JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Genetics

Two Infection Assays to Study Non-Lethal Virulence Phenotypes in C. Albicans using C. Elegans

Published: May 17th, 2021

DOI:

10.3791/62170

1Graduate Program in Genetics & Molecular Biology, Emory University, 2Department of Biology, Emory University

Fungal opportunist pathogens can cause life-threatening as well as minor infections, but non-lethal phenotypes are frequently ignored when studying virulence. Therefore, we developed a nematode model that monitors both the survival and reproduction aspects of host to investigate fungal virulence.

While pathogens can be deadly to humans, many of them cause a range of infection types with non-lethal phenotypes. Candida albicans, an opportunistic fungal pathogen of humans, is the fourth most common cause of nosocomial infections which results in ~40% mortality. However, other C. albicans infections are less severe and rarely lethal and include vulvovaginal candidiasis, impacting ~75% of women, as well as oropharyngeal candidiasis, predominantly impacting infants, AIDS patients and cancer patients. While murine models are most frequently used to study C. albicans pathogenesis, these models predominantly assess host survival and are costly, time consuming, and limited in replication. Therefore, several mini-model systems, including Drosophila melanogaster, Danio rerio, Galleria mellonella, and Caenorhabditis elegans, have been developed to study C. albicans. These mini-models are well-suited for screening mutant libraries or diverse genetic backgrounds of C. albicans. Here we describe two approaches to study C. albicans infection using C. elegans. The first is a fecundity assay which measures host reproduction and monitors survival of individual hosts. The second is a lineage expansion assay which measures how C. albicans infection affects host population growth over multiple generations. Together, these assays provide a simple, cost-effective way to quickly assess C. albicans virulence.

Candida albicans is an opportunistic fungal pathogen of humans residing in different niches, including the oral cavity, gastrointestinal, and urogenital tracts1. While typically commensal, C. albicans causes both mucosal and bloodstream infections, the latter of which can be deadly. The severity of C. albicans infection is dependent on host immune function, with immunocompromised individuals more susceptible to infection than healthy individuals1. In addition to host-related factors, C. albicans has several virulence traits which include, hyphae, biofilm formation, and production of se....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

1. Preparatory steps for the experiments

  1. Preparing C. albicans and Escherichia coli cultures
    NOTE: The strains used in this study are listed in Table 1.
    1. Maintain C. albicans and E. coli strains as glycerol stocks at −80 °C.
    2. Using a sterile toothpick, streak desired C. albicans strain onto solid yeast peptone dextrose (YPD) (1% yeast extract, 2% bactopeptone, 2% glucose, 1.5% agar, 0.004% adenine, 0.008% urid.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Here we present two assays that measure C. albicans virulence as a non-lethal phenotype using C. elegans as an infection model. The first assay, fecundity, monitors how C. albicans infection impacts single hosts for progeny production and survival. The second assay, lineage expansion, measures how C. albicans infection impacts population growth over multiple generations.

The fecundity assay has multiple measures of h.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Here, we present two simple assays that measure fungal virulence. Both assays leverage C. elegans as a host system that includes monitoring for both lethal and non-lethal host phenotypes. For example, fecundity assays investigate the reproductive success of individual infected hosts while also measuring individual survival. The daily monitoring provides not only total brood size, but also reproductive timing, and time of death. The lineage expansion assay was developed as a simplified version of the fecundity as.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

We thank Dorian Feistel, Rema Elmostafa, and McKenna Penley for their assistance in developing our assays and data collection. This research is supported by NSF DEB-1943415 (MAH).

