• Candida albicans;
  • flow-cytometry;
  • macrophages;
  • pathogenesis;
  • survival;
  • trehalose


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Accumulation of trehalose by yeast is an important protective mechanism against different stress conditions. This study examined the effect of trehalose on several growth features, as well as its association with the intracellular survival of yeasts exposed to macrophages. A tps1/tps1 mutant and its parental counterpart, CAI4, exhibited similar growth rates and preserved their dimorphic conversion and agglutination ability. However, electron-microscopy of cell-wall architecture showed a partial loss of material from the outer cell-wall layer in the tps1/tps1 mutant. Flow-cytometry revealed that the mutant had lower auto-fluorescence levels and a higher fluorescein isothiocynate staining efficiency. When co-cultured with macrophages, a slight reduction in binding to macrophages and slower ingestion kinetics were revealed for the tps1/tps1 mutant, but these did not interfere significantly with the amount of yeast ingested by macrophages after co-incubation for 2 h. Under the same conditions, CAI4 cells were more resistant to macrophage killing than was the tps1 null mutant, provided that the macrophages had been stimulated previously with interferon-γ. Measurement of trehalose content and the anti-oxidant activities of yeast cells recovered after phagocytosis revealed that the trehalose content and the glutathione reductase activity were increased only in CAI4 cells, whereas levels of catalase activity were increased similarly in both strains. These results suggest that the presence of trehalose in Candida albicans is a contributory factor that protects the cell from injury caused by macrophages.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The opportunistic pathogen Candida albicans is a component of commensal microbiota, and causes either superficial or potentially lethal systemic infections in humans. C. albicans is currently the fourth most common cause of bloodstream infection (candidaemia) in the USA [1]. The high morbidity and mortality provoked by C. albicans in immunocompromised individuals, e.g., AIDS patients, transplant recipients and individuals subjected to aggressive surgery or prolonged chemotherapy [2,3], makes this yeast one of the most prominent human pathogens. Nowadays, although effective drugs are available for the treatment of candidiasis, the increasing prevalence of resistant strains requires a search for new, more specific and potent, antifungal agents [4,5].

Yeast cells accumulate large amounts of the non-reducing disaccharide trehalose, both as a reserve compound and as a protector of cellular integrity against different conditions of nutritional deprivation and environmental injury (e.g., oxidative and osmotic stress, heat-shock or xenobiotics) [6–8]. Trehalose accumulation plays an important role in yeast physiology, but this sugar is absent from mammalian cells [8]. Hence, the enzymes involved in the trehalose biosynthetic and hydrolytic pathways could be considered as potential targets for antifungal therapy [9]. In C. albicans, trehalose-6-phosphate synthase activity, and subsequent storage of trehalose, is induced in response to oxidative treatments, and mutants incapable of trehalose synthesis (tps1/tps1) are highly susceptible to oxidative stress exposure in vitro[10]. Moreover, the tps1 gene is involved in virulence, as the tps1 null mutant displays a clearly lower infection rate than its parent strain (CAI4) when inoculated into mice [11].

Studies using murine models of disseminated candidiasis have demonstrated that macrophages play an important role in host resistance [12–14]. Macrophages are phagocytic cells of the immune system that are able to recognise and eliminate microorganisms crossing the epithelial barrier. Although these cells express the machinery for antigen presentation, their main contribution to antifungal defence involves the phagocytosis and killing of fungi [14–16].

The recognition of C. albicans by macrophages is based on components of the yeast cell-wall, and is mediated through receptors present on the cellular membrane of macrophages [17]. The yeast cells are then endocytosed and destroyed by the combined action of different enzymes, nitrogen compounds and reactive oxygen species (ROS), including ·OH, O2 and H2O2[14]. Fungi have evolved various mechanisms or putative virulence factors to evade phagocytosis, escape destruction and enable them to survive inside macrophages. Such mechanisms include the synthesis of specific molecules and/or the activation of enzymes with a protective effect [18–20], the dimorphic conversion from unicellular yeast forms to mycelial structures [21], and the triggering of macrophage apoptosis [22].

