Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

Summary

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.

Abstract

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Introduction

Microbes have evolved over time to occupy and thrive in different ecological niches such as the open physical boundaries of the soil and water, among others¹. In these niches, microbes often engage in the direct competition for limited resources; importantly, for nutrients that they use for supporting their growth or space, which they need to accommodate the expanding population^2,3. In certain instances, some holozoic organisms like amoeba may even predate on cryptococcal cells as a way of extracting nutrients from their biomass^4,5. In turn, this allows such organisms to establish territorial dominance via controlling the population numbers of its prey. Because of this predatory pressure, some prey may be selected to produce microbial factors, such as the cryptococcal capsule⁶, to reconcile the negative effects of the pressure. However, as an unintended consequence of this pressure, some microbes acquire factors that allow them to cross the species barrier and seek out new niches to colonize⁷, like the confined spaces of the human body that are rich in nutrients and have ideal conditions. The latter may explain how a terrestrial microbe like Cryptococcus (C.) neoformans can transform to become pathogenic.

To this end, it is important to study the initial contact that cryptococcal cells may have with amoeba and how this may select them to become pathogenic. More specifically, this may give clues on how cryptococcal cells behave when acted upon by macrophages during infection. It is for this reason that amoeba was chosen as a model for macrophages here, as it is relatively cheap and easy to maintain a culture of amoeba in a laboratory⁸. Of interest was to also examine how cryptococcal secondary metabolites viz. 3-hydroxy fatty acids^9,10 influence the interaction between amoebae and cryptococcal cells.

A simple way of perceiving the interaction between amoeba and its prey with the naked eye is to create a lawn using its prey on the surface of an agar plate and spot amoeba. The visualization of plaques or clear zones on the agar plate depicts areas where amoeba may have fed on its prey. However, at this macro level, only the outcome of the process is noted, and the process of phagocytosis is mechanized cannot be observed. Therefore, to appreciate the process on a cell-to-cell basis, there are several microscopic methods that can be used^11,12. For example, an inverted microscope with an incubation chamber can be used to video record a time-lapse of events between a phagocytic cell and its target¹³. Unfortunately, due to the cost of a microscope with a time-lapse functionality, it is not always possible for laboratories to purchase such a microscope, especially in resource poor-settings.

To circumvent the above limitation, this study presents a sequential exploratory design that evaluates the interaction of C. neoformans viz C. neoformans UOFS Y-1378 and C. neoformans LMPE 046 with Acanthamoeba castellani. First, a qualitative method is used that precedes a quantitative method. Still images are captured using an inverted fluorescence microscope, as well as a transmission electron microscope to depict amoeba-Cryptococcus interactions. This was followed by quantifying fluorescence using a plate reader to estimate the efficiency of amoeba to internalize cryptococcal cells. When reconciling findings from these methods during the data-interpretation stage, this may equally reveal as much critical information as perusing a phagocytosis time-lapse video.

Protocol

Cryptococcus neoformans and some Acanthamoeba castellanii strains are regarded as biosafety level-2 (BSL-2) pathogens; thus, researchers must take proper precautions when working with these organisms. For example, laboratory personnel should have specific training and personal protective equipment (PPE) such as lab coats, gloves, and eye protection. A biological safety cabinet (level-2) should be used for procedures that can cause infection¹⁴.

