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Keywords:

  • chitin;
  • flow cytometry;
  • paradoxical effect;
  • echinocandins

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Acknowledgements
  6. Literature Cited
  7. Supporting Information

The conventional methods used to evaluate chitin content in fungi, such as biochemical assessment of glucosamine release after acid hydrolysis or epifluorescence microscopy, are low throughput, laborious, time-consuming, and cannot evaluate a large number of cells. We developed a flow cytometric assay, efficient, and fast, based on Calcofluor White staining to measure chitin content in yeast cells. A staining index was defined, its value was directly related to chitin amount and taking into consideration the different levels of autofluorecence. Twenty-two Candida spp. and four Cryptococcus neoformans clinical isolates with distinct susceptibility profiles to caspofungin were evaluated. Candida albicans clinical isolate SC5314, and isogenic strains with deletions in chitin synthase 3 (chs3Δ/chs3Δ) and genes encoding predicted GlycosylPhosphatidylInositol (GPI)—anchored proteins (pga31Δ/Δ and pga62Δ/Δ), were used as controls. As expected, the wild-type strain displayed a significant higher chitin content (P < 0.001) than chs3Δ/chs3Δ and pga31Δ/Δ especially in the presence of caspofungin. Ca. parapsilosis, Ca. tropicalis, and Ca. albicans showed higher cell wall chitin content. Although no relationship between chitin content and antifungal drug susceptibility phenotype was found, an association was established between the paradoxical growth effect in the presence of high caspofungin concentrations and the chitin content. This novel flow cytometry protocol revealed to be a simple and reliable assay to estimate cell wall chitin content of fungi. © 2013 International Society for Advancement of Cytometry

Chitin is a β-1,4-homopolymer of N-acetylglucosamine that is synthesized by chitin synthase enzymes (1). This polysaccharide is present in most fungi and together with β-1,3-glucan, plays a fundamental role in maintaining fungal cell integrity and conferring structural rigidity during growth and morphogenesis (1–3). Mutations in glucan synthase genes reduce glucan levels in the cell wall while stimulating salvage pathways, leading to increased chitin synthesis. This pathway restores the strength of the cell wall matrix and prevents antifungal action (4). This compensatory increase in cell wall chitin synthesis enables some Candida species to grow at high caspofungin concentrations, a phenomenon termed paradoxical growth effect. As chitin is not present in human cells, inhibition of chitin synthesis has been proposed as a potential, selective antifungal target.

The assessment of cell wall chitin content based on glucosamine release through acid hydrolysis has been used extensively; however, this method is very laborious and time consuming (5–7). Epifluorescence microscopy has also been widely used to quantify chitin levels in fungi stained by Calcofluor White (CFW), a specific chitin dye (7–12). However, with this approach, only a limited number of yeast cells can be analyzed and the quantification of the fluorescence emitted cannot be performed accurately (7, 13, 14). Flow cytometry represents an efficient and fast approach for the analysis of cell architecture and functional phenotypes, with considerable advantages over conventional methods (15–18).

Here, we describe a fast and reliable protocol to measure cell wall chitin content in yeasts cells based on flow cytometric analysis after CFW staining.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Acknowledgements
  6. Literature Cited
  7. Supporting Information

Strains

Twenty-two Candida spp. and four Cryptococcus neoformans clinical isolates with well-characterized susceptibility profiles to caspofungin (antifungal chosen as representative of echinocandin class), were used in this study (Table 1). SC5314 (19) with wild-type chitin levels (used as positive control), chs3Δ/chs3Δ (Myco 3) (used as negative control) (20), pga62Δ/Δ (21), and pga31Δ/Δ (21) were used as control strains as their chitin contents were previously determined using other methodology (21).