Based on these antecedents, the present study examined the hypothesis that C. albicans mutants defective in trehalose synthesis might exhibit increased susceptibility to the lytic action of ROS generated by macrophages. Likewise, an additional analysis of phenotypic features related to virulence (i.e., dimorphic conversion, growth efficiency or cell-wall composition) was performed to examine the hypothetical connection between the absence of trehalose and other physiological processes involved in resistance to macrophage killing.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Reagents and antibodies

Hydroethidine and fluorescein isothiocyanate (FITC) were obtained from Molecular Probes (Eugene, OR, USA). All other reagents were obtained from Sigma (St Louis, MO, USA).

Cell lines and culture

The murine macrophage-like tumour cell line J774 (ECACC 85011428, from a female BALB/c mouse) was obtained from ATCC (Rockville, MD, USA). Adherent cells were cultured at 37°C in an atmosphere containing CO2 5% v/v in RPMI medium (BioWhittaker, Verviers, Belgium) supplemented with heat-inactivated fetal calf serum 10% v/v (Gibco, Grand Island, NY, USA), 5 mM L-glutamine (Seromed Biochrom, Berlin, Germany), streptomycin 100 mg/L and penicillin 50 mg/L (Flow Laboratory, Irvine, UK), hereafter referred to as ‘complete medium’. Before use, cells were washed twice in complete medium at room temperature, gently scraped off, counted in a Neubauer haemacytomer chamber, and either used directly for suspension cell assays, or dispensed into 24-well tissue culture plates at a concentration of 4 × 105 cells/mL or into Petri plates (Sarsted, Granollers, Spain) at 6 × 105 cells/mL for assays with adherent cells. Viability was assessed by trypan blue exclusion (Gibco) and was >95%.

Isolation of peritoneal macrophages

Mouse peritoneal macrophages were obtained from BALB-c or Swiss CD-1 mice, aged 6–8 weeks, by injecting 2 mL of phosphate-buffered saline (PBS) containing protease peptone broth 10% w/v. After 4 days, the mice were killed humanely and peritoneal macrophages were harvested by injecting 4 mL of sterile RPMI. After centrifugation and washing, the cells were resuspended in complete medium and cultured for 24 h in 75-cm2 culture flasks. For co-culture experiments, adherent cells were recovered by gentle scraping and transferred into 24-well microtitre plates at 4 × 105 cells/well in a final volume of 1 mL of complete medium. Animal experiments were approved by the Animal Care Committee of the University of Murcia.

Yeast strains and growth conditions

The parental strain of C. albicans, CAI4 (ura3Δ::imm434/ura3Δ::imm434) (TPS1) [23], and its isogenic trehalose-deficient derivative tps1/tps1[11], were used throughout. For some experiments, Saccharomyces cerevisiae strain MCY1264 (maα, gal, his3Δ, leu2::HIS3, lys2-801, ura-3-52) [24] was also used. The cultures were grown in medium containing peptone 2% w/v, yeast extract 1% w/v and galactose 2% w/v (YPGal). The yeasts were maintained by periodic subculturing on solid YPGal, or were harvested, washed, aliquoted and stored at −80°C in medium containing glycerol 25% v/v until required. Before use, yeast cells (blastoconidia) were transferred to fresh liquid YPGal medium and incubated for <14 h at 28°C with shaking. Yeast cells were then recovered, washed with PBS, counted in a Neubauer haemacytomer chamber, and transferred to RPMI.

Transmission electron-microscopy

Cells were pelleted (3000 g, 10 min, 4°C) and immediately fixed in glutaraldehyde 2.5% v/v (Serva, Heidelberg, Germany) in 0.1 M cacodylate buffer, pH 7.2–7.4, for 1 h at 4°C, post-fixed in OsO4 1% w/v and embedded in Epon before sectioning on a Reichert Jung ultramicrotome [25]. Ultra-thin sections were stained with uranyl acetate and lead citrate and examined with a Zeiss EM 10C electron-microscope.

