Adhesion of Candida parapsilosis to Bovine Serum Albumin under Fluid Shear
Summary
Adhesion is an important first step in colonization and pathogenesis for Candida. Here, 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.
Abstract
C. parapsilosis (Cp) is an emerging cause of bloodstream infections in certain populations. The Candida clade, including Cp, is increasingly developing resistance to the first and the second line of 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 the 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 allow for the fluorescent quantification of individual isolates. We also demonstrate 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.
Introduction
Candida spp. are common commensal organisms on human skin and mucosae that can lead to invasive diseases among the immunocompromised with substantial associated morbidity, mortality, and cost1,2,3. 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 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 conditions5,6. These studies have helped define the structure and functions of fungal adhesins in disease7,8,9. However, adhesion in the bloodstream, gastro-intestinal (GI) tracts, 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 produced due to the movement of liquids10,11. The C. albicans adhesin, Als5 has been shown to facilitate shear dependent adhesion12,13. CpAls7 (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 the animal models16,17,18. The most frequently used models are murine mucosal and bloodstream infections but invertebrate models, such as Galleria larvae, are increasingly being used because of the 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 yeast responses to the host environment. In contrast, in vitro adhesion assays permit the 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 the standard yeast growth medium, yeast-peptone-dextrose (YPD), but switch to strong adhesion when grown for a few hours at 37 °C in the tissue-culture medium 199 (M199)15,19. A detailed protocol is provided here for a medium throughput assay that permits the measurement of adhesion of multiple yeast samples that run in parallel, under defined conditions of growth, fluid shear, temperature, and substrate. The assay has been designed to maximize reproducibility, and to allow for the use of clinical isolates of Cp, as well as strains that have been experimentally manipulated in the lab. The assay as described here, for Cp adhesion to a bovine serum albumin (BSA) substrate, demonstrates that clinical isolates exhibit a range of adhesion, whereas two commonly used lab strains, CDC317 and CLIB214 show poor adhesion.
Protocol
Representative Results
Discussion
The data resulting from the above protocol can be analyzed using a standard spreadsheet software. 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 cove…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This work was supported by a grant from the William and Mary Oh-William and Elsa Zopfi Professorship in Pediatrics for Perinatal Research, the Kilguss Research Core, and an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P30GM114750.
Materials
| 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 | ||
| Dulbecco’s Phosphate-Buffered Saline (DPBS+) | 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 |
References
- Pittet, D., Monod, M., Suter, P. M., Frenk, E., Auckenthaler, R. Candida colonization and subsequent infections in critically ill surgical patients. Annals of Surgery. 220 (6), 751-758 (1994).
- Lau, A. F., et al. Candida colonization as a risk marker for invasive candidiasis in mixed medical-surgical intensive care units: development and evaluation of a simple, standard protocol. Journal of Clinical Microbiology. 53 (4), 1324-1330 (2015).
- Lionakis, M. S. New insights into innate immune control of systemic candidiasis. Medical Mycology. 52 (6), 555-564 (2014).
- Maubon, D., Garnaud, C., Calandra, T., Sanglard, D., Cornet, M. Resistance of Candida spp. to antifungal drugs in the ICU: Where are we now. Intensive Care Medicine. 40 (9), 1241-1255 (2014).
- Sheppard, D. C., et al. Functional and structural diversity in the Als protein family of Candida albicans. Journal of Biological Chemistry. 279 (29), 30480-30489 (2004).
- Liu, Y., Filler, S. G. Candida albicans Als3, a multifunctional adhesin and invasin. Eukaryotic Cell. 10 (2), 168-173 (2011).
- Filler, S. G. Can host receptors for fungi be targeted for treatment of fungal infections. Trends in Microbiology. 21 (8), 389-396 (2013).
- Oh, S. H., et al. Agglutinin-Like Sequence (ALS) genes in the Candida parapsilosis species complex: Blurring the boundaries between gene families that encode cell-wall proteins. Frontiers in Microbiology. 10, 781 (2019).
- Willaert, R. G. Adhesins of yeasts: Protein structure and interactions. Journal of Fungi (Basel). 4 (4), 119 (2018).