1. Cultivation and standardization of fungal cells (modified from Madu et al. ¹⁵ )

1. Streak out the test fungal strains (i.e., C. neoformans UOFS Y-1378 and C. neoformans LMPE 046) from stock cultures (no older than 9 months) on yeast-peptone-dextrose (YPD) agar plates. Information on YPD agar's ingredients can be found in Table 1.
   NOTE: C. neoformans UOFS Y-1378 has been shown to produce 3-hydroxy fatty acids, while C. neoformans LMPE 046 does not produce 3-hydroxy fatty acids. Refer to Supplemental File 1 for information on how the presence of these molecules is determined.
2. Incubate the agar plates for 48 h at 30 °C.
   NOTE: A plate can be stored for up to 2 months at 4 °C before it can be discarded or used to make a stock culture¹⁶.
3. Scrape off a loopful of cryptococcal cells (C. neoformans UOFS Y-1378 or C. neoformans LMPE 046) from the 48 h-old plate and inoculate into a 250 mL conical flask containing 100 mL of the chemically-defined YNB broth (6.7 g/L) supplemented with 4% (w/v) glucose. Information on YNB broth's ingredients can be found in Table 2.
4. Incubate the flasks at 30 °C for 24 h while agitating at 160 rpm on a rotary shaker.
5. After a 24 h incubation period, count the fungal cells using a hemocytometer and adjust the cell number to 1 x 10⁶ cells/mL with PBS at pH 7.4.
   NOTE: The prepared C. neoformans UOFS Y-1378 inoculum was used in steps 3.1 and 3.2, while C. neoformans LMPE 046 inoculum was only used in step 3.2.

2. Cultivation and standardization of amoeba cells (modified from Madu et al. ¹⁵ )

1. Thaw a stock culture of Acanthamoeba castellanii and bring it to room temperature (RT).
   NOTE: Amoeba was prepared based on the modified protocols of Axelsson-Olsson et al.⁸ and Schuster¹⁷.
2. Pipette 1 mL of the thawed culture and inoculate it into a 50 mL centrifuge tube containing 15 mL of ATCC medium 712. Information on ATCC medium 712's ingredients can be found in Table 3.
3. Manually shake it gently and immediately centrifuge for 5 min at 400 x g and 30 °C.
4. Aspirate the supernatant.
5. Resuspend the cells in 15 mL of ATCC medium 712 and incubate the tube at 30 °C for 14 days.
   NOTE: Periodically check the cells, using a simple light microscope, to determine if they are in a trophozoite state. Once they are in a trophozoite state, start a fresh culture.
6. Pipette 1 mL from a culture that shows cells in a trophozoite state and use it to inoculate a sterile 50 mL centrifuge tube containing 15 mL of fresh, sterile ATCC medium 712.
7. Incubate the tube at 30 °C for 1 week while agitating at 160 rpm on a rotary shaker.
8. After a week, count the amoeba cells using a hemocytometer and adjust the cell number to 1 x 10⁷ cells/mL with fresh, sterile ATCC medium 712.
9. Perform a viability assay using a trypan blue stain as detailed by Strober¹⁸. Proceed further with the cultures that show at least 80% viability.