Co-culture of yeast cells with mammalian cells

After culture for 18 h, J774 cells or peritoneal macrophages were washed with complete medium. For co-culture studies, adherent plated cells were incubated with yeasts at a ratio of 10:1 yeasts/macrophage. After incubation, the cultures were washed with PBS to remove unbound yeast cells, and the samples were then harvested for either biochemical analysis or fungicidal assays.

Analysis of phagocytosis by flow-cytometry

Phagocytosis experiments were performed with cells in suspension. J774 macrophages (1.5 × 106 cells/mL) were stained with hydroethidine (59 µM) for 1 h at room temperature, washed and resuspended in complete medium. In turn, yeasts (5 × 106 cells/mL) were labelled with FITC (0.75 mg/mL in PBS) for 30 min at 37°C, washed with PBS, and resuspended in complete culture medium at appropriate concentrations. Binding and phagocytic assays were performed in 5-mL tubes (Falcon; Becton Dickinson, Madrid, Spain) by mixing 100 µL of hydroethidine-labelled macrophages and 100 µL of FITC-labelled yeast cells in appropriate concentrations to obtain different macrophage/yeast cell ratios, centrifuging (1000 g, 5 min, 20°C) and culturing in suspension at 37°C for 30 min. The reaction was stopped by adding 500 µL of ice-cold PBS. The fluorescence of extracellular cells (i.e., free yeast cells and yeast cells adhering to phagocytes, but not internalised) was quenched by addition of 40 µL of trypan blue (0.4% w/v in PBS) per sample. The samples were then immediately mixed gently and analysed in a fluorescence-activated cell sorter. Binding experiments were analysed after co-incubation for 5 min without quenching.

Flow-cytometry was performed using an FC500 cytometer (Beckman Coulter, High Wycombe, UK) equipped with an argon ion laser with an excitation power of 15 mW at 488 nm. Forward scatter and side scatter were analysed on linear scales, while green (FL1) and red fluorescence (FL2) intensities were measured on logarithmic scales. Standard samples of FITC-labelled C. albicans cells or J774 macrophages were included in each phagocytosis assay. Samples incubated at 4°C were used as negative controls for phagocytosis. To select the macrophage population, analysis gates were set around debris and intact cells on a forward scatter vs. side scatter dot plot, and around red fluorescence on FL1 vs. FL2 dot plots. The fluorescence histograms of 5000 macrophage cells were generated using the gated data. The percentage of phagocytosing macrophages was the percentage of cells with one or more ingested yeast cells (fluorescent cells simultaneously green-FL1 and red-FL2) among the total macrophage cell population (5000 events displaying FL-2). The percentage of ingested yeasts/cell was calculated from the mean FL1 fluorescence intensity of the cells. The percentage of adhesion was expressed as the number of cells associated with yeasts among the total cell population. Data acquisition and analysis were performed using WINMDI software v.2.8 (

Quantification of macrophage fungicidal activity

Quantification experiments were performed with adherent cells. After monolayer cells were challenged with C. albicans, the medium was removed at the indicated times and the monolayers were washed thoroughly twice with cold PBS. The mixtures were then resuspended in 1 mL of sterile distilled water at 37°C for 5 min and agitated vigorously with a micropipette until the macrophages were completely lysed. Inspection of the initial lysate revealed only individual yeast cells, 98% of which were still in the yeast phase. Finally, the concentration of yeast cells in the lysates was determined and appropriate dilutions were seeded on YPGal plates. After incubation at 28°C for 48 h, CFUs were counted and the percentage of surviving yeast was calculated in comparison with the CFUs of yeast grown in the same conditions without macrophages.