- Isberg, R. R., Barnes, P. Dancing with the host; flow-dependent bacterial adhesion. Cell. 110 (1), 1-4 (2002).
- Thomas, W. Catch bonds in adhesion. Annual Review of Biomedical Engineering. 10, 39-57 (2008).
- Chan, C. X., Lipke, P. N. Role of force-sensitive amyloid-like interactions in fungal catch bonding and biofilms. Eukaryotic Cell. 13 (9), 1136-1142 (2014).
- Lipke, P. N., Klotz, S. A., Dufrene, Y. F., Jackson, D. N., Garcia-Sherman, M. C. Amyloid-like beta-aggregates as force-sensitive switches in fungal biofilms and infections. Microbiology and Molecular Biology Reviews. 82 (1), 00035 (2018).
- Bertini, A., et al. Targeted gene disruption in Candida parapsilosis demonstrates a role for CPAR2_404800 in adhesion to a biotic surface and in a murine model of ascending urinary tract infection. Virulence. 7 (2), 85-97 (2016).
- Neale, M. N., et al. Role of the inducible adhesin CpAls7 in binding of Candida parapsilosis to the extracellular matrix under fluid shear. Infections and Immunity. 86 (4), 00892 (2018).
- Clancy, C. J., Cheng, S., Nguyen, M. H. Animal models of candidiasis. Methods in Molecular Biology. 499, 65-76 (2009).
- MacCallum, D. M. Mouse model of invasive fungal infection. Methods in Molecular Biology. 1031, 145-153 (2013).
- Segal, E., Frenkel, M. Experimental in vivo models of candidiasis. Journal of Fungi (Basel). 4 (1), 21 (2018).
- Bliss, J. M., et al. Transcription profiles associated with inducible adhesion in Candida parapsilosis. mSphere. 6 (1), 01071 (2021).
- Hochmuth, R. M., et al. Surface adhesion, deformation and detachment at low shear of red cells and white cells. Transactions – American Society for Artificial Internal Organs. 18, 325-334 (1972).
- Munn, L. L., Melder, R. J., Jain, R. K. Analysis of cell flux in the parallel plate flow chamber: implications for cell capture studies. Biophysical Journal. 67 (2), 889-895 (1994).
- Shaw, S. K., Bamba, P. S., Perkins, B. N., Luscinskas, F. W. Real-time imaging of vascular endothelial-cadherin during leukocyte transmigration across endothelium. Journal of Immunology. 167 (4), 2323-2330 (2001).
- Shaw, S. K., et al. Coordinated redistribution of leukocyte LFA-1 and endothelial cell ICAM-1 accompany neutrophil transmigration. Journal of Experimental Medicine. 200 (12), 1571-1580 (2004).
- Grubb, S. E., et al. Adhesion of Candida albicans to endothelial cells under physiological conditions of flow. Infection and Immunity. 77 (9), 3872-3878 (2009).
- Finkel, J. S., et al. Portrait of Candida albicans adherence regulators. PLoS Pathogens. 8 (2), 1002525 (2012).
- Gulati, M., Ennis, C. L., Rodriguez, D. L., Nobile, C. J. Visualization of biofilm formation in Candida albicans using an automated microfluidic device. Journal of Visualized Experiments. (130), e56743 (2017).
- Shaik, S. S. Low intensity shear stress increases endothelial ELR+ CXC chemokine production via a focal adhesion kinase-p38β MAPK-NF-κβ pathway. Journal of Biological Chemistry. 284 (9), 5945-5955 (2009).
- dela Paz, N. G., Walshe, T. E., Leach, L. L., Saint-Geniez, M., D’Amore, P. A. Role of shear-stress-induced VEGF expression in endothelial cell survival. Journal of Cell Science. 125, 831-843 (2012).
- Bliss, J. M., Basavegowda, K. P., Watson, W. J., Sheikh, A. U., Ryan, R. M. Vertical and horizontal transmission of Candida albicans in very low birth weight infants using DNA fingerprinting techniques. Pediatric Infectious Disease Journal. 27 (3), 231-235 (2008).
- Bliss, J. M., et al. Candida virulence properties and adverse clinical outcomes in neonatal candidiasis. Journal of Pediatrics. 161 (3), 441-447 (2012).
.