3. Fluorescence staining of cells to study phagocytosis (modified from Madu et al. ¹⁵ )

1. Gathering qualitative data through use of fluorescence microscope
   NOTE: Perform this assay with Acanthamoeba castellanii and C. neoformans UOFS Y-1378.
   1. Dispense a 200-µL suspension of standardized amoebae (1 x 10⁷ cells/mL in ATCC medium 712) into chamber wells of an adherent slide and incubate for 2 h at 30 °C for cells to adhere to the surface.
   2. While amoeba cells are settling down to adhere, stain the standardized C. neoformans UOFS Y-1378 cells that were adjusted to 1 x 10⁶ cells/mL (in 999 µL of PBS) with 1 µL of fluorescein isothiocyanate in a 1.5 mL plastic tube.
      NOTE: Prepare the stain by dissolving 1 mg of fluorescein isothiocyanate in 1 mL of acetone.
   3. Gently agitate the C. neoformans UOFS Y-1378 cells on an orbital shaker set at 50 rpm for 2 h at RT and in the dark.
   4. After 2 h, centrifuge at 960 x g for 5 min at 30 °C to pellet the cells.
   5. Aspirate the supernatant to remove the PBS with the stain.
   6. Add 1 mL of PBS to the tube for washing the cell pellet. Wash the cells by gently pipetting.
   7. Centrifuge the cells at 960 x g for 5 min at 30 °C. Discard the supernatant. Repeat the washing step one more time.
   8. Resuspend the washed cells in 1 mL of PBS.
   9. Dispense a 200 µL suspension of the stained C. neoformans UOFS Y-1378 cells to chamber wells containing the unstained amoeba cells.
   10. Incubate the prepared co-culture at 30 °C for an additional 2 h period.
       NOTE: The co-culture can be incubated for different time points to suit the purpose of the experiment.
   11. At the end of the co-incubation period, aspirate the contents of the wells.
   12. Add 300 µL of PBS to the wells to wash the chamber wells and to remove any unbound co-cultured cells. Do this by gentle pipetting. Aspirate the contents of the wells. Repeat the washing step one more time.
   13. Prepare the 3% glutaraldehyde solution by adding 3 mL of glutaraldehyde to 97 mL of distilled water.
   14. Fix the co-cultured cells by adding 250 µL of 3% solution to the chamber wells and incubating for 1 h.
   15. Aspirate the fixative and wash the chamber wells as detailed from step 3.1.13.
   16. Dismantle the chamber wells using a tool that was provided with the chamber slides.
   17. Add a drop of the antifade compound, 1,4-diazabicyclo-[2.2.2]-octane to the slide to prevent auto-bleaching. Cover with a coverslip and seal the sides with a nail polish to prevent evaporation.
   18. View the co-cultured cells using the 100x objective lens (with oil) of a confocal laser-scanning microscope.
       NOTE: It is important to take pictures in bright-field and fluorescence to view interaction between amoeba and cryptococcal cells. Wherever possible, the fluorescence can be super-imposed onto the bright-field images. Amoeba cells are typically larger in size (i.e., 45-60 µm), and the trophozoite cells have an irregular shape. Cryptococcal cells are 5-10 µm in diameter and have a globose to ovoid shape. When exposed to a laser, it is possible that unstained amoeba cells may emit auto-fluorescence. Refer to Beisker and Dolbeare¹⁹ and Clancy and Cauller²⁰ for methods to reduce autofluorescence.
2. Acquiring quantitative data through use of fluorescence plate reader
   NOTE: Perform this assay with Acanthamoeba castellanii and C. neoformans UOFS Y-1378 or C. neoformans LMPE 046.
   1. Dispense a 100 µL suspension of standardized amoebae (adjusted to 1 x 10⁷ cells/mL in ATCC medium 712) into a black, adherent 96 well microtiter plate.
   2. Incubate the plate for 2 h at 30 °C to allow amoeba cells to adhere to the surface.
   3. While amoeba cells are settling down to adhere, stain the standardized C. neoformans UOFS Y-1378 cells that were adjusted to 1 x 10⁶ cells/mL (in 999 µL of PBS) with 1 µL of pHrodo Green Zymosan A BioParticles in a 1.5 mL microcentrifuge tube. Stain C. neoformans LMPE 046 cells as well in a separate tube.
      NOTE: The dye, unlike FITC, selectively stains cells that are trapped inside the acidic environment of a phagocytic cell^21,22. For this technique, it is important to maintain the cryptococcal cells in a medium with a neutral pH (PBS) and amoeba in a medium with a neutral pH (ATCC medium 712). A medium with an acidic environment will result in a false positive reading of the relative fluorescence units, implying that a greater number of cryptococcal have been internalized.
   4. Gently agitate cryptococcal cells on an orbital shaker set at 50 rpm for 2 h at RT and in the dark.
   5. After 2 h, centrifuge the microcentrifuge tube at 960 x g for 5 min at 30 °C to pellet the cells. Aspirate the supernatant to remove PBS with the stain.
   6. Add 1 mL of PBS to the tube to wash the pelleted cells. Wash the cells by gentle pipetting.
   7. Centrifuge the cells at 960 x g for 5 min at 30 °C. Discard the supernatant. Repeat the washing step one more time.
   8. Resuspend the pellet of washed cells in 1 mL of PBS.
   9. Dispense a 100 µL suspension of stained cryptococcal cells to wells containing unstained amoeba cells.
   10. Incubate the prepared co-culture at 30 °C for an additional 2 h period.
       NOTE: The co-culture can be incubated for different timepoints to suit the purpose of the experiment.
   11. At the end of the co-incubation period, measure the fluorescence on a microplate reader. Convert logarithmic signals to relative fluorescence units.
       NOTE: The dye's excitation is at 492 nm and emission is at 538 nm. Consult Beisker and Dolbeare¹⁹ and Clancy and Cauller²⁰ for methods to reduce autofluorescence.