Preparation of cell-free extracts

Cell-free extracts were prepared as described previously [26]. In brief, purified intracellular yeasts were harvested, washed with cold water, and resuspended at known densities (usually 10–15 mg/mL, wet weight) in 10 mM 4-morpholine-ethanesulphonic acid buffer, pH 6.0, plus 5 mM cysteine and 0.1 mM phenylmethanesulphonyl fluoride. The cellular suspensions were transferred into small pre-cooled tubes (0.5 cm in diameter) with 1.5-g Ballotini glass beads (0.45 mm in diameter). The cells were broken by vigorous vibration of the tubes in a vortex mixer for six cycles, each of 45 s. The crude extract was then centrifuged at 10 000 g for 5 min and the supernatant fraction was filtered through Sephadex G-25 NAP columns (Amersham Pharmacia Biotech, Uppsala, Sweden) that had been equilibrated previously with 50 mM potassium phosphate buffer, pH 7.8, in order to remove low molecular mass compounds interfering with enzymic activities.

Enzyme assays

Catalase activity was determined at 240 nm by monitoring the removal of H2O2, and glutathione reductase (GR) activity was assayed by measuring the oxidised glutathione-dependent oxidation of NADPH, as described previously [26].

Other measurements

Intracellular trehalose was extracted from 20–50 mg of yeast samples in 2 mL of boiling water and the concentration was measured with commercial trehalase (Sigma), using the method described by Blázquez et al. [27], except that glucose was estimated by the glucose oxidase–peroxidase procedure. Parallel controls were run to correct for the basal levels of glucose.

Growth was monitored by measuring the optical density of cultures at 600 nm in a Shimadzu UV spectrophotometer or by direct cell counting with a haemacytometer. Protein was estimated by the method of Lowry et al. [28], with bovine serum albumin as standard.

Statistical analysis

Data are represented as averages (±SE) and were analysed by one-way analysis of variance (anova) and the unpaired Student's t-test.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Outer cell-wall compositions

Disruption of both chromosomal copies of the tps1 gene causes pleiotropic effects, e.g., an inability to grow on glucose and fructose at certain temperatures, and a lack of hyphal formation at 37°C on glucose and at 42°C on all carbon sources [11]. These growth defects can be prevented by substituting glucose with galactose as the carbon source (YPGal medium). Growth features of the parental (CAI4) and trehalose-deficient (tps1/tps1) strains were therefore analysed in order to discount the existence of other effects in the mutant that could bias resistance to macrophage killing. The growth rates and generation times of both strains were roughly equivalent, the stationary phase being reached after incubation for c. 18 h. Moreover, microscopic analysis of blastoconidia, cultured with fetal calf serum at 37°C, revealed identical behaviour in agglutination reactions and a similar degree of hyphal formation (data not shown). Hence, growth defects or a failure to form filaments could be excluded as possible mechanisms of evading macrophage killing. These results also confirmed that the tps1 null mutant was competent to enter the morphogenetic programme, and that trehalose mobilisation is not required for this process [11,29].

As shown in Fig. 1(a), the morphology of tps1/tps1 populations analysed by flow-cytometry was more homogeneous than that of the parental strain CAI4, which showed a higher percentage of aggregated yeast cells. Histograms representing dose–response to FITC staining showed that the tps1 null mutant had lower auto-fluorescence levels, a higher intensity of green fluorescence for all concentrations of FITC investigated, and a cell population that was more homogeneously stained (Fig. 1(b),(c)). These data suggest the existence of differences between the strains at the external cell-wall level.


Figure 1.  Analysis of morphology and efficiency of fluorescein isothiocyanate (FITC) staining by flow-cytometry. Yeasts were cultured for 12 h on YPGal medium, washed with phosphate-buffered saline and stained with FITC for 30 min. (a) Cellular size (side scatter (SSC)) with respect to the cellular complexity (forward scatter (FSC)) of the yeast population in a bitmap. (b) Cell number (Events) with respect to the FITC fluorescence intensity (FL1-H) of 5000 cells from a previously selected population stained with FITC at different concentrations. (c) Fluorescence intensity in strain CAI4 (squares) and the tps1/tps1 mutant (triangles) with respect to the FITC concentration of the samples shown in (b). The data are the results of a single experiment that was representative of two independent experiments performed in duplicate.