Table 1.  In vitro antifungal susceptibility and paradoxical effect of caspofungin (CFS) against Candida spp. and C.ryptococcus neoformans clinical isolatesa. Minimal inhibitory concentrations (MIC; μg ml-1) were determined using prominent inhibition as an end point, corresponding to 50% (MIC50), according to CLSI protocol.
YeastStrain codeSourceCFS MIC50 (μg mL-−1) / phenotypeParadoxical growth (mean values) Start point/ end point (μg mL-−1)
  • a

    Minimal inhibitory concentrations (MIC; μg ml-1) were determined using prominent inhibition as an end point, corresponding to 50% (MIC50), according to CLSI protocol.

Ca. glabrataCg1Blood≫32/ NSNF
Cg2Blood0.125/ SNF
Cg3Peritoneal fluid32/ NSNF
Cg4Fecal0.125/ SNF
Cg5Peritoneal fluid0.5/ SNF
Cg6Blood0.25/ SNF
Ca. parapsilosisCp1Peritoneal fluid4/ NSNF
Cp2Blood2/ SNF
Cp3Blood4/ NSNF
Cp4Blood4/ NS16/64
Cp5Blood0.5/ SNF
Ca. tropicalisCt1Blood0.5/ S8/16
Ct2Peritoneal fluid0.5/ SNF
Ct3Pus4/ NS16
Ct4Pus4/ NS16
Ca. kruseiCk1Urine1/ SNF
Ck2Blood1/ SNF
Ck3Bronchial secretions1/ SNF
Ck4Bronchial secretions1/ SNF
Ca. albicansCa1Blood0.5/ S16/32
Ca2Blood0.5/ S16/32
Ca3Blood0.5/ S16/32
C. neoformansCn1Blood16/ NSNF
Cn2Blood16/ NSNF
Cn3Blood16/ NSNF
Cn4Blood32/ NSNF

Measurement of Cell Wall Chitin Content

Wild-type and mutant yeast cells were grown in YPD broth medium at 35°C, 150 rpm, until late logarithmic phase, and used to optimize flow cytometric protocol. A 106 yeast cells mL−1 suspension in sterilize distilled water was stained with 0 (autofluorescence), 2.5, 6.25, 12.5, and 25 μg CFW mL−1 (Fluka, St. Louis, MO), a specific chitin dye (excitation at 355 nm and emission at 433 nm), for 15 min at room temperature. In parallel, yeast cells were treated with minimal inhibitory concentration (MIC) values of caspofungin during 2 h, and stained with CFW. The yeast cells were washed twice and blue fluorescence (Pacific blue channel: 405-450/50 nm) emitted by 50,000 cells simple gated in FSC versus SSC parameters were quantified, using a BD FACSCanto™ II (Becton Dickinson, San Jose, California, USA) flow cytometer manufacturer by BD Biosciences (Supporting Information Fig. S1). BD FACSCanto™ II system consists of an excitation source with three lasers: blue (488-nm, air-cooled, 20-mW solid state), red (633-nm, 17-mW HeNe), and violet (405-nm, 30-mW solid state). The mean intensity of fluorescence (obtained from three independent experiments) emitted from stained (positive population) and nonstained (autofluorescence or negative population) yeast cells was analyzed and processed with FACSDiva software (version 6.1). In each experiment, a staining index (SI) was calculated as follows: (mean intensity of fluorescence of positive population − mean intensity of fluorescence of negative population)/2 × standard deviation of the mean intensity of fluorescence of negative population (22). The chitin content of the 26 clinical isolates was assessed according to the described protocol, after staining with 2.5 μg CFW mL−1 and the SI was calculated.

Epifluorescence Microscopy

To confirm flow cytometry results, epifluorescence microscopy analysis was performed. Yeast cells were grown and prepared as described for flow cytometric assays and stained with 25 μg CFW mL−1 for 15 min. After staining, 30 μL of the cell suspension was placed on a glass slide and overlapped with vectashield fluorescence mounting medium (Vector Laboratories, Peterborough, United Kingdom) and observed under an epifluorescence microscope (400×) Axioplan Zeiss, coupled with acquisition image system AxioVision (Zeiss, Barcelona, Spain).

