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 toxin^3,4. 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 inoculum^5,7. 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 screens^6,7,8. C. elegans has been extremely powerful for high-throughput screening of antifungal drugs^9,10, 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 species^14,15,16.

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 viability^17,18, 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 hosts^19,20. 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

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% uridine) and grow overnight at 30 °C.
      ​NOTE: If the C. albicans strain is auxotrophic, supplement the media with the necessary amino acids.
   3. Using a sterile loop or toothpick, inoculate a single C. albicans colony into 2 mL of liquid YPD. Incubate at 30 °C with shaking for 24 h.
   4. Using a sterile toothpick, streak E. coli (OP50) onto Luria Broth (LB; 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, 15 g/L agar) agar plates. Incubate overnight at 30 °C.
   5. Inoculate a single OP50 colony into 50 mL of LB. Incubate at 30 °C with shaking overnight.
2. Nematode growth media preparation
   1. In a 2 L flask, add 29 g of nematode growth media powder (NGM; 17.5 g/L agar, 3.0 g/L Sodium Chloride, 2.5 g/L Peptone, .005 g/L cholesterol) to 1L of water and mix with a stir bar.
   2. To inhibit E. coli overgrowth and allow C. albicans proliferation supplement NGM with 0.2 g/L streptomycin sulfate after autoclaving.
      ​NOTE: If using NGM for nematode maintenance, streptomycin is not required.
3. Maintaining nematode populations
   1. Spread 300 µL of overnight E. coli culture onto prepared NGM agar plates using a metal spreader. This technique will be referred to as seeding.
   2. Allow plates to dry at room temperature (RT). Grow plates overnight at 30 °C.
   3. Maintain nematode population by chunking every 3-4 days onto a newly seeded NGM plate with E. coli. Store at 20 °C. Chunking is a technique used to quickly transfer a random population of nematodes to a newly seeded plate, which allows for populations to proliferate. To do this, use a sterile spatula to cut a small square (1 x 1 inch) out of the NGM agar plate. Carefully transfer the square to a new seeded NGM plate with the side with the nematodes facing down on the new agar⁸.
      NOTE: C. elegans maintained at 20 °C will produce offspring ~48 h later which is useful to consider when synchronizing a population of nematodes. C. elegans maintained at 25 °C will develop and reproduce faster and populations maintained at 15 °C will have slower growth.
4. Nematode population synchronization
   1. Begin with an existing nematode population maintained on NGM/OP50.
   2. Pipette ~3 mL of M9 buffer onto the NGM plate containing nematodes. Wash nematodes eggs off the plate and gently use the tip of the pipette to scrape eggs off the agar (they tend to stick). Using a P1000 pipette, transfer the liquid containing both eggs and worms to a 15 mL conical tube. To assess the number of eggs still on the plate, use a dissecting microscope to look at the agar.
   3. Centrifuge conical for 2 min at 279 x g and RT.
   4. Remove the supernatant, being careful not to disturb the nematode pellet.
   5. Add 3 mL of 25% bleach solution ("CAUTION" when handling).
   6. Invert tube for 2 minutes. Check that the nematodes are dead using the dissecting microscope - they will be stick-straight and non-motile.
      NOTE: This will only kill the existing nematodes. The integrity of the eggs will not be affected.
   7. Centrifuge for 2 min at 279 x g and RT.
   8. Remove the supernatant and resuspend the pellet in 3 mL M9.
   9. Centrifuge for 2 min at 279 x g and RT.
   10. Remove the supernatant and resuspend the pellet in 300 µL of M9.
   11. Using a dissecting microscope, check the concentration of eggs by pipetting 5 µL of eggs onto a small Petri plate. The ideal concentration should be between 20-100 eggs. If the culture is too dilute, concentrate the solution by centrifugation and removal of excess liquid. If the culture is too concentrated, add more M9 until the desired concentration is reached.
5. Prepare C. albicans and E. coli cultures for nematode infection (seeding).
   1. Prepare a blank solution. In a cuvette, combine 900 µL of ddH₂O and 100 µL of liquid YPD.
   2. Insert the cuvette into the spectrophotometer. Set the wavelength to 600 nanometers using the up arrow. Click on the button "0 ABS 100% T" to set the blank solution.
   3. In a new cuvette, combine 900 µL of ddH₂O and 100 µL of overnight yeast culture. Take the blank solution out of the spectrophotometer and add the cuvette containing the yeast solution. Record the optical density shown on the screen (do not press any buttons). Multiply the reading by 10 (the yeast solution measured was a 1 in 10 dilution).
   4. Normalize culture to 3.0 OD₆₀₀/mL with ddH₂O in a 1.5 mL microcentrifuge tube. 1 OD₆₀₀ is approximately 3 x 10^-7 CFU/mL²¹.
      ​NOTE: If the OD₆₀₀ reading is 6.7, 3 OD/6.7 OD= 0.447 mL, add 447 µL of C. albicans culture to the microcentrifuge tube. Centrifuge at maximum speed (16, 873 x g) for 30 s. Remove supernatant and resuspend in 1 mL of ddH₂O.
   5. Transfer the overnight E. coli culture to a 50 mL conical tube.
   6. Centrifuge the culture at 279 x g for 2 min at RT.
   7. Aspirate a majority of the supernatant, leaving ~1 mL.
   8. Resuspend the pellet in the remaining supernatant and transfer to a pre-weighed 1.5 mL microcentrifuge tube.
   9. Spin down the microcentrifuge tube at maximum speed for 30 s.
   10. Using a p1000 pipette, remove the supernatant and weigh the final pellet.
   11. Dilute E. coli to 200 mg/mL in ddH₂O.
   12. Use master mix calculations (Table 2) and scale appropriately.

