Adhesion of Candida parapsilosis to Extracellular Matrix Proteins under Fluid Shear

Abstract

C. parapsilosis (Cp) is an emerging cause of bloodstream infections in certain populations. The Candida clade, including Cp, is increasingly developing resistance to first- and second-line antifungals. Cp is frequently isolated from hands and skin surfaces as well as the GI tract. Colonization by Candida predisposes individuals to invasive bloodstream infections. To successfully colonize or invade the host, yeast must be able to rapidly adhere to body surfaces to prevent elimination by host defense mechanisms. Here we describe a method to measure adhesion of Cp to immobilized proteins under physiologic fluid shear, using an end-point adhesion assay in a commercially available multichannel microfluidic device. This method is optimized to improve reproducibility, minimize subjectivity, and to allow fluorescent quantification of individual isolates. We also show that some clinical isolates of Cp show increased adhesion when grown in conditions mimicking a mammalian host, whereas a frequently used lab strain, CDC317, is non-adhesive under fluid shear.

SUMMARY:

Adhesion is an important first step in colonization and pathogenesis for Candida. An in vitro assay is described to measure adhesion of C. parapsilosis isolates to immobilized proteins under fluid shear. A multichannel microfluidics device is used to compare multiple samples in parallel, followed by quantification using fluorescence imaging.

INTRODUCTION:

Candida spp. are common commensal organisms on human skin and mucosae that can lead to invasive disease among the immunocompromised with substantial associated morbidity, mortality and cost^1–3. Although C. albicans remains an important cause of these infections, non-albicans species such as C. parapsilosis, C. glabrata, C. krusei, C. tropicalis, and C. auris are being increasingly recognized, especially in vulnerable populations and with frequent resistance to available antifungal drugs⁴ Non-albicans species present distinct elements of biology and pathogenesis that are still under active investigation.

Adhesion is an important first step in colonization and pathogenesis. Interference with this step may therefore offer an opportunity to stop disease progression at an early stage. Studies of Candida adhesion and invasion have been predominantly focused on static conditions^5,6. These studies have helped define the structure and functions of fungal adhesins in disease^7–9. However, adhesion in the bloodstream, GI and urinary tracts, and in catheters, must occur under conditions of fluid shear flow which places unique constraints upon adhesion. Adhesion under shear requires rapid catch bond formation and the ability to withstand strong pulling forces from liquid movement^10,11. The C. albicans adhesin, Als5 has been shown to facilitate shear dependent adhesion^12,13. Als7 (CpALS4800) has been previously shown to mediate adhesion of Cp to epithelial cells, and a knockout showed decreased virulence in a urinary tract infection model¹⁴. We demonstrated that CpALS4800 promotes adhesion under physiologically relevant fluid shear conditions¹⁵.

Candida colonization and pathogenesis have been extensively studied in animal models^16–18. The most frequently used models are murine mucosal and bloodstream infections, but invertebrate models such as Galleria larvae are increasingly being used because of low cost, rapid throughput and simplicity. Animal models recapitulate many steps of the human disease process in both the pathogen and host, including the host adaptive and innate immune responses, interactions of yeast with tissues and the microbiota, and the yeast responses to the host environment. In contrast, in vitro adhesion assays permit focus specifically on the adhesion step, and on the experimental manipulation of variables such as shear force, growth conditions of yeast, and adhesion to specific substrates.

Because Cp is capable of growth in both humans and environmental sources, it is likely to be capable of sensing and responding to different environments. In support of this notion, multiple clinical isolates of Cp show low adhesion under fluid shear when grown in standard yeast growth medium, yeast-peptone-dextrose (YPD), but switch to strong adhesion when grown for a few hours at 37°C in tissue-culture medium 199 (M199)^15,19. A detailed protocol is provided here for a medium throughput assay that permits measurement of adhesion of multiple yeast samples run in parallel, under defined conditions of growth, fluid shear, temperature, and substrate. The assay has been designed to maximize reproducibility, and to allow use of clinical isolates of Cp as well as strains that have been experimentally manipulated in the lab. The assay demonstrates that clinical isolates exhibit a range of adhesion, whereas two commonly used lab strains, CDC317 and CLIB214 show poor adhesion.

PROTOCOL:

1. Growth and induction of clinical strains

1.1 Streak Cp strains on 1% (m/v) yeast extract, 2% (m/v) peptone, 2% (m/v) dextrose YPD agar plates, and grow at 22°C.

Note. Plates may be stored on the lab bench, and re-used over the following week. Caution: Candida spp. are classified as Biosafety Level 2 organisms and should be handled using appropriate precautions.