4. Use of transmission electron microscopy to study phagocytosis (modified from van Wyk and Wingfield ²³ )

1. Add a 5 mL suspension of amoebae (adjusted to 1 x 10⁷ cells/mL in ATCC medium 712) to a 15 mL centrifuge tube and allow them to settle for 30 min at 30 °C.
2. Add a 5 mL suspension of C. neoformans UOFS Y-1378 cells (adjusted to 1 x 10⁶ cells/mL in PBS) to the same centrifuge tube that contains 5 mL of standardized amoeba cells.
3. Allow the tube stand for 2 h at 30 °C.
4. Centrifuge the tube at 640 x g for 3 min at 30 °C to pellet the co-cultured cells. Aspirate the supernatant. Do not wash the co-cultured cells.
5. Fix the co-cultured cells by resuspending the pellet in 3 mL of 1.0 M (pH = 7.0) sodium phosphate-buffered 3% glutaraldehyde for 3 h.
6. Centrifuge the tube at 1,120 x g for 5 min at 30 °C to pellet the co-cultured cells. Aspirate the supernatant.
7. Add 5 mL of sodium phosphate buffer to the centrifuge tube to wash the pelleted cells. Wash by gently pipetting the contents of the tube for 20 s.
8. Centrifuge the tube at 1,120 x g for 5 min at 30 °C to pellet the co-cultured cells.
9. Repeat steps 4.8-4.10. Aspirate the supernatant.
10. Fix the co-cultured cells again by resuspending the pellet in 3 mL of 1.0 M (pH = 7.0) sodium phosphate-buffered 1% osmium tetroxide for 1.5 h.
11. Remove the fixative (osmium tetroxide) by washing the co-cultured cells in a similar manner to removing 3% glutaraldehyde.
12. Dehydrate the TEM material (also known as the co-cultured cells) in a graded acetoneseries of 30%, 50%, 70%, 95% and two changes of 100% for 15 min each, respectively. To do so, add 3 mL of the acetone solution to the pelleted cells and let it stand for 15 min. Then, centrifuge at 200 x g for 10 min at RT Discard the supernatant and add the higher percentage of the acetone solution.
13. Prepare the epoxy of normal consistency according to the protocol by Spur²⁴.
    NOTE: The epoxy resin is used for sectioning.
14. Embed the TEM material into the freshly prepared epoxy resin. To do so, follow the steps below.
    1. Add 3 mL of the freshly prepared epoxy to a tube that contains the TEM material resuspended in 3 mL of 100% solution of acetone. Allow the tube to stand for 1 h.
    2. Centrifuge the tube at 200 x g for 10 min at 30 °C. Aspirate the epoxy-acetone solution.
    3. Add 6 mL of the freshly prepared epoxy to the pellet in the tube. Allow the tube to stand for 1 h.
    4. Centrifuge at 200 x g for 10 min at 30 °C. Aspirate all the epoxy-acetone solution.
    5. Add 3 mL of the freshly prepared epoxy to the tube. Allow the tube to stand for 8 h.
    6. Centrifuge at 200 x g for 10 min at 30 °C. Aspirate all the epoxy solution
    7. Add 3 mL of the freshly prepared epoxy to the tube. Keep the TEM material in the epoxy solution overnight in a vacuum desiccator.
       CAUTION: Epoxy resin is a radioactive material. Use PPE to handle the epoxy resin. The epoxy resin should also be handled in a fume hood. Researchers should follow safety regulations for discarding such material as specified by each country²⁵.
15. Polymerize the TEM material for 8 h at 70 °C.
16. On the ultramicrotome, trim small sections of approximately 0.1 mm x 0.1 mm and 60 nm thickness from the epoxy-embedded material with a mounted glass knife. Assemble sections on a grid and place the grids in a TEM sample holder box before staining.
17. Stain the sections with a drop of 6% uranyl acetate for 10 min in the dark. Ensure the sections are completely covered.|
    NOTE: Reconstitute the stain (6 g) in 100 mL of distilled water.
    CAUTION: Uranyl acetate is a radioactive material. Use PPE to handle uranyl acetate. Uranyl acetate should also be handled in a fume hood. Researchers should follow safety regulations for discarding such material as specified by each country²⁵.
18. Rinse sections by dipping them five times into a beaker that contains 100 mL of distilled water.
    NOTE: The distilled water should be disposed accordingly as it contains traces of uranyl acetate.
19. Stain the section with a drop of lead citrate for 10 min in the dark. Ensure the sections are completely covered.
    NOTE: Lead citrate should be prepared according to the protocol by Reynold²⁶.
20. Rinse sections by dipping them five times into a beaker that contains 100 mL of distilled water.
21. Individually assemble the grids with stained sections on a TEM sample holder box.
22. View sections with a transmission electron microscope.

