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 se....

Protocol

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.......

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.......

Materials

Name	Company	Catalog Number	Comments
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