2. Fecundity assay

NOTE: Representative data in shown in Supplementary Table 1 and a schematic in shown in Figure 1A.

1. Obtain or prepare the following: 35 mm x 10 mm Petri plates, NGM supplemented with 0.2 g/L streptomycin sulfate, E. coli OP50 culture, LB, C. albicans culture, YPD, Wire Pick, M9 buffer (3.0 g/L KH₂PO₄, 6.0 g/L Na₂HPO₄, 0.5 g/L NaCl, 1.0 g/L NH₄Cl), 15 mL conical tubes.
   Two Days Prior to Experiment
2. Inoculate C. albicans strains in 2 mL of YPD and E. coli (OP50) in 50 mL of LB and grow overnight at 30 °C.
3. Prepare 35 mm x 10 mm Petri plates with NGM supplemented with 0.2 g/L streptomycin sulfate. The number of plates prepared should last the whole experiment. The recommended number of replicates is 10 per treatment. For 10 replicates, 70 plates will be used. 1 L of NGM will make ~250 plates.
   One Day Prior to Experiment
4. Seed 35 mm x 10 mm NGM agar plates supplemented with streptomycin for Day 0, Day 2 & Day 3 according to the seeding protocol described above with the mastermix concentration (Table 2). Pipette the appropriate amount of mastermix onto the center of the plate. Spreading the culture is not necessary because a single spot of microbial growth is sufficient for host feeding and allows for us to easily identify hosts outside of the seed. Incubate the plates overnight at RT.
   NOTE: The Day 0 mastermix contains 50 µL of "seed" per replicate for each experimental treatment. Days 2 -7 include 10 µL of "seed" per replicate for each experimental treatment. There is no Day 1 plate because nematodes will reach the L4 stage 48 h after being synchronized onto a Day 0 plate. Once they reach L4, individual nematodes will be transferred to Day 2 plates.