Representative Results

Microbes are microscopic organisms that cannot be perceived with the naked eye. However, their impact may result in observable clinically evident illnesses, such as skin infections. When studying certain aspects of microbes, ranging from their morphology, byproducts, and interactions, being able to provide pictorial and video evidence is of the utmost importance.

We first sought to visualize the interaction between cryptococcal cells and amoeba. For this purpose, bright-field images that showed 2 h co-incubated cells were studied first. One image revealed a cryptococcal cell that was in the close proximity to amoeba. One of the amoeba cells was seen with extended pseudopodia to capture a cryptococcal cell (Figure 1A). Next, a corresponding image in fluorescence was captured for referencing (Figure 1B). The green fluorescence on the surface of the stained cells aided in confirming the presence of cryptococcal cells. The unstained amoeba also auto-fluoresced. This, in addition to the apparent difference size and morphology, assisted in further distinguishing the two cell types.

Autofluorescence is a quality often observed when biological structures naturally emit light that they have absorbed (e.g., following exposure to a laser during confocal laser scanning microscopy)²⁷. In Figure 1C, cryptococcal cells were noted (at the same timepoint of 2 h) that were already internalized by amoeba. The corresponding image in fluorescence was also captured for referencing (Figure 1D). Based on the evidence at hand, it is tempting to conclude that the amoeba killed the two trapped cells. However, phagocytosis is a dynamic process wherein the host, predator and pathogen, and prey employ different strategies to destroy or evade each other²⁸. The act of cryptococcal cells evading phagocytic cells is elegantly demonstrated by vomocytosis^29,30, which is a non-lytic expulsion process of trapped cells from macrophages. This daring move has been captured in time-lapse videos^29,30. Unfortunately, this highlights the limitation of studying still images of fixed cells, as in our study, to elucidate a dynamic process like phagocytosis. To the point, a researcher may miss the interval when a cell escapes from its capturer.

To compensate for the above, the reading of relative fluorescence units was considered. In the current study, readings were taken after a 2 h co-incubation period and helped to compare the response of the two test cryptococcal strains [i.e., one that produces 3-hydroxy fatty acids (C. neoformans UOFS Y-1378) and the other that does not (C. neoformans LMPE 046)]. It was hypothesized that 3-hydroxy fatty acids may act as a virulence determinant that impair the uptake of cryptococcal cells, including phagocytosis by amoeba. For more information on the influence of 3-hydroxy fatty acids on amoeba, it is advised to refer to Madu et al.^15,31. Figure 2 shows the amount of cryptococcal cells that were internalized based on the reading of fluorescence units. When comparing the two cryptococcal isolates, it was clear that cells that produce the 3-hydroxy fatty acids were internalized less frequently compared to cells that do not produce 3-hydroxy fatty acids.