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To uncover possible structural differences that could be involved in the adhesive capacity and susceptibility to intracellular lysis by macrophages, the morphology of both yeast strains was studied by transmission electron-microscopy (Fig. 2). The number and size of internal vesicles (dense particles in the cytoplasm) depended on the functional stage of each cell, and there were no significant variations between the two strains. However, CAI4 showed a continuous and dense structure at the outer cell-wall level, while these cell-wall components appeared to be detached and surrounding the cell of the tps1/tps1 mutant. Although further analysis is required to define the composition of this material [25], its presence might affect further binding and internalisation by macrophages.


Figure 2.  Analysis of morphology by electron-microscopy. CAI4 (a) or tps1/tps1 (b) yeasts were cultured for 12 h in YPGal medium at 28°C, processed, and analysed by electron-microscopy (×5000). Bars represent 2 µm. The micrographs represent the results obtained in two independent experiments, each analysing 50 yeast cells/strain.

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Adherence and ingestion by macrophages

The first step in phagocytosis involves the recognition of pathogen-associated molecular patterns on the microbial surface by pattern recognition receptors expressed on the membrane of phagocytes, and this is followed by adhesion to the pathogen mediated by these receptor–ligand interactions. The capacity of both yeast strains to adhere to macrophages, as well as their susceptibility to ingestion, was examined by flow-cytometry in phagocytic assays after co-culturing J774 cells with C. albicans yeast strains at different yeast/macrophage ratios.

Both strains adhered to the membrane of macrophages in a dose–response fashion after a short challenge period of 5 min (Fig. 3(a)). However, the tps1/tps1mutant displayed a lower adherence capacity than CAI4, and the differences became greater as the number of yeast cells increased. These results suggest the existence of a chemical difference in the cell-wall composition of mutant cells that could be involved directly in the interaction with the recognition receptors of macrophages. However, despite the different rate of binding to yeasts, macrophages showed similar phagocytic ability for both strains after incubation for 30 min, reaching saturation at a 10:1 yeast/macrophage ratio (Fig. 3(b)). Further phagocytic assays were performed at this ratio, since macrophages displayed their maximal phagocytic capacity, and therefore phagocytosed yeasts were recovered at high density for enzymic measurements.


Figure 3.  Analysis of binding and phagocytosis as a function of the yeast/macrophage cell ratio and time for J774 hydroethidine-stained cells and CAI4 (squares) or tps1/tps1 (triangles). Fluorescein isothiocyanate-stained yeasts were co-cultured in suspension at 37°C for 30 min. (a) Percentages of adhesion after 5 min. (b) Percentages of phagocytosing macrophages after 30 min at different yeast/cell ratios. (c) Percentages of phagocytosing macrophages at a 1:10 cell/yeast ratio at different time-points. (d) Percentages of ingested yeasts at a 1:10 cell/yeast ratio at different time-points. Percentages were calculated by analysing the data obtained by flow-cytometry, and are the means of three independent experiments, each using duplicates. Asterisks represent statistically significant differences (p <0.05).

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When the time course of phagocytosis at a 10:1 yeast/macrophage ratio was monitored, no significant differences were observed in the percentage of macrophages with ingested tps1/tps1 or CAI4 blastoconidia (Fig. 3(c)). However, the number of mutant cells engulfed by the macrophages tested was lower for up to 30 min with respect to the parental strain (Fig. 3(d)), indicating that the reduced adherence rate is accompanied by slower ingestion.

In addition, adherent J774 cells were co-cultured for 60 or 120 min at a 1:10 cell/yeast ratio, and the difference between the number of ingested and bound yeasts was analysed by phase-contrast microscopy. Despite the longer incubation time, some macrophages remained free of intracellular C. albicans cells, regardless of strain, while the number of yeasts inside a particular macrophage was also variable (Fig. 4). However, after co-incubation for 1 h, more extracellular yeasts were observed in tps1/tps1 cultures, which is consistent with the flow-cytometry data for shorter periods (Fig. 4). Nonetheless, when incubation was prolonged for 2 h, the phagocytosis of both yeast strains was complete. These results confirm the altered adhesive capacity of tps1/tps1 and the slower kinetics of ingestion, although the ability of macrophages to engulf either strain after they were in contact with the macrophage membrane was similar.


