Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

Summary

The polysaccharide capsule is the primary virulence factor in Cryptococcus neoformans, and its size correlates with strain virulence. Capsule diameter measurements are used in phenotypic testing and to gauge therapeutic efficacy. Here a standard method of capsule induction is presented, and two methods of staining and measuring diameter are compared.

Abstract

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic yeast. Capsule size can vary widely between strains, has the ability to grow rapidly when introduced to stressful or low nutrient conditions, and has been positively correlated with strain virulence. For these reasons, the size of the capsule is of great interest to C. neoformans researchers. The growth of the C. neoformans capsule is induced during phenotypic testing to help understand the effects of different treatments on the yeast or size differences between strains. Here we describe one of the standard methods of capsule induction and compare two accepted methods of staining and measuring capsule diameter: (i) India ink, a negative stain, used in conjunction with conventional light microscopy and (ii) co-staining with fluorescent dyes of both the cell wall and capsule followed by confocal microscopy. Finally, we show how measurement of capsule diameter from India ink-stained samples can be automated using computational image analysis.

Introduction

Affecting a quarter million people every year and resulting in more than 180,000 deaths annually, Cryptococcus neoformans is a pathogenic, intracellular yeast and the causative agent of cryptococcosis^1,2,3. Hardest hit are HIV-positive patients in poor countries who do not have ready access to antiretroviral therapy, making them acutely susceptible to the illness^4,5,6. Data from the CDC indicate that in sub-Saharan Africa, C. neoformans kills more people than tuberculosis annually and more every month than any Ebola outbreak on record¹. The most common route of exposure occurs from inhaling desiccated spores that are commonplace in the environment⁷. Upon entering the lungs, there are several virulence factors that contribute to the success of C. neoformans within infected individuals. The polysaccharide capsule is considered the microbe's primary virulence factor, as acapsular strains are not virulent⁸.

The cryptococcal capsule is made of up three principle components: glucuronoxylomannan (GXM), galactoxylomannan (GalXM), and mannoproteins (MPs)⁹. While MPs are a relatively minor cell wall-associated component of the capsule, they are immunogenic and can promote a mostly pro-inflammatory response^9,10. In contrast, GXM and GalXM make up the bulk of the capsule (>90% by weight) and have immunosuppressive effects¹¹. In addition to its immunomodulatory effects, the rapid enlargement of the capsule in vivo creates a mechanical barrier to ingestion by host phagocytic cells (i.e., neutrophils and macrophages)¹². The C. neoformans capsule and its synthesis are complex, but overall, increased capsule diameter is correlated with increased virulence^6,13,14. Given this, it is important for C. neoformans researchers to be able to quickly and accurately quantify capsule measurements.

Both the C. neoformans cell and its polysaccharide capsule are dynamic structures and show changes over time¹⁵. The capsule can change in density, size, and assembly in response to changes in the host environment^16,17,18. Low iron or nutrient levels, exposure to serum, the human physiological pH, and increased CO₂ are known to initiate capsule growth^16,18,19,20. Further, researchers have shown structural changes resulting in significant differences in immunoreactivity during an infection, lending an advantage to C. neoformans over its host^21,22. This is known because the architecture of the C. neoformans capsule has been analyzed in a variety of ways. Electron microscopy, for example, has revealed that the capsule has a heterogeneous matrix with an inner electron-dense layer underneath an outer, more permeable layer²³. Light scattering and the use of optical tweezers have allowed researchers to further elucidate its macromolecular properties²⁴. Analyzing the results from both static and dynamic light scattering measurements, we know that the polysaccharide capsule has a complex branching structure²³. Optical tweezers have been used to test the rigidity of the structure as well as evaluate its antibody reactivity²⁴. However, by far the most frequently employed analysis of the C. neoformans capsule is the measurement of its size.

To quantify capsule size, researchers use what should be a simple measurement: the linear diameter of the capsule. Digital microscopes are used to capture images of multiple C. neoformans cells (generally hundreds) stained with either India ink or fluorescent dyes. The size of each cell body and surrounding capsule is measured. The data are compiled, and the average diameter of the capsule is calculated by subtracting the cell body diameter from the whole cell diameter (cell body + capsule). Up until this point, these measurements have been done manually. While generally accurate, this method has drawbacks for researchers. Large data sets can take days or even weeks to analyze by hand. And because these measurements are done manually, subjectivity and human error may affect the result.

Automated computational image analysis has become an indispensable tool for researchers in many areas of molecular cell biology, enabling faster and more reliable analysis of biological images ^25,26,27. Precise image analysis techniques are necessary to mine quantitative information from what are often complex and immense data sets. However, some measurements, especially the measurement of C. neoformans capsule, have been difficult to automate. Accurately identifying the interface between the cell wall and capsule, which generally appears as a dark ring when imaged by phase-contrast microscopy, can be troublesome to resolve using a simple threshold. Further, C. neoformans cells in culture tend to clump together and accurate segmentation of the cells is necessary for accurate measurements.

The aim of this project was to (i) illustrate one of the standard protocols for capsule induction in C. neoformans, (ii) compare and contrast India ink and fluorescence staining as they pertain to capsule diameter measurements, (iii) develop simple, computational methods to measure capsule diameter using images of India ink stained cells using an image analysis software, and, (iv) assess the benefits and limitations of measuring capsule diameter manually and using software automation. We find that of the two staining methods, fluorescent labeling of the cell wall and capsule, while more time-consuming, provided the most consistent results between experiments. However, both methods enabled us to successfully distinguish between lab and clinical C. neoformans strains exhibiting different capsule sizes. Further, we were able to automate the measurement of capsule diameter from India ink stained images and found that this was a viable alternative to manual measurement of capsule.

Protocol

NOTE: C. neoformans is a Biosafety Level 2 (BSL-2) pathogen and researchers working with it must take proper precautions. Detailed procedures on how to safely work with BSL-2 pathogens can be found on the Center for Disease Control's (CDC) website, but it is important to note that all persons coming into contact with C. neoformans should be properly trained in handling pathogenic agents and should always wear appropriate personal protective equipment (PPE), generally latex or nitrile gloves. Further, rotors on centrifuges should be sealed to prevent aerosolization of samples and any spills cleaned up immediately using a 10% bleach solution²⁸.

1. Capsule Induction

NOTE: All steps should be performed at room temperature unless otherwise noted.

1. Culture C. neoformans in yeast peptone dextrose (YPD) broth (pH 6.5 +/-0.2) and incubate at 37 °C with shaking until they are in log phase (approximately 18 - 36 h).
2. Centrifuge cells at 870 x g for 5 min at 21 °C and wash three times with phosphate buffered saline (PBS).
3. Use a hemocytometer (or an automated cell counter) to count the cells and resuspend at 2 x 10⁵ cells/2 mL in Dulbecco's minimal essential media (DMEM).
4. To induce the capsule, incubate at 37 °C and 5% CO₂ for 18 h.
   NOTE: The combination of the absence of nutrients in the media and the presence of CO₂ stimulates C. neoformans capsule growth.
5. Post-incubation, harvest the cells by centrifuging (as above) and removing all but 5-10 µL supernatant. At the same time, harvest a corresponding un-induced cell sample for each strain to be measured. Use these as negative controls.
   NOTE: The cell pellets will be very small and may be difficult to see. Be careful when aspirating supernatant.
6. Stain with either India ink (Section 2.1) or fluorescent dyes (Section 2.2).

2. Staining

1. Staining with only India ink
   1. To stain the yeast with India ink, resuspend the pellet in the remaining supernatant and add 4μL (approximately half) of cell suspension and 4μL of India ink onto a glass microscope slide. Gently mix and avoid foaming.
   2. Apply a 13-mm glass coverslip (#1.5 thickness) and seal edges with a non-toxic sealant (clear nail polish) to prevent the sample from drying out.
2. Staining with only fluorescent dyes
   1. To stain the yeast with a fluorescent dye, resuspend the cell pellet in 50 µL PBS with 1% bovine serum albumin (BSA).
   2. Incubate the culture with an equal volume of Calcofluor white (CFW) (1 µg/mL) and capsule mAb 18B7 (see Table of Materials) conjugated to AlexaFluor488 (AF488) (10 µg/mL) for 30 min at 37 °C.
   3. Centrifuge cells at 870 x g for 5 min at 21 °C post-incubation and aspirate supernatant.
   4. Resuspend cell pellet in 10 µL PBS with 1% bovine serum albumin (BSA).
   5. Pipette 8 µL of cell suspension onto a glass microscope slide.
   6. Apply a 13-mm glass coverslip (#1.5 thickness) and seal edges with a non-toxic sealant (clear nail polish) to prevent the sample from drying out.
3. Staining with both India ink and fluorescent dyes
   1. To more directly compare the efficacy of India ink staining to that of fluorescent dyes, co-stain the same cell population with both types of stain. To do so, first incubate cells with both a fluorescent capsule and cell wall stain, as per steps 2.2.1 - 2.2.3.
   2. Next, pipette equal volumes (4 µL) of the fluorescently stained cell suspension and India ink onto a glass slide. Gently mix.
   3. Apply a 13-mm glass coverslip (#1.5 thickness) and seal the edges with a non-toxic sealant (clear nail polish) to prevent the sample from drying out.

3. Image Acquisition/Microscopy

1. Imaging cells stained with India ink.
   1. Acquire images using a standard light microscope (100X magnification).
   2. Image a minimum of 50 cells per condition.
      NOTE: The number of cells per field will vary, and this is acceptable. It is important, however, that overlapping cells be kept at a minimum, as these are difficult to measure (both manually and with automated software). For these experiments, images were acquired at a depth of 16 bits with exposure times optimized to maximize image contrast while minimizing the blur caused by small movements of the cells.
2. Imaging cells stained with fluorescent dyes
   1. To image the cells by fluorescence microscopy, open the microscope imaging software and select the appropriate excitation and emission wavelengths for the fluorophores used (CFW and AF488 are excited using the 405nm and 488 nm lights, respectively). Acquire images using a fluorescence microscope.
   2. Image a minimum of 50 cells per condition.
      NOTE: The number of cells per field will vary, and this is acceptable. It is important, however, that overlapping cells be kept at a minimum as these are difficult to measure (both manually and with automated software).
   3. Acquire a z-stack image series of the cells to ensure that the maximum capsule diameter for each cell within a given field is obtained.
      1. In the microscope control software, click on the "z-stack" bar to open up the "z-stack" control panel, then select the "First/Last" option in the top left corner of the panel.
      2. Use the fine-focus control on the microscope to focus at a level that is below the widest point of a single yeast cell and the capsules for all within the current field of view and click “Set First”.
      3. Use the fine-focus control to focus above the widest point of the cell body of the same cell and of the capsule for all of the cells within the current field of view and click “Set Last”.
      4. Then, click “Start Experiment” on the “Acquisition” tab to acquire the z-stack for the current position. Repeat the process at multiple positions until z-stack images of a minimum of 50 cells have been acquired.
         NOTE: For these experiments, the confocal pinhole was set to 1.0 AU and an overlapping z-series of 10-20 slices covering 0.7 µM each were acquired at the selected positions. AU = Airy unit.
   4. Next, convert each z-stack series into maximum intensity projections (MIP) using appropriate image analysis software.
      NOTE: MIPs project the greatest intensity at every plane of the stacked image²⁹. Creating MIPs allows one to produce a 2-D representation of images that correspond to the maximum cell and capsule diameter within the captured 3-D stack.
3. Imaging cells stained with both India ink and fluorescent dyes
   1. Acquire images using either a widefield (40X magnification) or confocal microscope (63X or 100X magnification). For cells stained with both India ink and fluorescent dyes, capture images using a widefield microscope equipped with a computer-controlled stage and oil immersion objective.
      NOTE: The microscope in use should be controlled using an imaging software CFW. fluorescence that is excited through a 340-380 nm excitation filter, and emitted light should be detected through a 435-485 nm barrier filter reflected from a 400-nm dichroic mirror. AF488 fluorescence can be excited through a 465-495 nm excitation filter, and emitted light can be detected through a 515-555 nm barrier filter reflected from a 505-nm dichroic mirror.
   2. Image a minimum of 50 cells per condition for each staining method.

4. Manual Measurement of Capsule

1. For cells stained with India ink or fluorescent stains, use a cell measurement software to manually measure capsule and cell diameter (see Table of Materials). To do this, go to "File" > "Open Image" > "Measure" and use the cursor to draw a straight line through the widest point of the whole cell (total diameter).
2. Next, click "Measure" again and draw a straight line through the cell body (cell body diameter).
   NOTE: Measurements are auto-saved onto the cells.
3. Repeat this process for all cells (minimum of 50/group).
4. To save images once they are measured, click "File" > "Save As" and name the images appropriately.
5. Select the newly measured files and export data to a spreadsheet software.
6. Calculate the average capsule diameter by using the equation: ([total diameter] - [cell body diameter]).

5. Automated Measurement of Capsule

NOTE: For successful automated measurement, use 16-bit images with a high level of contrast and that are properly focused along with an appropriate image analysis software (see Table of Materials). When imaging C. neoformans for capsule measurement, it is important to focus on the dark ring at the boundary between the cell wall and capsule. This allows the software to correctly delineate the cell and capsule.

1. Invert and create a copy of all India ink stained cell images using an appropriate image-editing software (see Table of Materials). To do this, click "File" > "Open" to open India ink stained images and then "Control" + "I" to invert the image. Click "File" > "Save As" to save the inverted image.
2. To import both the original and inverted images into the software click "File" > "Import Sequence" > "Load Image" > "Define Sequence (Manually)" > "Dimensions (Channel)" > "Import" > "Apply".
   NOTE: The C. neoformans cell bodies and capsules can be identified and masked using specific algorithms - referred to as 'recipes' - included in the software.
3. Use the 'Colony Analyzer (Fluorescence)' recipe on the inverted image to detect and mask the cell body, then click "apply". In the inverted image, the dark ring defining the boundary of the C. neoformans cell becomes brighter than the surrounding capsule.
4. Use the 'Colony Analyzer (Fluorescence)' recipe on the inverted image to segment the C. neoformans cell bodies from their capsules, then click "apply". This creates a count mask. Rename this count mask "Cell Body" by right-clicking the tab labeled "count mask".
   NOTE: The recipe consists of multiple image processing steps: background removal, object detection, object separation, and subset filtering.
5. Next, use the 'Cell Proliferation' recipe on the original image to detect and mask the capsule, and click "apply". This creates a count mask. Rename this count mask "Capsule" by right-clicking the tab labeled "count mask".
6. Using the recipe, ‘Capsule Partition’, create a new mask onto the original image to partition adjacent capsules from one another. To do this, click “Capsule Partition” from the recipe dropdown menu. In the Capsule Partition Box, choose the now renamed count mask “Capsule” (5.5) for Capsule Region. Choose the now renamed count mask “Cell Body” for Crypto Cells (5.4). Choose “Original” for the input channel, and “Create Mask for Partitioned Capsule. Click “Apply
   NOTE: The recipe generates cell and capsule diameter, defined as the average of the longest and shortest axes crossing the center of the cell and capsule, and area measurements from detection masks. Find the output that is located under the spreadsheet tab in this software. Repeat this process for all images (minimum of 50 cells/group). The recipe's parameters could be tuned to optimize detection of individual images or optimized for batch detection of multiple images. Additional measurements can be calculated by the recipe for comprehensive characterization of the cell and capsule's morphology.
7. Export data to a spreadsheet software of choice for further analysis.

Results

To illustrate capsule induction, cell staining, imaging, and measuring techniques, we used three strains of C. neoformans: the common, well-characterized laboratory strain, H99S³⁰, and two clinically isolated strains of previously unknown capsule diameter, B18 and B52³¹.

The workflow of capsule induction, staining, and image acquisition using India ink is shown in

Discussion

For decades, the capsule has been a major focus of research for both mycologists and clinicians interested in C. neoformans and cryptococcosis due to its role as a major virulence factor for the pathogen. Using microscopy to measure differences in capsule size between strains and under different growth conditions can provide important information about the pathogen and its responses to various stimuli (i.e., different environmental conditions, potential drug treatments, etc.) Here, we have outl...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Capsule Induction
C. neoformans cells			The clinical lab strain, H99S, was a kind gift from Dr. John Perfect (Duke University).  The clinical strains, B18 and B52, were kind gifts from Dr. Greg Bisson (University of Pennsylania).
Yeast Peptone Dextrose Broth (YPD)	Fisher Scientific	DF0428-17-5
Phosphate Buffered Saline (PBS)			This is made in the lab using standard recipe (137mM NaCl, 2.7 mM KCl, 10mM Na2HPO4O, 2 mM Kh2PO4O)
DMEM/high-glucose with L-glutamine, without sodium pyruvate	GE Life Sciences	SH30022.01
6-well plates	Falcon	CL5335-5EA
Shaking incubator	Thermo Scientific		MaxQ6000
CO2 incubator	Fisher Scientific		Isotemp
Centrifuge	Thermo Scientific		Legend XTR
Staining
Microcentrifuge	Thermo Scientific		Legend Micro 21R
India ink	Fisher Scientific	14-910-56
Calcofluor white	Sigma-Aldrich	18909-100ML-F
18B7 mouse anti-GXM antibody conjugated to Alexafluor 488			A kind gift from Dr. Arturo Casadevall (Johns Hopkins University)
PBS with 1% Bovine Serum Albumin (BSA)			PBS is the same recipe listed above (line 4) with 1% BSA added and filter sterilized.
Bovine Serum Albumin	Sigma-Aldrich	A9418
Superfrost microscope slides	Fisher Scientific	12-550-143
Glass coverslips	Corning	2855-18	#1.5 thickness
Clear nail polish or other non-toxic sealant
Image Acquisition
Immersion oil	Cargille	16484
Light microscope with immersion oil objective	Zeiss		Zeiss Axio A1 with a Plan - NEOFLUAR 100x oil immersion NA 1.30 objective
Light microscope camera	Zeiss		Zeiss Axiocam ErCD camera
Confocal microscope with oil immersion objective	Zeiss		LSM 700 laser scanning confocal equipped with a Plan-Apochromat 63X NA 1.4 oil immersion DIC M27 objective.
Confocal microscope software	Zen 2009
Confocal microscope camera	Nikon		Nikon Ti-Eclipse with a Intensilight epifluorescence illuminator (Nikon), CoolSNAP MYO microscope camera (Photometrics), Plan Apo 60x NA 1.40 oil immersion objective (Nikon) and 1.5x magnification changer.
Widefield imaging software	Nikon Elements (Nikon)
Capsule Measurement
Image editing software	Photoshop (Adobe)
Microscope software for manual measurement	Axiovision (Carl Zeiss)
Image analysis software for automated meesurement	Aivia (DRVision Technologies)
Spreadsheet software	Excel (Microsoft)