To enhance the qualitative data, transmission electron microscopy was included in the analysis (Figure 3A). Here, it was noted that the strain that produces 3-hydroxy fatty acids (C. neoformans UOFS Y-1378) had spiky protuberances on the capsule (Figure 3B), which may be used by the cell to release 3-hydroxy fatty acids to the outside environment.

It is important to note that the data (in Figure 1, Figure 3) convey the fate of cryptococcal cells as being internalized and not killed/phagocytized. To determine if the cells survived the phagocytic event, it is recommended to include an additional assay in which the researcher lyses the amoeba cells and prepares a spread plate agar to enumerate the cryptococcal colony forming units (CFU). By counting CFUs, Madu et al.¹⁵ reported that cryptococcal cells producing 3-hydroxy fatty acids were also resistant to the phagocytic action of amoeba following internalization. Thus, these cells yielded a significantly higher survival rate when compared to cells that do not produce 3-hydroxy fatty acids.

Figure 4 shows the importance of TEM sample preparation and examination. In this instance, C. neoformans UOFS Y-1378 sections were purposefully overexposed to electron bombardment. At the end, the captured image cannot be used, as it compromises the quality of information that can be deduced. Taken together, the obtained information shows that by combining these different techniques, a researcher is able to deduce sufficient information to determine the fate of cryptococcal cells when co-cultured with amoeba.

Ingredient	Quantity
bacteriological peptone	20 g/L
yeast extract	10 g/L
glucose	20 g/L
agar	15 g/L

Table 1: Ingredients for making YPD agar. Add the required amount all the ingredients in 1 L of water. Heat while stirring to dissolve the ingredients completely. Once done autoclave prior to the use.

Ingredient	Quantity
ammonium sulfate	5 g/L
biotin	2 μg/L
calcium pantothenate	400 μg/L
folic acid	2 μg/L
inositol	2000 μg/L
niacin	400 μg/L
p-aminobenzoic acid	200 μg/L
pyridoxine hydrochloride	400 μg/L
riboflavin	200 μg/L
thiamine hydrochloride	400 μg/L
boric acid	500 μg/L
copper sulfate	40 μg/L
potassium iodide	100 μg/L
ferric chloride	200 μg/L
manganese sulfate	400 μg/L
sodium molybdate	200 μg/L
zinc sulfate	400 μg/L
monopotassium phosphate	1 g/L
magnesium sulfate	0.5 g/L
sodium chloride	0.1 g/L
calcium chloride	0.1 g/L

Table 2: Ingredients for making YNB broth. Add the required amount all the ingredients in 1 L of water. Heat while stirring to dissolve the ingredients completely. Once done autoclave prior to the use.

Part I: Basal medium.
Ingredient	Quantity
proteose peptone	20 g/L
yeast extract	1 g/L
agar (if needed)	20 g/L
Part II: Supplements.
Ingredient (stock solutions)	Quantity
0.05 M CaCl2	8 mL
0.4 M MgSO4 x 7H2O	10 ml
0.25 M Na2HPO4 x 7H2O	10 mlL
0.25 M KH2PO4	10 mL
Na Citrate x 2H2O	1 g
0.005 M Fe(NH4)2(SO4)2 x 6H2O	10 mL

Table 3: Ingredients for making ATCC medium 712. Prepare the basal medium in 900 mL of water. Prepare the supplements separately and add to the basal medium. Once done adjust the pH to 7.4 with 1 N HCl or 1 N NaOH and autoclave. Filter sterilize 50 mL solution of 2 M glucose (18 g/50 mL) and add it aseptically to the complete medium prior to use.

Figure 1: Bright-field and corresponding fluorescent micrographs showing amoeba-Cryptococcus interactive moments. (A) An amoeba cell in close proximity to a C. neoformans UOFS Y-1378 cell can be seen. The corresponding fluorescent image is shown in (B). (C) Depiction of two C. neoformans UOFS Y-1378 cells that are trapped inside the amoeba food vacuole. The corresponding fluorescent image is shown in (D). This figure has been modified from Madu et al.¹⁵. A = amoeba; C = C. neoformans. Please click here to view a larger version of this figure.