For 1 replicate	E. coli (OP50) control condition			C. albicans & E. coli (OP50) treatment condition
	OP50	H2O	Total	OP50	C. albicans	H2O	Total
Day 0	6.25 ul	43.75 ul	50 ul	6.25 ul	1.25 ul	42.5 ul	50 ul
Days 2-7	1.25 ul	8.75 ul	10 ul	1.25 ul	.25 ul	8.5 ul	10 ul

Table 2: Mastermix volumes of E. coli and C. albicans cultures needed to infect nematodes for the fecundity assay.

Day of Experiment (Day 0)

5. Synchronize nematodes and plate ~50 eggs onto each Day 0 replicate of the control plates (OP50 only) and treatment plates (C. albicans + OP50). Incubate at 20 °C for 48 h.
   Day 2
6. Transfer a single L4 nematode by picking⁸ from the Day 0 plate to each of the replicate Day 2 seeded plates. L4 hosts can be identified by a small pocket in the middle of the dorsal side of their body²². Transfer the nematodes hatched and matured from the same type of seeded plate (i.e., L4 nematodes from D0 control plates must be transferred to Day 2 control plates).
7. Inoculate C. albicans cultures needed in 2 mL of YPD and E. coli (OP50 strain) in 50 mL of LB.
   Day 3
8. Transfer nematodes from Day 2 plates to Day 3 seeded plates, keeping track of each replicate (i.e., Replicate A from Day 2 must be moved to the Replicate A Day 3 plate).
9. Incubate Day 2 (only containing eggs) and Day 3 (containing the single adult) plates at 20 °C for 24 h.
10. Seed 35 mm x 10 mm NGM supplemented with streptomycin plates for Days 4 & 5 using 10 µL of mastermix per plate (Table 2) and incubate plates at RT for 24 h.
    Day 4
11. Transfer nematodes from Day 3 plates to Day 4 seeded plates.
12. Incubate Day 3 and Day 4 plates at 20 °C for 24 h.
13. Using a dissecting scope, count the viable progeny for each Day 2 plate. Note any replicates that died or are no longer on the plate (censored). Once the number of progeny are recorded, discard Day 2 plates.
    NOTE: Censored refers to nematodes that disappear on the plate. This can occur when nematodes crawl off the plate. Although less common during the 24 h window, dead nematodes' carcasses disintegrate into the agar also resulting in censorship. Censored data is not included in the final progeny and survival data analysis.
14. Inoculate new cultures of C. albicans strains in 2 mL of YPD and E. coli (OP50) in 50 mL of LB.
    Day 5
15. Transfer nematodes from Day 4 plates to Day 5 seeded plates.
16. Incubate Day 4 and Day 5 plates at 20 °C for 24 h.
17. Count the viable progeny for each Day 3 plate. Note any replicates that died or are no longer on the plate (censored). Once the number of progeny are recorded, discard Day 3 plates.
18. Seed 35 mm x 10 mm NGM supplemented with streptomycin plates for Days 6 & 7 using 10 µL of mastermix per plate (Table 2) and incubate plates at room temperature for 24 h.
    Day 6
19. Transfer nematodes from Day 5 plates to Day 6 seeded plates.
20. Incubate Day 5 and Day 6 plates at 20 °C for 24 h.
21. Count the viable progeny for each Day 4 plate. Note any replicates that died or are no longer on the plate (censored). Once the number of progeny are recorded, discard Day 4 plates.
    Day 7
22. Transfer nematodes from Day 6 plates to Day 7 seeded plates.
23. Incubate Day 6 and Day 7 plates at 20 °C for 24 h.
24. Count the viable progeny for each Day 5 plate. Note any replicates that died or are no longer on the plate (censored). Once the number of progeny are recorded, discard Day 5 plates.
    Day 8
25. Count the viable progeny for each Day 6 plate. Note any replicates that died or are no longer on the plate (censored). Once the number of progeny are recorded, discard Day 6 plates.
    Day 9
26. Count the viable progeny for each Day 7 plate. Do not count the largest nematode (parent). Note any replicates that died or are no longer on the plate (censored). Once the number of progeny are recorded, discard Day 7 plates.
    ​NOTE: This assay can also be used to assess survival. Record when each nematode died. At the end of the experiment, the percentage of nematodes that survived over the seven-day experiment can be compared for each treatment. Nematodes will sometimes crawl away from the food/pathogen source and try to climb the slides of the Petri plate. Check all areas of the plate before moving on. Dead nematodes will generally leave behind a carcass. Censor any nematode that cannot be located and do not count that nematode as dead. Do not include censored data in the final analysis of progeny and survival.
27. Analyze data for brood size and late reproduction using either one-way ANOVA or Kruskal-Wallis, depending on the normality of the data sets, as well as post-hoc Tukey/Dunn's multiple testing to identify significant differences between the treatment groups using GraphPad Prism software. Detect differences between survival curves using the Wilcoxon log-rank test.

