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Keywords:

  • Candida albicans;
  • Mutant strain;
  • Virulence

Abstract

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

The virulence of Candida albicans 92′, a morphological mutant unable to filament, was assayed in an experimental model of systemic candidiasis in three strains of mice with different susceptibilities to the infection. The mortality parameters studied pointed to the low virulence of this mutant strain. Study of the fungal load of C. albicans 92′ in kidneys and brain revealed the presence of low numbers of CFUs and a high percentage of clearance, particularly in the brain. Adhesion studies demonstrated a reduced capability of the mutant to adhere to human epithelial cells. This strain can be considered a potential tool for cloning genes involved in virulence.


1Introduction

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

Candida albicans is an opportunistic pathogen of increasing relevance in the incidence of candidiasis in immunocompromised hosts [1]. A number of factors are thought to influence the virulence of this fungus [2,3]. Among them are its ability to undergo the dimorphic switch from a budding yeast to a hyphal form [4] and its adhesion capacity [5,6]. Although some strains of C. albicans are partially defective in hyphal development [7–9] or adhesion [10,11] and its virulence has been assayed [9–11], only two mutant strains that fail to form filaments in response to serum or other inducers of filamentous growth have been considered avirulent in a mouse infection model [7,8].

In this work, we study the virulence of the morphological mutant C. albicans 92′, a non-filamenting strain isolated in our laboratory [12]. To assess the relationship between the low virulence of the mutant and its adhesion ability, we evaluated the adhesion of C. albicans 92′ in comparison to the wild-type. Adhesion was tested in vitro by flow cytometry.

2Materials and methods

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

2.1Microorganisms, culture and infection conditions

C. albicans 1001 (also ATCC 64385) was obtained from the Spanish Type Culture Collection (Department of Microbiology, University of Valencia, Spain).

C. albicans 92′ is a morphological mutant obtained in an earlier screening in which C. albicans 1001 cells were treated with UV light (640 erg mm−2); the survival rate was 80%[12]. Sugar utilisation patterns determined with an API 20C AUX gallery (API system) showed that C. albicans 1001 and 92′ had the same pattern of sugar fermentation. The mutation is genetically stable over long periods of time (>200 generations) and the strain maintains its phenotype after serial passage in broth culture and after recovery from the organs of mice.

The morphological mutant C. albicans 92′ gave rise to pointed colonies, different from the wild-type ones (Fig. 1A,B), and failed to produce mycelia under the conditions usually employed to induce hyphal morphogenesis in wild-type strains, such as Lee medium or media with inducers such as N-acetylglucosamine or proline (Fig. 1C,D). 92′ blastospores were unable to form hyphae in horse/human serum at 37°C. No further phenotypic alteration in the usual pattern of blastoconidial development by budding was observed (data not shown). The growth rates (measured as glucose consumption [13]) of both 1001 and 92′ were similar when incubated in human serum at 37°C (data not shown).

image

Figure 1. Colonial and cellular morphology of C. albicans 1001 (A, C) and 92′ (B, D). Cells were incubated in foetal calf serum at 37°C for 5 h.

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For infection studies, parental and mutant strains were grown at 30°C in YED agar plates (1% Difco yeast extract, 2% glucose, and 2% agar). After 24 h, cells were harvested, washed twice with phosphate-buffered saline (PBS) and diluted to the desired density in the same buffer for injection into the lateral tail vein of the mice at a volume of 0.5 ml.

2.2Mice

The mouse strains used were inbred BALB/c, DBA/2 and the hybrid (BALB/c×DBA/2) CD2F1. Both male and female mice, ranging in age from 12 to 16 weeks and with a weight of about 18 g, were used in the virulence studies. All animal studies were carried out at the Animal Facilities of the School of Medicine at the Universidad Complutense of Madrid.