Figure 2: The results of the internalization assay of cryptococcal cells co-cultured with amoeba. The reading of relative fluorescence units allows for the interpretation and comparison of the efficiency of amoebae to internalize C. neoformans UOFS Y-1378 and C. neoformans LMPE 046. The error bars represent the calculated standard errors based on three biological replicates. This figure has been modified from Madu et al.¹⁵. Please click here to view a larger version of this figure.

Figure 3: Transmission electron micrographs showing amoeba-Cryptococcus interactions. TEM micrographs (A, B) confirm the observations in Figure 1C,D. (A) Shown is a C. neoformans UOFS Y-1378 cell trapped inside the amoeba food vacuole, while (B) is a close-up view of Figure 3A.This figure has been modified from Madu et al.¹⁵. A = amoeba cell; C = C. neoformans cell. The red arrow points at a capsular protuberance. Please click here to view a larger version of this figure.

Figure 4: A transmission electron micrograph showing C. neoformans UOFS Y-1378 cells. The cells are damaged and thus cannot provide meaningful data. Red arrows indicate points where the section is torn. Please click here to view a larger version of this figure.

Supplemental File 1.  Please click here to download this file.

Discussion

In the paper, different techniques were successfully employed to reveal the possible outcome that may arise when amoeba interact with cryptococcal cells. Also, we were interested to show the effects of 3-hydroxy fatty acids on the outcome of Cryptococcus-amoeba interactions.

The first technique used was confocal microscopy, which rendered still images. The major drawback of this technique here was that it only gave us information that is limited to a particular timepoint. Any conclusion that can be drawn based on the results lends itself to inductive reasoning, wherein one can arrive at a conclusion based on a set of observations³²_. However, just because one observes several situations in which a pattern exists does not mean that that pattern is true for all situations. Thus, in the study, it is shown and possibly cautioned how such limited information may lead to unfounded conclusions. To the point, in the absence of contradictory or supportive, complementary evidence, it may be concluded that internalization may have led to the phagocytosis of cells.

The pace of development in imaging brings new opportunities to make scientific discoveries, as was the case with the uncovering of vomocytosis^29,30. To illustrate this point without use of a microscope that can record time-lapse videos, this discovery would have not been possible. Therefore, a lack of access to such high-end instrumentation will always be an obstacle in resource poor-settings that are not at the forefront of uncovering such processes. One way to overcome this is to seek out new collaborations or discover innovative ways to address research questions. One welcome development has been the introduction and application of specialized stains such as the phagocytic stain used here^21,22. This stain is pH-sensitive and fluoresces only in acid environments such as in the lumen of amoeba food vacuole¹⁵. It is worthwhile to point out that the stain only gives information related to the internalization of cells. Determination if cells are eventually phagocytized in additional experiments may be required.

Importantly, such a stain also proved to be useful in the measurement of fluorescence. The latter allowed integration of quantitative data in an attempt to explain what happens biologically at one specific timepoint. Here, fate of cells was discerned (i.e., it was determined whether the presence of 3-hydroxy fatty acids impaired or promoted the internalization of cells) by extrapolating meaning from the readings of relative fluorescence units.

Unlike in this study, researchers may also opt to measure the fluorescence of cells over a time period. The obtained information is useful in determining the number of cells that are internalized at one timepoint and following how the amount changes over the period. Likewise, images can also be taken at corresponding timepoints.

This study shows the power of combining a number of methods to reach a reasoned conclusion. The approach of combining multiple approaches to monitor phagocytosis either to compare or complement an initial technique is not new. For example, Meindl and co-workers³³ compared three techniques (image analysis, fluorescence, and flow cytometry readings) to investigate how fluorescence-labelled particle size affects macrophage phagocytosis. The study proved that of the three techniques, plate reading may be the best option to monitor phagocytosis³³.