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

NameCompanyCatalog NumberComments
1.5 mL eppendorf microtubes 3810XMillipore SigmaZ606340
100 mm x 15 mm petri platesSigma-AldrichP5856-500EA
15 mL Falcon ConicalsFisher Scientific14-959-70C
50 mL Falcon ConicalsFisher Scientific14-432-22
AdenineMillipore SigmaA8626
Agar (granulated, bacterilogical grade)Apex BioResearch Produces20-248
Aluminum Wire (95% Pt, 32 Gauge)Genesee Scientific59-1M32P
Ammonium ChlorideMillipore Sigma254134
Bacterial Cell SpreaderSP Scienceware21TP50
BactoPeptoneFisher BioReagantsBP1420-500
Disposable Culture Tubes (20 x 150 mm)FIsherBrand14-961-33
Dissection Microscope (NI-150 High Intensity Illuminator)Nikon Instrument Inc.
E. coliCaenorhabditis Genetics CenterOP50
GlucoseMillipore Sigma50-99-7
Medium Petri Dishes (35 X 10 mm)Falcon353001
Metal SpatulaSP Scienceware8TL24
Nematode Growth Media (NGM)Dot ScientificDSN81800-500
Potassium Phosphate monobasicSigmaP0662-500G
Sodium ChlorideFisher ScientificBP358-1
Sodium PhosphateFisher ScientificBP332-500
Streptomycin SulfateThermo-Fisher Scientific11860038
TryptoneMillipore Sigma91079-40-2
UridineMillipore SigmaU3750
Wildtype C. elegansCaenorhabditis Genetics CenterN2
Yeast ExtractMillipore Sigma8013-01-2