1.2 The day prior to the adhesion assay, transfer approximately 6 colonies of each strain to a 250 mL Erlenmeyer (conical) flask containing 10 mL of YPD medium. Grow overnight in a microbiological shaker set to 250 rpm at 37 °C.

Note. Cp need to grow for 20–24 h to reach stationary phase in liquid culture at 37 °C.

1.3 The day of the adhesion assay, transfer 1 mL of liquid culture from the Erlenmeyer flask to a microfuge tube. Centrifuge the culture at maximum speed in a microfuge (16,000 × g) for 3 minutes. Resuspend in 1 mL of sterile water, and repeat this step for a total of three washes. At the end of the last wash, resuspend in 1 mL sterile water. Use wide-orifice pipet tips of 1 mL and 200 μL sizes for handling yeast suspensions in this and subsequent steps.

(Note. Many adhesive strains of Cp stick to the sides of the Erlenmeyer flask, and require mechanical scraping to remove).

1.4 Count the yeast using a hemocytometer or equivalent device. Dilute the yeast to achieve a reasonably countable concentration. Use a 500-fold dilution, putting 20 μL into 10 mL of water.

Note. Most strains grow to 10⁸ – 10⁹ cells/mL in overnight shaking culture at 37 °C.

1.5 Dilute the yeast into 2 mL of YPD or Medium 199 (M199), at a final concentration of 3 × 10⁶ cells/mL in a 2 mL microfuge tube. Incubate in a 37 °C water bath for 3 h.

2. Coating of microfluidic channels

2.1. Prepare in advance a 2% (m/v) solution of bovine serum albumin (BSA) in Hank’s Balanced Salt Solution containing calcium and magnesium (HBSS+). To do this, gently sprinkle 4 g of BSA powder on the surface of 200 mL of HBSS+, and incubate at 37 °C for 30–60 min undisturbed, to allow the protein to wet and then dissolve. Filter sterilize and store at 4 °C.

2.2. Warm 2.5 mL of the BSA to 37 °C for at least 1 hour, keeping it sterile.

Note. This step is necessary to reduce bubble formation in the microchannel plate.

2.3. While the BSA is warming, move the microfluidics controller to the tissue culture hood, turn on the device and start the software. Place the fluidics interface within the sterile field, and gently clean the silicone gasket with a lint-free tissue paper wetted with 70% (v/v) alcohol. Avoid prolonged or repeated contact of alcohol with the acrylic plate to prevent crazing or cracking of the plastic. Air dry the interface facing up in the hood airflow to remove all traces of alcohol.

2.4. Add 100 μL of pre-warmed BSA solution as a droplet in the central indentation of each “outlet” channel (Figure 1A) of a 48 well, 24-microchannel plate, using sterile technique.

Figure 1. Microfluidics assay layout.

(A) A pair of channels, showing reverse fluid flow from the “outlet” to the “inlet”. The consecutive tiled fields captured by the microscope are shown by dotted lines (1–10 for the upper channel, and 11–20 for the lower). (B) Setup of microfluidics controller software for reverse flow during BSA coating (Step 2.6). Screenshot reproduced here with permission from the manufacturer.

2.5. Place the interface on top of the microchannel plate, aligning the four bolts of the interface with the corresponding sockets in the plate. Tighten the bolts using gloved fingers. Be aware that resistance to hand tightening indicates misalignment. Lift up the plate slightly and reseat it, and resume fastening the bolts. When the bolts are finger tight, use the torque wrench to further tighten the interface.

2.6. Using the microfluidics software interface (Figure 1B), set the Mode to Manual. Use the default Fluid option (Water@19degC) for both sets of columns, and set shear to 1 dyn/cm^2. Activate “outlet” columns (#2,4,6,8) to pump liquid towards “inlets”. Run at room temperature for 30 min.

2.7. Visually inspect each “inlet” well by peering through the bottom of the microchannel plate to ensure that a droplet of liquid has pooled in each “inlet”, confirming that all 24 channels were successfully wetted and filled.

2.8. Unfasten the interface, and top up each well (“inlets” and “outlets”) with 250 μL of HBSS+ to prevent drying out of channels, and put the plate in a tissue culture incubator.

Note. A minimum of 48 hours is required for proteins to be uniformly adsorbed to the channel surfaces of the microchannel plate. After protein coating, plates may be stored for up to two weeks in a humid environment before use in adhesion assays. For longer periods, wrap plates in plastic film to prevent evaporation.