Figure 4.  Optical microscopy analysis of ingestion and binding of yeast cells by macrophages. J774 cells were co-cultured as monolayers in eight-well chamber slides for 60 or 120 min at a 1:10 cell/yeast ratio with the CAI4 or tps1/tps1 strains grown in exponential phase. Differences in the ingestion or binding of yeasts by macrophages were determined by analysing the samples with phase-contrast microscopy (×100). The micrographs represent the results obtained in two independent experiments in which 50 macrophages/sample were analysed.

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Resistance of tps1/tps1 to macrophage killing in interferon (IFN)-γ-pre-stimulated macrophages

Trehalose accumulation and expression of anti-oxidant enzymes in C. albicans have been described as mechanisms for defence against oxidative stress [10]. To assess the role of trehalose in the ability of C. albicans to survive the stress conditions generated during phagocytosis, the viabilities of the tps1/tps1 and CAI4 strains were compared after ingestion by either J774 cells (Fig. 5(a)) or peritoneal macrophages obtained from BALB-c (Fig. 5(b)) or Swiss CD-1 (not shown) mice. The non-pathogenic yeast S. cerevisiae was used as a control for the efficiency of killing by J774 cells. Fig. 5(a) shows that >75% of C. albicans cells survived after macrophage challenge for 2 h, with no significant differences between intracellular viability for the two strains. In contrast, S. cerevisiae MCY1264 showed a significant decrease in viability compared with C. albicans. Nevertheless, peritoneal macrophages from BALB-c mice were more efficient in eliminating C. albicans, although no clear distinction in the degree of viability between the strains was revealed (Fig. 5(b)). Similar results were achieved with peritoneal macrophages obtained from Swiss-CD1 mice (data not shown).


Figure 5.  Intracellular yeast survival after phagocytosis. J774 cells (a) and peritoneal macrophages (b) obtained from female BALB-c mice (aged 8 weeks), unstimulated or pre-stimulated with interferon (IFN)-γ (100 U, 18 h), were co-cultured at a 1:10 cell/yeast ratio with Candida albicans CAI4 (empty bar) or tps1/tps1(solid bar), or with Saccharomyces cerevisae MCY1264 (stacked bar), for 2 h at 37°C. Endocytosed yeasts were recovered from macrophages by osmotic lysis, and the number of CFUs was scored after 48 h. The results are expressed as a percentage of the yeast viability found in controls cultured in the same conditions in the absence of macrophages. Error bars represent the SE of four independent experiments performed in duplicate. Asterisks represent statistically significant differences (p <0.05) between CAI4 and tps1/tps1 or MCY1264.

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Furthermore, when the intracellular survival of yeast challenged with J774 cells (Fig. 5(a)) and that of yeasts challenged with BALB-c peritoneal macrophages stimulated previously with IFN-γ (100 U for 18 h) (Fig. 5(b)) were compared, the survival rate was c. 1.5-fold lower for tps1/tps1 than for CAI4 blastoconidia (Fig. 5(b)). Thus, the tps1/tps1 mutant was more susceptible to killing mediated by IFN-γ-stimulated macrophages than was its parental strain CAI4, probably as a result of deficient protection against oxidative stress within the phagolysosome as a consequence of an inability to synthesise trehalose.

Trehalose content and anti-oxidant enzymic activities

To analyse further the level of resistance to injury caused by IFN-γ-stimulated macrophages, the trehalose content and the activity of two specific anti-oxidant enzymes (i.e., catalase and GR) were determined directly in cells recovered after phagocytosis. As expected, the amount of endogenous trehalose in the tps1/tps1 strain did not vary significantly (Table 1), confirming that disruption of the tps1 gene had occurred [11]. However, the level of trehalose increased almost three-fold in parental cells within 2 h of being internalised by macrophages (Table 1). In turn, the levels of catalase and GR were higher for the tps1 null mutant in basal conditions, but were modulated differently in response to macrophage ingestion (Fig. 6). A similar rise in catalase activity was observed in both strains after phagocytosis (Fig. 6(a)), whereas GR activity only increased noticeably in engulfed CAI4 cells (Fig. 6(b)). Hence, activation of the anti-oxidant response cannot compensate for the absence of trehalose in the tps1/tps1 mutant defence against phagocytic lysis. These results suggest strongly that trehalose storage, as well as some anti-oxidant activities, are contributory elements in the C. albicans defensive system that ensures cellular protection against attack by activated macrophages.