3. Lineage Expansion Assay

NOTE: Representative data in shown in Supplementary Table 2 and a schematic is shown in Figure 2A.

1. Obtain or prepare the following: 100 mm x 15 mm Petri plates containing NGM agar supplemented with 0.2 g/L streptomycin sulfate, E. coli OP50 cultures, LB, C. albicans cultures, YPD, M9 buffer, Wire Pick, 15 mL conical tubes.
   Day -2
2. Inoculate C. albicans strains in 2 mL of YPD and E. coli (OP50) in 50 mL of LB and grow overnight at 30 °C.
   Day -1
3. Seed 100 mm x 15 mm NGM agar plates supplemented with streptomycin with 300 µL of mastermix per plate (Table 3). Spread the mastermix onto the plate using a sterile metal spreader. Incubate the plates overnight at 30 °C. Six replicates per treatment is recommended. Thus, prepare seven plates (One plate for synchronized nematodes, six plates for each adult nematode) per treatment.

	E. coli (OP50) control condition			C. albicans & E. coli (OP50). treatment condition
For 1 replicate	OP50	H2O	Total	OP50	C. albicans	H2O	Total
	1.25 ul	8.75 ul	10 ul	37.5 ul	7.5 ul	255 ul	300 ul

Table 3: Mastermix volumes of E. coli and C. albicans cultures needed to infect nematodes for the lineage expansion assay.

Nematode population growth: Day 0

4. Synchronize nematodes and plate 10-25 eggs on a single plate for each control (OP50 only) and treatment (C. albicans + OP50) and incubate at 20 °C for 48 h.
   Day 2:
5. Transfer a single L4 worm from each control and treatment Day 0 plate to seeded plates of the same treatment. L4 hosts can be identified by a small pocket in the middle of the dorsal side of their body²².
6. Incubate at 20 °C for 5 days (a total of one week following synchronization).
   Day 7:
7. Using a p1000 pipette, wash entire nematode population from each plate using 5 mL of M9 buffer and transfer to a 15 mL conical tube.
8. Store at 4 °C for 1 h to allow the nematodes to settle for easier counting.
9. Dilute each conical to final volume of 10 mL with M9 buffer.
10. For each biological replicate, count the number of nematodes in a 20 µL aliquot. Repeat this to obtain 6 technical replicates for each biological replicate. Back calculate to determine the total population size. If samples are too dilute (i.e., fewer than 10 nematodes per sample), concentrate the population in a smaller volume of M9 buffer.
    NOTE: Centrifugation of live nematodes will not harm the nematodes.
    Example calculation:
    70 Hosts           =               X (Total hosts)
    20 µL (aliquot)                  10,000µL (Total Volume)
11. Analyze data for lineage expansion using either one-way ANOVA or Kruskal-Wallis, depending on the normality of the data sets, as well as post-hoc Tukey/Dunn's multiple testing to identify significant differences between the treatment groups using GraphPad Prism software.

Results

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...

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