3. Adhesion Assay

3.1. While the yeast are incubating in YPD and M199 (step 1.5) aspirate inlet and outlet wells of the BSA coated microchannel plate. During this and following aspirations, avoid disturbing the channel that runs from the central indentation of each well (Figure 1A). Instead, aspirate from the edge of the well. Add 1 mL of HBSS+ to the “outlet” wells.

3.2. Attach the microfluidics interface as above (step 2.5). Using the default fluid setting (Water@19degC) Shear at 2 dyn/cm^2 at room temperature to wash the channel and remove unbound BSA. Wash for 2–3 hours at this flow rate.

3.3. Remove interface from microchannel plate. Turn on plate heater unit of microfluidics device, and make sure it is set to 37 °C. Aspirate medium from all wells on plate, and add 0.5 mL of induced yeast from step 1.5 to each pair of “outlets”. Resuspend yeast well by inverting the 2 ml tubes 3–6 times, and gently pipetting up and down immediately prior to addition to wells. Leave the “inlet” wells empty.

3.4. Fasten fluidics interface to microchannel plate as in step 2.5 above. Use device software to set Fluid to HBSS@37degC, and Shear to 5 dyn/cm^2. Activate “outlet” columns (#2,4,6,8) to pump liquid towards “inlets”. Run on plate heater at 37 °C for 30 min, to allow yeast to adhere to the BSA-coated channel.

3.5. During this time, prepare a wash buffer of 30 mL of Dulbecco’s phosphate-buffered saline containing calcium and magnesium (DPBS+) containing 5 μM calcofluor, which is included to render the yeast fluorescent for detection. Warm in a bath at 37 °C.

3.6. At the end of 30 min adhesion, Pause flow using the software interface without altering other flow conditions. Unfasten interface, and aspirate all wells (“inlets” and “outlets”).

3.7. Add 1 mL of wash buffer to “outlets”. Reattach fluidics interface, and resume flow using the software for another 10 minutes. This step is designed to wash away non-adherent and loosely bound yeast, and simultaneously fluorescently stain yeast in the channel.

3.8. After 10 minutes, remove fluidics interface and replace with lid. Gently clean bottom of microchannel plate with a lint-free wipe, and proceed to imaging.

4. Imaging and quantification

4.1. Place the microchannel plate in an appropriate microscope stage holder. Use a 20x objective lens, which will allow a field of view such that the channel fills approximately half of the image height. Locate the left end of channel 1 (in “inlet” 1). Adjust the stage so that the channel is positioned as in Figure 1A, position 1.

4.2. Acquire a single brightfield image of the channel. Measure the area of the channel area in μm², using the rectangle measuring tool in oreder to normalize area measurements during data analysis (step 5.2 below).

4.3. Switching to the DAPI fluorescent channel (excitation 395 nm, emission 440/40 nm) adjust focus to adherent yeast on the bottom surface of the channel. Lock the autofocus on this plane. Set fluorescence excitation intensity to 1.5% and camera exposure conditions (Binning 2×2, 15 ms exposure, 12-bit, Gain 4) to avoid saturation of the image sensor.

4.4. Using the motorized stage and ND Acquisition>XY Imaging in the microscope controller software, automatically capture a consecutive series of non-overlapping images spaced one field of view apart (666 μm) of first channel pair (1/2). Collect 10 images from left to right for the upper channel, shift down 666 μm and collect another 10 from right to left for the lower channel (as shown schematically in Figure 1A). Use the Relative XY option, so that once image positions are defined, a similar series can be triggered for each channel pair, after the start of the channel is manually defined. Collect images in the DAPI channel (ND Acquisition> λ>DAPI).

4.5. Monitor images to confirm that the channel remains within the field of view as the motorized stage moves the plate.

Note. Each set of 20 images from a channel pair will be automatically saved with a consecutive file name by ND acquisition.

4.6. Use the autostep function (XYZ Navigation>XY step) to move 25750 μm down to “inlet” 3. Fine tune channel position manually as in step 4.1. Capture the next set of images (ND Acquisition) of channel pair 3/4.

4.7. Repeat this process, until all 12 channel pairs have been imaged, with results saved in 12 files. Follow the manufacturer’s ordering of channels from 1–24.

4.8. Merge all 12 sets of fluorescent images into a single file (File>Merge ND Documents). Confirm that the file order matches the order in which the 12 sets of images were captured.

4.9. Use Binary>Threshold to separate yeast from background based on their fluorescence level. Apply the same threshold to the entire stack of images.