Table 1.   Trehalose content of Candida albicans CAI4 and the tps1/tps1 mutant
 Trehalose (nmol/min/mg protein)
  • a

    Increasing factor of trehalose content with respect to the control (1.0) is shown in parentheses.

Control4.97 (1.0)a1.53 (1.0)
Phagocytosed13.7 (2.7)1.62 (1.05)

Figure 6.  Effect of phagocytosis on the enzymic activity of catalase and glutathione reductase in Candida albicans. J774 cells were co-cultured at a 1:10 cell/yeast ratio, with C. albicans CAI4 or the tps1/tps1 mutant for 2 h at 37°C. The catalase (a) and glutathione reductase (b) enzymic activities were measured in yeast recovered from macrophages (solid bar) or yeast cultured in the same conditions in the absence of macrophages (empty bar). The data are the results of a single experiment that was representative of three independent experiments performed in duplicate. ‘a’ indicates a statistically significant difference (p <0.05) between yeast in the presence and absence of macrophages, and ‘b’ indicates a statistically significant difference between CAI4 and the tps1/tps1 mutant.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

A powerful approach for the study of host–pathogen relationships is to analyse the in-vitro interaction between a particular microorganism and purified host mammalian immune cells. This approach has proved particularly useful for studying the infectivity and resistance of mutant microbial strains that are defective in specific molecular pathways. In the present study, this approach was used to study the resistance of a C. albicans tps1 null mutant (defective in trehalose synthesis) and its parental strain CAI4 to killing mediated by both J774 and resident peritoneal murine macrophages.

Preliminary experiments revealed no significant variations in the growth rates, dimorphic conversion and agglutination ability of these strains. Therefore, these factors do not appear to be involved in the decreased virulence observed previously in mice [11,30]. In contrast, the differences detected with the tps1/tps1 mutant concerning cell-wall architecture, auto-fluorescence levels and FITC staining efficiency indicate the existence of structural and/or chemical modifications in the composition of the cell-wall. Since FITC forms a stable thio-urea bond through free amino groups of proteins or peptides [31], the higher FITC-staining efficiency of the tps1/tps1 mutant could reflect a higher amount and/or exposure of amine groups in its cell wall. This could be explained, at least in part, by the loss of external cell-wall components that was observed by electron-microscopy. In this respect, it was also found that the mutant displayed a slightly lower capacity to adhere to macrophages and kinetics of ingestion than did CAI4. These results support the hypothesis that the tps1/tps1 mutant has differences in its cell-wall composition, probably as a consequence of a pleiotropic effect of TPS1 disruption. Nevertheless, the ability of macrophages to engulf either strain after contact with the macrophage membrane was the same, and all the yeast cells present in both cultures were completely phagocytosed by the macrophages after co-incubation for 2 h.

The C. albicans cell-wall is a complex structure, composed mainly of glycans that play an important role in the continuous interchange that regulates the balance between saprophytism and parasitism [17]. Masuoka and Hazen [32] showed that the hydrophobicity of the C. albicans cell-wall is dependent on the level of glycosylation. Highly hydrophobic cells have longer and more abundant β-1,2-oligomannosyl side-chains, and seem to be more adherent to a variety of host tissues and more resistant to phagocytic killing than hydrophilic cells [32]. Glycan variation in the outer cell-wall could therefore provide a possible explanation of the differences between the two strains at the cell-wall surface.