Paradoxical Effect of Caspofungin

The ability of the clinical isolates to grow in the presence of high caspofungin (Merck, Rahway, NJ levels, termed paradoxical growth, was tested over a range of concentrations varying from 0.03 to 256 μg mL−1 and MICs were determined using prominent inhibition as an endpoint corresponding to 50% (MIC50) (23, 24). The paradoxical effect was defined as a progressive increase in cell growth occurring at least two drug dilutions above the MIC, following 48-h incubation (23).

Data Analysis

The SI mean values displayed by the different isolates after CFW staining were compared using the Student's t-test. Significant effects were accepted at P < 0.05. The SPSS Statistics 17.0 Software for Windows was used to perform the statistical analysis. All experiments were performed in triplicate.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Acknowledgements
  6. Literature Cited
  7. Supporting Information

The cytometric protocol was optimized using four Candida albicans strains with known differences in chitin contents: chs3Δ/chs3Δ, pga31Δ/Δ, pga62Δ/Δ, and the reference strain SC5314. These strains are deleted in genes that are involved in chitin synthesis (chs3Δ/chs3Δ) or encode GPI proteins that are involved in cell wall biosynthesis or in cell wall salvage pathways (pga31Δ/Δ and pga62Δ/Δ) (7, 21). A range of CFW concentrations was tested and 2.5 μg CFW mL−1 revealed to be the concentration to achieve the best resolution to differentiate the chitin content of the four strains used as controls. The positive control strain (wild-type SC5314) showed higher intensity of fluorescence than the negative control strain (chs3Δ/chs3Δ) (Figs. 1B and 1E, respectively). After treating the cells with caspofungin, a significant increase of the intensity of fluorescence was observed only in the reference strain (Figs. 1C and 1F). The reference strain SC5314 had significantly higher SI values (P < 0.001) than strains chs3Δ/chs3Δ and pga31Δ/Δ and had lower values when compared to the pga62Δ/Δ strain (Fig. 2). Flow cytometry chitin measurements were concordant with the chitin levels determined by the quantification of glucosamine released by acid hydrolysis, previously obtained by others (7, 20, 21). Caspofungin treatment of the reference strain SC5314 led to a significant increase in chitin content (P < 0.001), contrasting with the mutant strains where caspofungin did not produce any effect (Fig. 2). The chitin levels obtained after caspofungin exposure of the reference and chs3Δ/Δ strains are in agreement with those achieved by the classic method performed by other authors (7).

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Figure 1. Histograms representing the variation of fluorescence emitted by the positive (SC5314) and the negative (chs3Δ/chs3Δ) control strains after 2.5 μg CFW mL−1 staining: (A and D) nonstained cells (autofluorescence); (B and E) cells stained with CFW; and (C and F) cells stained with CFW after treatment with MIC values of caspofungin during 2 h. The wild-type strain showed higher intensity of fluorescence (B) than the deleted strain (E). [Color figure can be viewed in the online issue which is available at wileyonlinelibrary.com]

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Figure 2. Cell wall chitin content of reference and chs3Δ/chs3Δ, pga62Δ/Δ, and pga31Δ/Δ strains. A suspension of 106 cells mL−1 was stained with 2.5 μg CFW mL−1 and the intensity of fluorescence was quantified by flow cytometry. The SI mean values displayed by the different strains were determined after three independent experiments. Reference and mutant strains had significantly differences (P < 0.001) in SI mean values, revealing diverse chitin levels in the cell wall. After caspofungin exposure, only reference strains showed a significant increase in chitin levels (P < 0.001).