Note. A 12-bit greyscale intensity low threshold of 500, and a high threshold set to the maximum of 4095 is typical.

4.10. Measure the Threshold area (Measure>Perform Measurement>All Frames). Open the report (Analysis Controls>Automated Measurement Results) and check the data.

Note. There should result a table of measurements for 240 images (20 images from each of 12 channel pairs), and threshold area for each image is listed in the column labeled BinaryArea [μm²].

4.11. Export data to a tab delimited text file (Export).

REPRESENTATIVE RESULTS:

Using the methods described in the Protocol section, the adhesion of 6 strains of Cp was compared (Table 1). Four of the strains were recent clinical isolates at low passage number^29,30, and CLIB214 and CDC317 are commonly used strains that have been in laboratory culture for many years. A wide range of Adhesion Indices were observed, from 0.2% to 91% (Figure 2). Three clinical isolates (JMB81, JMB77 and Ro75) showed strong adhesion when grown in M199. Interestingly, both lab strains showed relatively poor adhesion in either growth medium. The third clinical isolate, WIH04, resembled the lab strains with relatively poor adhesion.

Table 1.

Strains of Candida parapsilosis used in this study.

Strain	Description	Reference/Source
JMB81	Invasive clinical isolate from infant blood culture	29
JMB77	Invasive clinical isolate from infant blood culture	29
Ro75	Commensal clinical isolate from infant stool	30
WIH04	Invasive clinical isolate from infant blood culture	29
CLIB214	Case of Sprue, Puerto Rico	ATCC (#22019)
CDC317	Health care worker’s hand	ATCC (#MYA-4646)

Figure 2. Adhesion assay comparing 6 isolates of Candida parapsilosis.

Isolates were grown for 3 h in YPD or M199 medium prior to adhesion assay as described in protocol. Graph represents the mean, and error bars the standard error of the mean from four consecutive experiments, with duplicate channels in each experiment. Adhesion index represents the percentage of the flow channel surface that was covered with yeast. Comparisons were made with analysis of variance (ANOVA). Between-group comparisons were made with the Holm-Sidak test. *, P < 0.001. YPD-M199 comparisons not significant for WIH04, CLIB214 and CDC317.

The results shown here are from four consecutive experiments, run on different days. They demonstrate the reproducibility of the adhesion assay.

DISCUSSION:

The data resulting from the above protocol can be analyzed using standard spreadsheet office software such as Microsoft Excel or LibreOffice Calc. Data are expressed as “adhesion index”, which is calculated as follows: The BinaryArea value for each set of 10 images (representing the yeast coverage for a single channel) is summed across the images, and the mean and standard deviation are calculated for the summed area of each channel pair.

The channel area measured in step 4.2 represents the maximum possible area in a single field of view that might ever be covered in yeast. In this protocol, the entire length of the channel is recorded in 10 consecutive images, which represents an area of nearly 2.5 mm².

This area is multiplied by 10 to represent the length of the channel, and the value is used to normalize the Averages and Standard Deviations to express them as percentages of Adhesion Index. Effectively this measures the surface area of a channel that is covered in yeast, with an Adhesion Index of 100% indicating that the entire length of the channel was carpeted with yeast from edge to edge, an area of nearly 2.5 mm².

Cell adhesion under defined fluid shear was first measured using parallel plate flow chambers²⁰. These custom-built flow chambers typically used microscope slides or cover slips as bases, and offered a single channel for measurement^21–23. Using a commercial version of such a flow chamber, the yeast form of two C. albicans strains was found to bind more strongly to endothelium than the hyphal or pseudohyphal form²⁴.

The development of a multichannel flow chamber system opened up the possibility of higher throughput adhesion assays. The flow chamber hardware and disposable supplies are costly, but they offer a uniformity of manufacture that decreases experimental variability. Finkel et al. used this system to measure adhesion of C. albicans to the silicone material used to make one face of the channel as a model of intravenous catheter associated disease²⁵. Another group used it to measure adhesion of C. albicans and Saccharomyces cerevisiae to BSA-coated channels, followed by phase contrast (brightfield) imaging of a single field of view, followed by software-based cell counting¹². A third group used the same multichannel system to assess biofilm formation in C. albicans interacting with uncoated channels, also using brightfield imaging over several hours²⁶.

In the current protocol, the multichannel flow system was used to measure specifically the adhesion step of Cp. Several modifications help increase accuracy of quantification, maximize dynamic range, reduce the potential for biohazard spills, and reduce variation.

