Two Infection Assays to Study Non-Lethal Virulence Phenotypes in C. Albicans using C. Elegans
Summary
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.
Abstract
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.
Introduction
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 secretory aspartyl proteinases (SAPs), which function to promote adhesion and invasion of C. albicans into host epithelial cells2, and candidalysin, a cytolytic peptide toxin3,4. Together, this suggests that C. albicans virulence is a complex phenotype resulting from an interaction between the pathogen and its host environment. Therefore, investigating virulence is best studied using model organisms that serve as host environments, in contrast to in vitro approaches.
Several host models, including both vertebrate and invertebrate organisms, have been developed to study C. albicans infection. The murine model, considered the gold standard, is often used for its adaptive and innate immune system, and ability to monitor disease progression both systemically and in specific organs5. However, there are significant limitations to this host model, including maintenance costs, small number of offspring, and decreased power and reproducibility associated with small sample sizes5. Therefore, other, more simple model organisms such as zebrafish (Danio rerio), fruit fly (Drosophila melanogaster), wax moth (Galleria mellonella), and nematode (Caenorhabditis elegans) have been developed. These non-mammalian model organisms are smaller, require less laboratory maintenance and larger sample sizes allow for greater power and reproducibility compared to murine models. Each of these models have specific advantages and disadvantages that need to be considered when choosing an infection model. G. mellonella offers the most physiologically similar environment to humans as it can be grown at 37 °C and has various phagocytic cells7. Furthermore, this model allows for the direct injection of a specific inoculum7. However, there is no fully sequenced genome, and no established method of creating mutant strains. Similar to G. mellonella, the D. rerio model allows for direct injection of a specific inoculum5,7. It also has both adaptive and innate immune system5, which is unique to this non-mammalian model, yet requires aquatic breeding tanks to maintain. D. melanogaster and C. elegans have similar advantages and disadvantages, which include fully sequenced genomes that are easy to manipulate and generate mutant strains7 but do not have adaptive immunity or cytokines7. Of all these non-mammalian models, C. elegans has the most rapid life cycle, self-fertilize to generate large numbers of genetically identical offspring, and are the most amenable to large-scale screens6,7,8. C. elegans has been extremely powerful for high-throughput screening of antifungal drugs9,10, characterizing virulence factors7, and identifying C. albicans-specific host defense networks11. The innate immune system in C. elegans has multiple components that are highly conserved with humans12. Host innate defenses include production of antimicrobial peptides13 (AMPs) and reactive oxygen species14,15,16.
The severity of C. albicans infection is predominantly measured by host survival but cannot capture non-lethal virulence phenotypes. An often-overlooked aspect of host fitness is reproduction, but several studies suggest that C. albicans impacts reproduction by reducing sperm viability17,18, suggesting that this may be an important aspect of host fitness to study. Therefore, the impact of C. albicans infection on host fecundity is a useful way to study non-lethal virulence phenotypes. We have developed two infection assays using C. elegans to investigate both survival and reproduction phenotypes in healthy hosts19,20. Here we describe both the fecundity and lineage expansion assays. Fecundity measures both progeny produced and survival of single hosts, and lineage expansion assesses the consequences of infection over three host generations. We demonstrate how these assays can be utilized to screen C. albicans deletion mutants to capture both dramatic and subtle differences in lethal and non-lethal virulence phenotypes.
Protocol
Representative Results
Discussion
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…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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).
Materials
| 1.5 mL eppendorf microtubes 3810X | Millipore Sigma | Z606340 | |
| 100 mm x 15 mm petri plates | Sigma-Aldrich | P5856-500EA | |
| 15 mL Falcon Conicals | Fisher Scientific | 14-959-70C | |
| 50 mL Falcon Conicals | Fisher Scientific | 14-432-22 | |
| Adenine | Millipore Sigma | A8626 | |
| Agar (granulated, bacterilogical grade) | Apex BioResearch Produces | 20-248 | |
| Aluminum Wire (95% Pt, 32 Gauge) | Genesee Scientific | 59-1M32P | |
| Ammonium Chloride | Millipore Sigma | 254134 | |
| Bacterial Cell Spreader | SP Scienceware | 21TP50 | |
| BactoPeptone | Fisher BioReagants | BP1420-500 | |
| Disposable Culture Tubes (20 x 150 mm) | FIsherBrand | 14-961-33 | |
| Dissection Microscope (NI-150 High Intensity Illuminator) | Nikon Instrument Inc. | ||
| E. coli | Caenorhabditis Genetics Center | OP50 | |
| Glucose | Millipore Sigma | 50-99-7 | |
| Medium Petri Dishes (35 X 10 mm) | Falcon | 353001 | |
| Metal Spatula | SP Scienceware | 8TL24 | |
| Nematode Growth Media (NGM) | Dot Scientific | DSN81800-500 | |
| Potassium Phosphate monobasic | Sigma | P0662-500G | |
| Sodium Chloride | Fisher Scientific | BP358-1 | |
| Sodium Phosphate | Fisher Scientific | BP332-500 | |
| Streptomycin Sulfate | Thermo-Fisher Scientific | 11860038 | |
| Tryptone | Millipore Sigma | 91079-40-2 | |
| Uridine | Millipore Sigma | U3750 | |
| Wildtype C. elegans | Caenorhabditis Genetics Center | N2 | |
| Yeast Extract | Millipore Sigma | 8013-01-2 |
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