  1. Underhill, D. M., Iliev, I. D. The mycobiota: interactions between commensal fungi and the host immune system. Nature Reviews Immunology. 14 (6), (2014).
  2. Ibrahim, A. S., Filler, S. G., Sanglard, D., Edwards, J. E., Hube, B. Secreted Aspartyl Proteinases and Interactions of Candida albicans with Human Endothelial Cells. Infection and Immunity. 66 (6), 3003-3005 (1998).
  3. Calderone, R. A., Fonzi, W. A. Virulence factors of Candida albicans. Trends in Microbiology. 9 (7), 327-335 (2001).
  4. Mayer, F. L., Wilson, D., Hube, B. Candida albicans pathogenicity mechanisms. Virulence. 4 (2), 119-128 (2013).
  5. Chin, V., Lee, T., Rusliza, B., Chong, P. Dissecting Candida albicans Infection from the Perspective of C. albicans Virulence and Omics Approaches on Host-Pathogen Interaction: A Review. International Journal of Molecular Sciences. 17 (10), 1643 (2016).
  6. Elkabti, A., Issi, L., Rao, R. Caenorhabditis elegans as a Model Host to Monitor the Candida Infection Processes. Journal of Fungi. 4 (4), 123 (2018).
  7. Arvanitis, M., Glavis-Bloom, J., Mylonakis, E. Invertebrate models of fungal infection. Biochimica et biophysica acta. 1832 (9), 1378-1383 (2013).
  8. Issi, L., Rioux, M., Rao, R. The Nematode Caenorhabditis Elegans - A Versatile Vivo</em> Model to Study Host-microbe Interactions. Journal of Visualized Experiments. (128), e56487 (2017).
  9. Breger, J., et al. Antifungal Chemical Compounds Identified Using a C. elegans Pathogenicity Assay. PLoS Pathogens. 3 (2), 18 (2007).
  10. Okoli, I., et al. Identification of antifungal compounds active against Candida albicans using an improved high-throughput Caenorhabditis elegans assay. PloS one. 4 (9), 7025 (2009).
  11. Pukkila-Worley, R., Ausubel, F. M., Mylonakis, E. Candida albicans Infection of Caenorhabditis elegans Induces Antifungal Immune Defenses. PLoS Pathogens. 7 (6), 1002074 (2011).
  12. Kim, D. H., Ausubel, F. M. Evolutionary perspectives on innate immunity from the study of Caenorhabditis elegans. Current Opinion in Immunology. 17 (1), 4-10 (2005).
  13. Kim, D. H., et al. A Conserved p38 MAP Kinase Pathway in Caenorhabditis elegans Innate Immunity. Science. 297 (5581), 623-626 (2002).
  14. van der Hoeven, R., McCallum, K. C., Cruz, M. R., Garsin, D. A. Ce-Duox1/BLI-3 Generated Reactive Oxygen Species Trigger Protective SKN-1 Activity via p38 MAPK Signaling during Infection in C. elegans. PLoS Pathogens. 7 (12), 1002453 (2011).
  15. van der Hoeven, R., Cruz, M. R., Chávez, V., Garsin, D. A. Localization of the Dual Oxidase BLI-3 and Characterization of Its NADPH Oxidase Domain during Infection of Caenorhabditis elegans. PLOS ONE. 10 (4), 0124091 (2015).
  16. Chávez, V., Mohri-Shiomi, A., Garsin, D. A. Ce-Duox1/BLI-3 Generates Reactive Oxygen Species as a Protective Innate Immune Mechanism in Caenorhabditis elegans. Infection and Immunity. 77 (11), 4983-4989 (2009).
  17. Vander, H., Prabha, V. Evaluation of fertility outcome as a consequence of intravaginal inoculation with sperm-impairing micro-organisms in a mouse model. Journal of Medical Microbiology. 64, 344-347 (2015).
  18. Castrillón-Duque, E. X., Suárez, J. P., Maya, W. D. C. Yeast and Fertility: Effects of In Vitro Activity of Candida spp. on Sperm Quality. Journal of Reproduction & Infertility. 19 (1), 49-55 (2018).
  19. Feistel, D. J., et al. A Novel Virulence Phenotype Rapidly Assesses Candida Fungal Pathogenesis in Healthy and Immunocompromised Caenorhabditis elegans Hosts. mSphere. 4 (2), (2019).
  20. Feistel, D. J., Elmostafa, R., Hickman, M. A. Virulence phenotypes result from interactions between pathogen ploidy and genetic background. Ecology and Evolution. 10 (17), 9326-9338 (2020).
  21. Mitchell, B. M., Wu, T. G., Jackson, B. E., Wilhelmus, K. R. Candida albicans Strain-Dependent Virulence and Rim13p-Mediated Filamentation in Experimental Keratomycosis. Investigative Ophthalmology & Visual Science. 48 (2), 774-780 (2007).
  22. Altun, Z. F., Hall, D. H. WormAtas Hermaphrodite Handbook - Introduction. WormAtlas. , (2006).
  23. Yuan, X., Mitchell, B. M., Hua, X., Davis, D. A., Wilhelmus, K. R. The RIM101 Signal Transduction Pathway Regulates Candida albicans Virulence during Experimental Keratomycosis. Investigative Ophthalmology & Visual Science. 51 (9), 4668-4676 (2010).
  24. Davis, D., Edwards, J. E., Mitchell, A. P., Ibrahim, A. S. Candida albicans RIM101 pH Response Pathway Is Required for Host-Pathogen Interactions. Infection and Immunity. 68 (10), 5953-5959 (2000).
  25. Chamilos, G., et al. Candida albicans Cas5, a Regulator of Cell Wall Integrity, Is Required for Virulence in Murine and Toll Mutant Fly Models. The Journal of Infectious Diseases. 200 (1), 152-157 (2009).
  26. Bruno, V. M., et al. Control of the C. albicans Cell Wall Damage Response by Transcriptional Regulator Cas5. PLoS Pathogens. 2 (3), 21 (2006).
  27. Davis, D. Adaptation to environmental pH in Candida albicans and its relation to pathogenesis. Current Genetics. 44 (1), 58 (2003).
  28. Jain, C., Yun, M., Politz, S. M., Rao, R. P. A Pathogenesis Assay Using Saccharomyces cerevisiae and Caenorhabditis elegans Reveals Novel Roles for Yeast AP-1, Yap1, and Host Dual Oxidase BLI-3 in Fungal Pathogenesis. Eukaryotic Cell. 8 (8), 1218-1227 (2009).
  29. De, A., Sahu, A. K., Singh, V. Bite size of Caenorhabditis elegans regulates feeding, satiety and development on yeast diet. bioRxiv. , 473256 (2018).
  30. Pukkila-Worley, R., Ausubel, F. M. Immune defense mechanisms in the Caenorhabditis elegans intestinal epithelium. Current Opinion in Immunology. 24 (1), 3-9 (2012).
  31. Smith, A. C., Hickman, M. A. Host-Induced Genome Instability Rapidly Generates Phenotypic Variation across Candida albicans Strains and Ploidy States. mSphere. 5 (3), 00433 (2020).
  32. Palominos, M. F., Calixto, A. Quantification of Bacteria Residing in Caenorhabditis elegans Intestine. BIO-PROTOCOL. 10 (9), (2020).
  33. Marsh, E. K., May, R. C. Caenorhabditis elegans, a Model Organism for Investigating Immunity. Applied and Environmental Microbiology. 78 (7), 2075-2081 (2012).
  34. Liberati, N. T., et al. Requirement for a conserved Toll/interleukin-1 resistance domain protein in the Caenorhabditis elegans immune response. Proceedings of the National Academy of Sciences of the United States of America. 101 (17), 6593-6598 (2004).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved