Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

Summary

Here, we demonstrate how image cytometry can be used for quantification of pathogenic fungi in association with host cells in culture. This technique can be used as an alternative to CFU enumeration.

Abstract

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans commonly employ infection of mammalian hosts or host cells (i.e. macrophages) followed by yeast quantification using colony forming unit analysis or flow cytometry. While colony forming unit enumeration has been the most commonly used method in the field, this technique has disadvantages and limitations, including slow growth of some fungal species on solid media and low and/or variable plating efficiencies, which is of particular concern when comparing growth of wild-type and mutant strains. Flow cytometry can provide rapid quantitative information regarding yeast viability, however, adoption of flow cytometric detection for pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Here, we demonstrate an image-based cytometric methodology using the Cellometer Vision (Nexcelom Bioscience, LLC) for the quantification of viable pathogenic yeasts in co-culture with macrophages. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome, and here, we quantitatively assess the growth of H. capsulatum yeasts in RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with image cytometry. Our method faithfully recapitulates growth trends as measured by traditional colony forming unit enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of live macrophages with a GFP-expressing strain of C. albicans. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens in association with host cells.

Introduction

Studies of pathogenic fungi in association with their hosts and/or host cells often require quantification of viable fungal cells over a time course or under different infection conditions. Enumeration of colony forming units (CFU) is the standard method by which the number of viable fungal cells has been measured, however, this technique has several drawbacks and limitations. First, many fungal species are slow-growing. Growth of visible colonies on solid media can take 1-2 weeks, significantly slowing the pace of research. Second, manipulation of samples during CFU plating is a laborious process, since several dilutions must be plated to ensure a countable number of colonies. Third, the number of CFU is typically lower than the number of viable organisms plated because the plating efficiency is well below 100%. For example, plating efficiencies for the dimorphic fungal pathogen Histoplasma capsulatum can be as high as 90%, but are routinely as low as 30% and are even lower (10%) for the related fungal dimorph Paracoccidioides brasiliensis ^{1, 2}. Plating efficiency for Candida albicans is also subject to variability ³. Finally, CFU analysis accounts for only live and actively dividing cells capable of establishing growth on solid media, whereas in many situations, it would be useful to determine the presence and concentration of dead and/or metabolically inactive cells.

Previously, flow cytometric methods for quantification of several pathogenic fungal species has been described ^4-6. However, due to biosafety containment issues involved with using biosafety level 2 (BSL2) or BSL3-level pathogens on shared flow cytometers, adoption of this technique has been limited. Like flow cytometry, image cytometry is a sensitive and rapid method of cell quantification. However, image cytometry can be performed at a fraction of the cost with comparable results ^7-11. Here, we describe methods for performing image cytometry of pathogenic fungi in association with host cells. We demonstrate our methods using two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum is a dimorphic fungal pathogen that causes respiratory disease; in humans, it grows as budding yeast and replicates within alveolar macrophages. Candida albicans is a human commensal species that occasionally causes candidiasis. We show that image cytometry allows rapid quantification of these yeasts, together with visualization capability.

Protocol

1. Infection of Macrophages with H. capsulatum, C. albicans

1. 16 hr prior to infection, seed macrophages at desired density in 24-well plates. In this protocol, a density of 3.0 x 10⁵ cells/well was used.
2. Add fungal cells in log-phase growth at a desired multiplicity of infection (MOI). This protocol can accommodate a range of MOI (0.2 - 5.0). For the infection of RAW264.7 macrophages with H. capsulatum, we used an MOI of 0.2.
3. Following 1.5 hr to allow for phagocytosis, wash macrophages three times with PBS to remove extracellular fungi.
4. Incubate for desired number of hours prior to sample analysis. At low MOI (0.2 in our experiment), infected macrophage cells remain viable for several days, and samples can be analyzed approximately every 12-24 hr.

2. Macrophage Lysis

1. To liberate fungi from macrophage cells, remove media, wash 3 times with PBS, and add 0.5 ml sterile water. Under these conditions, macrophages will lyse and fungal cells will remain intact.
2. Incubate for 5 min at room temperature.
3. Transfer lysate to sterile tube, keep on ice.
4. Transfer 20 μl lysate to a separate tube, then add 20 μl AO/PI solution. Proceed directly to step 5: "Sample Preparation for Image Cytometric Analysis"

3. CFU Plating

1. Perform ten-fold dilution of lysates (from step 2.3) in media.
2. Plate 100 μl of each dilution, in duplicate, on HMM-agarose plates. Incubate plates in a humidified chamber at 37 °C with 5% CO₂ for 7-8 days.
3. Manually count colonies on plates displaying a minimum of 100 and a maximum of 1,000 distinct colonies.

4. Visualization of Fungi within Live Macrophages

1. To collect live macrophages, wash cells 3 times with PBS. Add 0.5 ml PBS and incubate for 30 min at 4 °C.
2. To remove macrophages from tissue culture wells, gently pipette up and down several times. Transfer liquid to sterile tube, keep on ice.
3. Transfer 20 μl sample to a separate tube, then add 20 μl AO/PI solution. Proceed directly to step 5: "Sample Preparation for Image Cytometric Analysis".

5. Sample Preparation for Image Cytometric Analysis

1. Pipette the target sample thoroughly and then transfer 20 μl of sample into the disposable cell counting chamber.
2. Allow the cells to settle in the chamber for 30 sec.
3. Insert counting chamber into the image cytometer.

6. Cellometer Instrument Setup

1. Insert the Fluorescence Optics Modules: VB-535-402 and VB-660-502 into the system and make sure they are locked in place.
   1. VB-535-402 (excitation at 475 nm, emission at 535 nm) is used for acridine orange and GFP detection.
   2. VB-660-502 (excitation at 540 nm, emission at 660 nm) is used for propidium iodide detection.
2. Turn on the image cytometer and open the accompanying software.

7. Cellometer Software Setup

1. Select the preset "Assay Type" and "Cell Type" in the Assay Dropdown Menu.
   1. For viability, the assay is optimized for acridine orange and propidium iodide detection.
   2. For detection of Candida albicans infection, the assay is optimized for GFP detection.
2. Select "Options" on the top and click on "Take Background Image", and allow the operation to complete.
3. Click on "Preview Bright-Field Image".

8. Image Acquisition Procedure

1. Insert the prepared sample chamber into the image cytometer.
2. Use the focus knob and adjust the focus.
3. Once in focus, click on "Count", and allow the image acquisition operation to complete.
4. Remove the disposable counting chamber and dispose of appropriately.

9. Image Data Analysis

1. Concentration and viability measurement
   1. Once the counting is completed, the concentration and viability of the target cells are displayed in the result page.
   2. Click on "Export" to export the data into FCS Express 4 for cell population analysis of GFP in the Candida albicans infection experiment.
2. FCS Express analysis of Candida albicans-infected cells
   1. Import the ".NXDAT" file into FCS Express 4 and plot the results in a fluorescence histogram.
   2. Apply cell population gate to the histogram to determine the population percentages of Candida albicans-infected cells.

Results

We used the Cellometer Vision image cytometer to monitor growth of H. capsulatum in macrophage cells. RAW 264.7 cells were infected with H. capsulatum yeast cells and at various time points, samples were subjected to AO/PI staining followed by image-based cytometric analysis. In parallel, samples were analyzed by traditional CFU enumeration. At each time point, samples were incubated in water to lyse macrophages, and the liberated yeast cells were identified by the Cellometer Vision software (F...

Discussion

Image cytometry allows the user to capture high quality images and, using specialized software, perform rapid quantification of cells. One potential challenge to the adoption of image cytometry in the field of microbial pathogenesis is that the microbes to be counted are present in a mixed population of cells, including mammalian host cells. Here, we demonstrate that image cytometry can be used for the quantification of viable pathogenic yeasts during in vitro macrophage infection. Our methodology not only faith...

Materials

Name	Company	Catalog Number	Comments
			REAGENTS
DMEM	Life Technologies	11965-084
Fetal Bovine Serum	Life Technologies	16000044
AO/PI Solution	Nexcelom Bioscience	CSK-0102
Disposable Counting Chamber	Nexcelom Bioscience	CHT4-SD100
			EQUIPMENT
Cellometer Vision	Nexcelom Bioscience
Cellometer Vision Software	Nexcelom Bioscience