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Furthermore, the results obtained by flow cytometry were consistent with epifluorescence microscopy observations (data not shown). The flow cytometric protocol for chitin quantification is considerably less laborious and more accurate in comparison with the previously described methods as a large amount of cells (50,000) are randomly evaluated, without operator interference. Given the variation in yeast morphology such as cell shape and size, different species may emit different levels of autofluorescence, which arises from endogenous fluorophores. This autofluorescence emission analyzed under epifluorescence microscopy or flow cytometry results in a background “noise” which may interfere with the quantification of fluorescence emitted by stained cells (22). To avoid autofluorescence interference, especially when fluorescence emitted by cells from different species is compared, normalization of data is mandatory (22). This was achieved through the calculation of a SI ([mean intensity of fluorescence of positive population − mean intensity of fluorescence of negative population]/2 × standard deviation of the mean intensity of fluorescence of negative population) which provides sensitivity and reliability to the output data and enables comparison of the fluorescence emitted by cells with distinct morphologies. Not only this, but also the standardization of this protocol, in different labs and instruments. With this approach, we can expect a possible normalization in intra- and interlaboratory results.

The relationship between the CFW SI and the caspofungin susceptibility phenotype displayed by clinical strains is shown in Table 1 and Figure 3. Among the distinct species included in this study, Ca. parapsilosis, Ca. tropicalis, and Ca. albicans clinical isolates showed a higher CFW SI, and therefore a higher cell wall chitin content comparing to Ca. glabrata and Ca. krusei (Fig. 3). C. neoformans showed intermediary levels (Fig. 3).

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Figure 3. Cell wall chitin content of Candida spp. and C. neoformans clinical isolates in the absence and presence of caspofungin. Yeast cells were stained with 2.5 μg CFW mL−1 and fluorescence emitted was quantified by flow cytometry. Higher SI values were observed in strains that showed a paradoxical growth (*) in the presence of caspofungin. #Significantly different chitin levels after caspofungin treatment.

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Notably, several Ca. parapsilosis and Ca. tropicalis (Cp4, Ct1, Ct3, and Ct4) isolates showed a significant increase in chitin level (P < 0.001) in comparison with other isolates from the same species (Fig. 3). Interestingly, these strains, along with all Ca. albicans isolates tested, exhibited a paradoxical growth in the presence of high caspofungin concentrations. The ability to grow at high caspofungin concentrations has been frequently described among Ca. albicans, Ca. parapsilosis, and Ca. tropicalis (23) and has been suggested to relate to a compensatory increase in cell wall chitin (7, 25). This salvage mechanism strengths cell wall damaged by exposure to echinocandins. Chitin quantification by flow cytometry revealed to be a highly sensitive method. It enabled the detection of different chitin levels, which allowed us to validate the association between a higher amount of cell wall chitin and paradoxical growth in the presence of caspofungin concentrations well above the MIC. Also, when yeast cells were treated with caspofungin for 2 h, a significant increase in chitin levels was obtained, especially in strains that showed the paradoxical effect (Cp4, Ct1, Ct3, Ct4, Ca1, Ca2, and Ca3) (Fig. 3). The finding that caspofungin treatment stimulates chitin biosynthesis has also been described by other authors (21, 23, 25). The clinical relevance of this in vitro effect is yet uncertain. Although unrelated to resistance, the paradoxical effect may represent a drug tolerance mechanism and an adaptive response to the presence of caspofungin. In contrast, Ca. glabrata, Ca. krusei, and C. neoformans strains showed an absence of paradoxical growth, displaying the lowest chitin levels. Interestingly, the accuracy of this novel methodology can predict the occurrence of this paradoxical effect within a few minutes, representing a valuable tool for the detection of an antifungal compensatory mechanism in the presence of high antifungal concentrations, namely echinocandins.

The flow cytometry protocol described herein may constitute a useful tool to evaluate the inhibition of chitin synthesis by new drugs presently under development.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Acknowledgements
  6. Literature Cited
  7. Supporting Information

The authors thank Isabel Santos for excellent technical support.

Literature Cited

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Acknowledgements
  6. Literature Cited
  7. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Acknowledgements
  6. Literature Cited
  7. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
CYTO_22250_sm_SuppFig1.TIF185KSupporting Information Figure 1.
CYTO_22250_sm_SuppInfo.doc37KSupporting Information: MIFlowCyt

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