2.3Quantification of yeast cells in kidneys and brains and histological examination

Kidneys and brains from individual mice were removed aseptically and placed in a tissue homogeniser with 5 ml and 3 ml of PBS, respectively. The number of viable yeast units in the specimens (six to eight mice per group) was determined by a plate dilution method, using YED/chloramphenicol [14]. Results (means±S.D.) were expressed as log CFU per mouse. For histological studies, tissues were excised and immediately fixed in formaldehyde, and sections (3–4 μm) of paraffin-embedded tissues were stained with PAS reagent or haematoxylin/eosin [15].

2.4Adhesion analysis

The method used was a modification from the cytometric assay described by Polacheck et al. [16].

2.4.1Yeast labelling

Optimal labelling conditions were the following: 2×107 yeast cells ml−1 treated with 1μg ml−1 of FITC for 30 min. Then, cells were washed twice with PBS and diluted to the appropriate concentration.

2.4.2Epithelial cells preparation

Two human cell lines were used for the adherence assays: HeLa (ATCC CCL2) (cervical epithelial cells) and Henle-407 (ATCC CCL6) (intestinal epithelial cells). Henle-407 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) and HeLa cells were maintained in RPMI, both supplemented with 10% heat-inactivated fetal calf serum, 2 mM l-glutamine, 50 IU penicillin and 50 μg ml−1 streptomycin. Growth was performed at 37°C in a humidified 5% CO2-95% air incubator. Prior to exposure to the yeasts, cells were transferred to 100-mm Petri dishes and grown for 24 h, to 75% confluence. Cells were then washed three times with PBS and immediately prior to adherence assays, monolayers of cells were treated or not with cytochalasin D (5 μg ml−1) for 1 h at 37°C.

2.4.3Adherence reaction

In the adherence reaction, 5×105 epithelial cells were mixed with yeast cells at different epithelial cell:yeast ratios (1:10, 1:50 or 1:100). The yeast/epithelial cell mixtures and control tubes containing only yeasts or only epithelial cells were incubated at 37°C for 60 min. Afterwards, the adherence reaction mixtures were washed twice with PBS and fixed with 1% paraformaldehyde for 10 min and washed again twice with PBS. Then, cells were collected for flow cytometric analyses.

2.4.4Flow cytometry assays

A FACScan flow cytometer (Becton-Dickinson), calibrated routinely for immunofluorescence analysis, was used to determine the fluorescence intensities of yeasts and epithelial cells. The excitation wavelength was 488 nm, and emitted light was collected. Data were analysed with CellQuest software (Becton-Dickinson).

3Results

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

3.1Course of infection in BALB/c, CD2F1 and DBA/2 mice

Three inbred strains of mice (BALB/c, DBA/2 and the hybrid CD2F1) exhibiting different patterns of susceptibility to candidiasis were infected intravenously with four different doses of 1001 or 92′ blastospores: 1×107, 1×106, 1×105 and 5×104. The course of infection was monitored by the mortality parameters and the level of fungal load in the most representative organs: brain and kidney [17]. Fig. 2 shows that at the highest doses (1×107, 1×106) of 1001 all the mice died within a few days. At the lower doses, chronic infection was observed in strains BALB/c and CD2F1 but not in the susceptible DBA/2 mice, all of which died of an acute infection. No mortality was observed in BALB/c and CD2F1 mice upon inoculation with similar doses of C. albicans 92′, whereas 10–15% of DBA/2 mice died upon infection with the highest dose. Using the PRESTA statistical program (F.I.S. Spain) [18], the Cox proportional hazards model for survival data (Cox regression) was applied. After this statistical analysis, the results obtained can be assumed to be representative. The strain of C. albicans significantly modified survival; the risk of death was much lower with the mutant than the parental strain (RR-CI95: 0.001–0.011).

image

Figure 2. Curves of survival from the three strains of mice used when injected with C. albicans 1001 (♦) or 92′ (?).