Firstly, the fluidics system is usually run in the forward orientation, with fluid flowing from “inlet” to “outlet”. In addition to the viewing section of the channel, there is also a narrower serpentine region of the flow path which is used to offer hydraulic resistance to the flow. This region tends to trap yeast. In the current approach, yeast suspensions are run in reverse orientation, from “outlet” to “inlet” (Figure 1A). In this orientation the serpentine region falls downstream of the viewing section, reducing concern of trapping of yeast.

Growth conditions of Cp immediately prior to the adhesion assay strongly influence adhesion, as does coating of the channel with BSA or extracellular matrix proteins¹⁵. Growth in mammalian cell medium (M199) or serum leads to increased adhesion. It is important to note that unlike C. albicans, Cp does not elongate in the 5 hour duration of this assay. Shear rate strongly influences adhesion, with maximal shear observed at 5 dynes/cm², which is similar to that of blood in capillaries and post-capillary venules^27,28.

To reduce the potential for biohazard spills, yeast growth, adhesion under fluid shear, and washing are performed using Biosafety level 2 microbiology precautions at the laboratory bench. After these steps, the microfluidics plate is untethered from the hardware, the plate exterior is cleaned and covered, and the closed plate is imaged at the microscope stage. This approach reduces the risk of contamination of the microscope area.

This protocol uses calcofluor white to stain yeast cell walls. This approach is designed to allow fluorescent imaging of clinical isolates, without need for genetic manipulation to add fluorescent protein tags such as GFP. Fluorescent thresholding allows measurement of the surface area that is covered with yeast. To reduce potential interference with adhesion, the dye is added after the adhesion step, during washing.

Adhesion along the length of the viewing channel may vary. By summing fluorescence along the length of the channel, the variability and subjectivity offered by one or a small number of fields of view are reduced. The current approach instead extracts the maximum available information from the microfluidics plate by capturing the entire channel. Imaging is greatly facilitated by a motorized stage and autofocus mechanism for rapid tiled imaging of the length of the channel (Figure 1A) with relatively little manual input. Use of lower power objectives results in an increased field of view, which in turn allows for slight drift in position as the channel is scanned by the motorized stage. Because tolerances of plate manufacture results in slight variation in the precise locations of channels, it is not possible to image all 24 channels/240 images in a single operation. The approach used here (2 channels and 20 images at a time) is a compromise. Nevertheless, using this approach it is possible to complete imaging and quantification (steps 4.1–4.10) in approximately 15 minutes.

Using this setup to measure adhesion of clinical isolates, a wide range of adhesion indices from 0.2% to 91% was observed. Interestingly, two frequently used Cp strains, CLIB214 and CDC317 showed weak adhesion (Figure 2). These observations indicate that there is significant variation among Cp isolates, and that the assay can provide adhesion data across a wide dynamic range.

Potential variations of this assay include use of different species of fungi. Virtually any species that stains fluorescently with calcofluor may potentially be used in this assay, although they may require different substrates, shear forces, or growth conditions. It is also possible to grow monolayers of endothelial cells or epithelial cells in the flow channels, and measure adhesion of yeast to host cells. Mammalian cells tend to exclude calcofluor, and generally offer low fluorescent background, so that specific detection of yeast is still possible. However, it should be noted that adhesion assays between two cell types require substantially greater effort to maintain both fungal and mammalian cells in optimal physiologic condition.

Table 2.

Reagents and equipment used in this study.

Name of Material/ Equipment	Company	Catalog Number	Comments/Description
Bioflux 200	Fluxion	Bioflux 200
Bioflux Microfluidics plates, 48 well, low shear	Fluxion	910-0004
Bovine Serum Albumin (BSA) Fraction V	Fisher Scientific	BP1605
Calcofluor Fluorescent Brightener	Sigma-Aldrich	F3543
DAPI filter set 440/40	Nikon
DPBS+ (Dulbecco’s Phosphate-Buffered Saline)	Corning Cellgro	21-030-CM	With calcium and magnesium
Hank’s Balanced Salt Solution, 1X (HBSS+)	Corning Cellgro	21-023-CM	With calcium and magnesium, without phenol red
Inverted microscope with Perfect Focus	Nikon	Ti-E
M199 medium	Lonza	12-117Q	With Earle’s salts and HEPES
Motorized Stage	Nikon	Ti-S-E
Nikon 20x lambda Plan-Apo objective	Nikon
NIS-Elements software 5.02	Nikon
Spectra fluorescent LED light source	Lumencor	SPECTRA-X3
Zyla 4.2 sCMOS camera	Andor	Zyla 4.2