TEM is particularly a powerful tool, as it provides a bird's eye view into the lumen of the food vacuole. Often, this level of detail is frequently missed by confocal microscopy in the form of still images, including time-lapse videos. To this point of the TEM, it was interesting to visualize protuberances on the surfaces of the cryptococcal capsule. It was previously hypothesized that these cell surface structures are used as a channel to release 3-hydroxy fatty acids into the surrounding environment to possibly promote cell survival^9,10,15,31. The detail on the TEM micrograph further reveals that protuberances on the internalized cell are not distorted and have maintained their integrity. Thus (given the integrity of the protuberances), it is possible that they may deliver 3-hydroxy fatty acids into the food vacuole environment and alter internal conditions, leading to cell survival as reported by Madu et al.^15,31. A major limitation of using the electron microscope is that sample preparation is very laborious. Moreover, to avert destroying the samples as seen in Figure 4, the experimenter should be well-trained to manually operate the ultramicrotome and microscope.

In conclusion, it is envisaged that researchers will be encouraged by the prospect of studying phagocytosis simply by combining still fluorescent images with quantitative data. It is trusted that researchers can obtain enough information from this protocol and optimize it in their own studies. This may include the development of antibodies against targeted metabolites and applying this to immunofluorescence studies, including immuno-gold labelling during TEM examination.

Materials

Name	Company	Catalog Number	Comments
1,4-Diazabicyclo-[2.2.2]-octane	Sigma-Aldrich	D27802	-
1.5-mL plastic tube	Thermo Fisher Scientific	69715	-
15-mL Centrifuge tube	Thermo Fisher Scientific	7252018	-
50-mL Centrifuge tube	Thermo Fisher Scientific	1132017	-
8-Well chamber slide	Thermo Fisher Scientific	1109650	-
Acetone	Merck	SAAR1022040LC	-
Amoeba strain	ATCCÒ	30234TM	-
ATCC medium 712	ATCCÒ	712TM	Amoeba medium
Black 96-well microtiter plate	Thermo Fisher Scientific	152089	-
Centrifuge	Hermle	-	-
Chloroform	Sigma-Aldrich	C2432	-
Confocal microscope	Nikon	Nikon TE 2000	-
Epoxy resin:
[1] NSA	[1] ALS	[1] R1054	-
[2] DER 736	[2] ALS	[2] R1073	-
[3] ERL Y221 resin	[3] ALS	[3] R1047R	-
[4] S1 (2-dimethylaminoethanol)	[4] ALS	[4] R1067	-
Fluorescein isothiocyanate	Sigma-Aldrich	F4274	-
Formic Acid	Sigma-Aldrich	489441	-
Fluoroskan Ascent FL	Thermo Fisher Scientific	374-91038C	Microplate reader
Glucose	Sigma-Aldrich	G8270	-
Glutaraldehyde	ALS	R1009	-
Hemocytometer	Boeco	-	-
Lead citrate	ALS	R1209	-
Liquid Chromatography Mass Spectrometer	Thermo Fisher Scientific		-
Methanol	Sigma-Aldrich	R 34,860	-
Orbital shaker	Lasec	-	-
Osmium tetroxide	ALS	R1015	-
pHrodo Green Zymosan A BioParticles	Life Technologies	P35365	This is the pH-sensitive dye
Physiological buffer solution	Sigma-Aldrich	P4417-50TAB	-
Rotary shaker	Labcon	-	-
Sodium phosphate buffer:
[1] di-sodium hydrogen orthophosphate dihydrate	[1] Merck	[1] 106580	-
[2] sodium di-hydrogen orthophosphate dihydrate	[2] Merck	[2] 106345
Transmission electron microscope	Philips	Philips EM 100	-
Trypan blue	Sigma-Aldrich	T8154	-
Ultramicrotome	Leica	EM UC7	-
Uranyl acetate	ALS	R1260A	-
Vacuum dessicator	Lasec	-	-
Vial	Sigma-Aldrich	29651-U	-
YNB	Lasec	239210	-
YPD agar	Sigma-Aldrich	Y-1500	-