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3.2Quantitation of C. albicans viable units and histology

The degree of fungal load in kidneys and brains of infected mice is shown in Table 1. As can be observed, the number of CFUs recovered from the kidneys of the mice infected with 1×107 and 1×106 blastospores of strain 1001 was very high (log CFU 5–6) and was similar among the three strains of mice. Under these experimental conditions, all the mice died. At doses of 1×105 and 5×104, the degree of fungal load depended on the strain of mice (DBA/2>CD2F1>BALB/c). After challenge with 1×107 and 1×106 blastospores of strain 92′, clearance of infection was observed in more than 60% of the population, while the rest had log CFU values of 2–3, notably lower than that obtained in C. albicans 1001 infection. When the 1×105 and 5×104 doses were used, renal clearance was observed in 100% of the mice. These results were identical for all three strains of mice.

Table 1.  Course of 92′/1001 infection in mice of three different strainsa
  1. aAge-matched mice were intravenously challenged with various doses of C. albicans 1001 and 92′. Median survival times (MST, days) and number of dead mice at 60 days over total animal tested (D/T) are shown.

  2. bParallel groups of mice infected as above were killed the day before the MST for CFU enumeration in their kidneys and brains. Values are presented as log CFU±S.D. from mice that had not cleared infection. Clearance of infection: ***40%; **more than 60%; *100% of population, respectively. nt: not tested. Statistically different log CFU kidney (P= 0.000) and log CFU brain (P= 0.000) (ANOVA test by C. albicans strain; the following variables were significant in both kidney and brain (P<0.00015): time (>time<CFU), mouse strain (BALB/c<CFU), dose (>dose>CFU).

DoseC. albicans strainMTSD/Tlog CFUb
    KidneyBrain
BALB/c mice
1071001311/116.06±0.404.48±0.38
 92′>600/102.67±0.28***
1061001612/125.63±0.584.22±0.56
 92′>600/122.03±0.31***
1051001>605/102.99±1.211.89±0.24
 92′>600/11**
5×1041001>605/163.42±1.45****
 92′>600/12**
CD2F1 mice
107100138/86.81±0.124.68±1.04
 92′>600/92.37±0.37***
1061001712/125.71±0.133.90±0.14
 92′>600/122.25±0.20***
1051001>606/163.18±1.03***1.96±0.18***
 92′>600/9**
5×1041001>607/164.10±1.61**2.03±0.45**
 92′>600/8**
DBA/2 mice
1071001110/10ntnt
 92′>601/72.15±0.40***
1061001110/106.10±0.343.92±0.12
 92′>601/82.30±0.40***
105100156/65.85±0.532.84±1.12
 92′>601/101.95±0.24***
5×1041001815/155.47±0.482.02±0.28
 92′>600/15**

Challenge with the 1×107 and 1×106 doses of strain 1001 afforded log CFU values of 3–4 in brain, while a log CFU of about 2 was observed following inoculation of the low doses. By contrast, strain 92′, regardless of the dose or the strain of mice, produced clearance of the infection in the brains of all the mice inoculated (Table 1).

Histological analyses of the kidneys were performed at different days post challenge. On day +1, the 1001 strain was able to colonise the renal pelvis, a prominent inflammatory reaction being seen throughout the renal tubules together with disorganisation of the glomeruli. In contrast, with the inoculation of strain 92′, no alterations were observed (data not shown). Analyses on day +3 revealed the most striking differences: C. albicans 1001 induced very pronounced inflammatory foci, producing a severe destruction of renal tissue and the parenchyma, which killed the mice within 3 days (Fig. 3A1/A2). Contrariwise, at the same time of infection, the C. albicans 92′ strain produced no alterations and the kidneys remained healthy (Fig. 3A3/A4), this persisted even after 60 days of infection (data not shown).

