind throughout various organs following clearance from the bloodstream.

Activation of the cAMP cascade by steroidogenic hormones and glucagon in the pathogenic fungus Candida albicans

Incubation of Candida albicans yeast cells with human luteinizing hormone (hLH), human chorionic gonadotrophin (hCG) or glucagon produced a significant rise in cAMP total levels. The effect of these hormones in permeabilized cells of the fungus produced a 2–3 fold increase in the Mg 2+, GTP-dependent adenylyl cyclase activity as well as full activation of the cAMP-dependent protein kinase (PKA) activity. These results indicate that the interaction of the mammalian hormones with the fungus triggered the cAMP activation cascade in a similar way to that found in higher eukaryotic organisms.