As a general rule, survival of C. albicans inside phagocytic cells is determined by the offensive and self-protective abilities with regard to the harmful environment inside phagosomes [18–22]. Thus, trehalose accumulation and activation of anti-oxidant enzymes have been proposed as possible mechanisms for defence against oxidative stress in C. albicans. It has been shown previously that the trehalose-deficient mutant tps1/tps1 undergoes dramatic cell killing when exposed to acute oxidative stress (50 mM H2O2), despite the fact that the enzymic anti-oxidant response is activated [10,26]. Hence, in order to assess the role of trehalose in the ability of C. albicans to withstand the stress conditions generated within macrophages, the present study analysed and compared the survival rate, the content of endogenous trehalose, and the activity of the anti-oxidant enzymes catalase and GR, in the tps1/tps1 and CAI4 strains after phagocytosis mediated by J774 cells.

According to the in-vitro assays of basal candidacidal activity performed with J774 cells and murine resident macrophages, no significant differences in terms of intracellular viability were observed. In support of these results, it was reported that the susceptibility to phagocytosis mediated by macrophages of some C. albicans mutants did not correlate with C. albicans virulence in an in-vivo model of infection [33,34]. Nevertheless, when J774 cell lines or peritoneal macrophages were stimulated previously with IFN-γ, the tps1 null mutant was more susceptible to injury than its parent CAI4. This sensitivity is probably a result of deficient protection against oxidative stress within the phagolysosome as a consequence of an inability to synthesise trehalose. IFN-γ activates the lytic machinery of macrophages, enhancing ROS and NO production, and modulates the expression of certain membrane receptors associated with recognition and ingestion of C. albicans and antigen presentation [14,35,36]. Although some studies have suggested that the IFN-γ priming of mouse [34] or human [37] phagocytes is insufficient to enhance their candidacidal activity, the activating effect on the lytic activity mediated by ROS was sufficiently high to demonstrate the differences in sensitivity to macrophage killing between the CAI4 and tps1/tps1 strains.

It has been shown previously that oxidative stress produced by H2O2 induces the synthesis of trehalose without increasing TPS1 mRNA levels, suggesting that H2O2-induced trehalose-6-phosphate synthase activation might be caused by post-translational modification of the pre-existing enzyme [38,39]. Indeed, the present results demonstrated a marked increase in the content of endogenous trehalose in CAI4 cells in response to phagocytosis.

The levels of mRNA encoded by genes for catalase and GR in C. albicans, as well as their subsequent enzymic activation, are clearly increased following H2O2 exposure [26,40]. C. albicans also exhibits an obvious transcriptional anti-oxidant response to phagocytes, which includes genes coding for catalase, superoxide dismutase and cytochrome c peroxidase [19,20]. Thus, although the tps1 null cultures presented higher anti-oxidant activity levels in basal conditions, the rate of induction of catalase activity observed after phagocytosis was similar for both strains, while GR levels were raised noticeably only in CAI4 cells. Therefore, the lower resistance to phagocytosis exhibited by tps1 cells means that the induction of anti-oxidant activities cannot counteract the lack of trehalose accumulation. In contrast with results obtained after H2O2 treatment in vitro[26], the existence of a compensatory mechanism for the default in a trehalose-deficient mutant is only supported partially. A plausible explanation is that yeast cells need to develop a more elaborate and complex response inside the phagolysosomes against the different injurious and destructive agents that are secreted. The absence of GR stimulation in the tps1/tps1 mutant following ingestion may contribute, together with the absence of trehalose, to the drastic loss of viability caused by macrophage killing.

In conclusion, although the putative roles played by the distinct structures in the cell-wall organisation and/or composition of the CAI4 and tps1/tps1 strains require more extensive study, the present work suggests strongly that the presence of trehalose in C. albicans contributes to cell protection against the injury mediated by macrophages. Further studies of resistance to macrophage killing in mutants with other genetic defects in the trehalose metabolic pathways should confirm the importance of trehalose as a component protective against phagocytosis.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank C. Ferrer and A. Zuasti for helpful discussions concerning electron-microscopy. This work was supported by projects PB/07/FS/02 and PB/27/FS/02 of the Fundación Séneca, and BIO-BMC 06/01-003 of DGI, CARM (Murcia, Spain).


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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