image

Figure 3. Effects of infection on kidneys (A) and brains (B) from BALB/c mice infected with 1×106 cells of C. albicans 1001 and 92′. A1/A2: Tissue sections prepared from kidneys of mice infected with strain 1001 on days 1 (A1) and 3 (A2) post infection. A strong inflammatory reaction can be observed with the presence of C. albicans inside the tubules (haematoxylin/eosin; ×150). A3/A4: Renal sections from mice infected with 92′ on days 1 (A3) and 3 (A4) post infection. Neither lesions nor evidence of fungal growth can be observed (haematoxylin/eosin; ×150). B1/B2: Tissue sections prepared from brains of mice infected with strain 1001 on days 1 (B1) and 3 (B2) post infection. A considerable degree of tissue damage and a high degree of fungal growth can be seen. B3/B4: Tissue sections from brains of mice infected with 92′ on days 1 (B3) and 3 (B4) post infection. Neither lesions nor evidence of fungal growth can be observed (haematoxylin/eosin; ×150).

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Histological examination of the brains of the mice infected with the 1001 strain pointed to a slight inflammatory reaction on day +1 that was increased by day +3 (Fig. 3B1/B2), while the 92′ strain induced no alterations on days +1 and +3 (Fig. 3B3/B4).

3.3Adherence of C. albicans 1001 and 92′ to epithelial cells

A comparative adherence analysis of both C. albicans strains was performed with flow cytometry using two different epithelial cell lines (see Section 2.4). Prior to the adhesion reaction, the labelled yeasts and epithelial cells (not labelled) were analysed by flow cytometry, both affording a histogram with a single peak with higher and lower (basal) fluorescence, respectively (Fig. 4). In the adherence reaction, the adhesion of the labelled yeasts to the epithelial cells was reflected in a histogram with two peaks representing two subpopulations of epithelial cells: one without adherent yeasts (basal fluorescence of epithelial cells) and the other one with adherent yeasts, the latter population showing higher fluorescence intensity. Experiments were performed at different epithelial cell:yeast ratios: 1:10 (Fig. 4), 1:50 and 1:100. At the higher yeast cell concentrations (1:50 and 1:100), yeast aggregates were observed (data not shown).

image

Figure 4. Flow cytometric assay of adherence of C. albicans 1001 (A) and 92′ (B) to Henle-407 cells. Henle-407 (EC) and labelled yeasts, serving as controls, were incubated separately and analysed without gating. In the adherence reactions (EC: 1001 and EC: 92′), the parameters used to select the EC population were size and granularity (because of its higher size and complexity), ‘gating out’ non-adherent yeasts. Control experiments were defined as: 4°C (temperature of reaction); 56°C (inactivation of the yeast prior to the reaction) and w/o CytD (epithelial cells not treated with cytochalasin D).

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Study of C. albicans 1001 adhesion revealed a peak of the fluorescent subpopulation corresponding to 56% of the epithelial cells (Henle-407), while the proportion of the low-fluorescence subpopulation (epithelial cells alone) decreased correspondingly with respect to the controls (Fig. 4A). In the case of the 92′ strain, the percentage of epithelial cells bearing labelled yeasts was 36%, indicating a reduced adhesion compare to the wild-type (Fig. 4B). The results obtained with HeLa cells were comparable (data not shown).

In order to check the accuracy of this assay, control analyses were done under conditions known to decrease yeast adherence to epithelial cells [16]. These conditions involved incubation of the labelled yeasts for 30 min at high temperature (56°C), which possibly inactivates Candida adhesins [19] and an adherence reaction performed at a low temperature (4°C), where the kinetics are very slow. Both treatments resulted in a reduced adherence of the yeasts to the epithelial cells, as demonstrated by the unaltered proportion of the peak of the low-fluorescence subpopulation (Fig. 4). The similar behaviour observed with both yeast strains in all these control conditions reinforces the validity of these experiments. Another control involved carrying out the adherence reaction without cytochalasin D (Fig. 4). In this case, the decrease in the percentage of adhesion reflected a different susceptibility to phagocytosis by the epithelial cells, depending on the C. albicans strain used: 34% for 1001 and 46% for 92′.

4Discussion

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

The virulence of C. albicans 92′, a morphological mutant unable to form hyphae under all filament-inducing conditions (Fig. 1), was assayed in a mouse model of systemic candidiasis in inbred mice. Three strains of mice exhibiting different patterns of susceptibility to infection with C. albicans[20] were infected with four different doses of 1001 and 92′ blastospores. After statistical analysis of the results obtained (Fig. 2, Table 1) C. albicans 92′ can be considered to have extremely low virulence, significantly lower, even, than other strains classified as avirulent [7,8].

The level of colonisation of this mutant strain was analysed. The organs studied were the brain and the kidneys, whose involvement in disseminated Candida infections in humans and in mice has been previously described [3,17]. The number of log CFU recovered from the kidneys after inoculation with C. albicans 1001 increased with the dose and with the susceptibility of the mouse strain (Table 1), this kind of behaviour is consistent with the data on fungal load reported in the literature. In contrast, the percentage of renal clearance and the log CFU of the mice injected with C. albicans 92′ after 60 days (chronic infection) was always the same, regardless of the strain of mice used (Table 1). This type of behaviour is completely different from that observed by other authors [21], where C5-deficient mice showed higher levels of tissue colonisation. Histological analyses also revealed a fast clearance and the absence of tissue damage in both the kidneys and brains of the mice infected with strain 92′ (Fig. 3). The reduced virulence and the rapid clearance of C. albicans 92′ from kidneys observed in strains of mice with different Th-dependent responses suggest that recovery from 92′ infection may be independent of the role played by Th1/Th2 cytokine patterns.

The low virulence of C. albicans 92′ and its low level of organ colonisation might be due to poor tissue adhesion, as previously described for other mutant strains [11]. The vascular endothelium plays a critical role during the dissemination through the circulation, because blood-borne organisms probably adhere to and penetrate the endothelial cell lining of the blood vessels to gain access to the tissue parenchyma [22]. The results in Fig. 4 show that the 92′ strain was able to adhere to epithelial cells, although to a lower extent than strain 1001. The accuracy of the assay was confirmed using different conditions [16] that reduced the adhesion capacity of both C. albicans strains. The greatest advantage of this flow cytometric method is that very low epithelial:yeast cell ratios can be used, avoiding the flawed conclusions that could be derived from experiments performed at high ratios, where yeast coadhesion might occur and be mismeasured as adhesion to epithelial cells [23].

Alone, this reduced adhesion capacity of strain 92′ cannot explain the absence of virulence. The results obtained with cytochalasin D (an inhibitor of phagocytosis) [22], which allowed us to discern between internalisation-plus-killing and adhesion, indicated that epithelial phagocytosis as observed by flow cytometry was higher for the 92′ strain; this, together with reduced adhesion capability of this strain, might explain the reduced virulence and the lack of tissue damage in the infection by strain 92′. In forthcoming studies we plan to further investigate the phagocytosis of neutrophils and macrophages with both C. albicans strains (1001 and 92′).

Given the low virulence, the reduced adhesion capacity and, probably, the higher susceptibility to phagocytosis of the 92′ strain, we think that our mutant will be a good recipient for cloning genes involved both in morphological switching (in vitro) and in virulence (in vivo). In order to test this latter possibility, a preliminary experiment was carried out to mimic the injection of a pool of transformants from a library. Mice were injected with a low dose of strain 92′ (known to produce clearance of the infection) together with 100 and 1000 cells of the wild-type strain. The only cells recovered from the kidneys corresponded to the wild-type, which is able to colonise tissues. We think that, using 92′ as recipient strain, it might be possible to clone genes that allow yeast cells to maintain in different organs using a murine model of systemic candidiasis.

Acknowledgements

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

We thank Alicia Couso for her helpful technical assistance; Dr. José Luis González for his expert histological analysis; Dr. Rosalina Pomés for previous studies and Dr. Antonio Portolés for his help with the statistical analysis. This work was supported by the Fondo de Investigaciones Sanitarias (FISS) (Spain) with Grant 97/0047-01.

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