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

Amanda C. Smith; Judy Dinh; Meleah A. Hickman

doi:10.3791/62170

JoVE Journal > Genetics

Genética

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

Published: May 17, 2021

doi:

10.3791/62170

Amanda C. Smith¹, Judy Dinh², Meleah A. Hickman^1,2

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

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

1. Preparatory steps for the experiments

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.
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.
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.
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.
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 OD₆₀₀is approximately 3 x 10^-7 CFU/mL²¹.
  NOTE: If the OD₆₀₀reading 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.

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
Inoculate C. albicans strains in 2 mL of YPD and E. coli (OP50) in 50 mL of LB and grow overnight at 30 °C.
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
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
For 1 replicate	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)

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
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).
Inoculate C. albicans cultures needed in 2 mL of YPD and E. coli (OP50 strain) in 50 mL of LB.
Day 3
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).
Incubate Day 2 (only containing eggs) and Day 3 (containing the single adult) plates at 20 °C for 24 h.
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
Transfer nematodes from Day 3 plates to Day 4 seeded plates.
Incubate Day 3 and Day 4 plates at 20 °C for 24 h.
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.
Inoculate new cultures of C. albicans strains in 2 mL of YPD and E. coli (OP50) in 50 mL of LB.
Day 5
Transfer nematodes from Day 4 plates to Day 5 seeded plates.
Incubate Day 4 and Day 5 plates at 20 °C for 24 h.
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.
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
Transfer nematodes from Day 5 plates to Day 6 seeded plates.
Incubate Day 5 and Day 6 plates at 20 °C for 24 h.
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
Transfer nematodes from Day 6 plates to Day 7 seeded plates.
Incubate Day 6 and Day 7 plates at 20 °C for 24 h.
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
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
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.
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.

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
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
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
For 1 replicate	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

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:
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.
Incubate at 20 °C for 5 days (a total of one week following synchronization).
Day 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.
Store at 4 °C for 1 h to allow the nematodes to settle for easier counting.
Dilute each conical to final volume of 10 mL with M9 buffer.
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)
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.

Representative 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 host fitness during C. albicans infection. To assess how C. albicans infection impacts two distinct measures of host fitness, survival, and reproduction, we developed the fecundity assay¹⁹, which monitors individual hosts. Briefly, single hosts are isolated to control plates (uninfected) or C. albicans treatment plates and monitored every day for seven days for survival, and the number of daily progeny produced (Figure 1A). As this assay encompasses multiple measures of host fitness, it is an efficient way to assess C. albicans virulence. First, we evaluated total brood size by calculating the sum of daily progeny per host. Hosts infected with wildtype C. albicans produce a significantly smaller brood size on average compared to uninfected hosts (Figure 1B, black vs grey bars, p < 0.0001, Kuskal-Wallis test, and post-hoc Dunn's multiple comparison test). By monitoring daily progeny production, we can also detect differences in the timing of reproduction. We previously demonstrated that C. albicans infected hosts have delayed reproduction, with a large fraction of their progeny produced later in their adulthood compared to uninfected hosts¹⁹. The fraction of late reproduction is calculated by dividing the progeny produced on Days 4-7 by the total number of progeny produced. Uninfected hosts produce ~20% of their progeny during this late reproduction window whereas wildtype C. albicans infected hosts have 60% of their total offspring in this late reproduction window (Figure 1C).

The fecundity assay not only provides data to assess non-lethal virulence phenotypes, the data can also be used to assess host survival in a seven-day period. We plotted host survival (Figure 1D) and observed a decrease in survival for wildtype C. albicans infected hosts compared to uninfected hosts (grey vs. black lines, Figure 1D), but this difference was not statistically significant (p = 0.687, Log-rank test). This can be attributed to the short seven-day window of time this experiment covers. We previously showed it takes eight days for C. albicans infected hosts to reach 50% mortality¹⁹. Thus, this assay can only detect differences in host survival at very early time points.

Fecundity assays capture how C. albicans infection alters host fitness during early adulthood. We used this assay to screen for differences in virulence across reproductive and survival phenotypes using two C. albicans deletion strains (Table 1) previously identified to decrease virulence in murine, D. melanogaster, and C. elegans infection models¹¹^,¹⁹^,²³^,²⁴^,²⁵. CAS5 is a gene that encodes a transcription factor that regulates cell wall homeostasis, adherence, and stress response²⁶. Here, hosts infected with C. albicans cas5Δ/Δ have significantly larger brood sizes and a smaller fraction of late reproduction compared to wildtype C. albicans infected hosts (Figure 1B,C, orange bars, p < 0.0001, Kruskal-Wallis test, and post-hoc Dunn's multiple comparison test). Additionally, hosts infected with C. albicanscas5Δ/Δ do not have significantly different brood sizes or fraction of late reproduction compared to uninfected hosts (p > 0.9999, Kruskal-Wallis test, and post-hoc Dunn's multiple comparison test), indicating that cas5Δ/Δ strains are avirulent. Furthermore, we found that cas5Δ/Δ reduces host mortality compared to wildtype C. albicans (Figure 1D, orange), although this difference is not statistically significant (p = 0.687, Log-rank test), likely due to the short timeframe in which host fitness is evaluated. RIM101 is a gene that encodes a transcription factor required for alkaline-induced hyphal growth²⁷. Hosts infected with rim101Δ/Δ C. albicans had a significantly smaller fraction of late reproduction compared to wildtype C. albicans (Figure 1C, blue bar, p = 0.0184, Kruskal-Wallis test, and post-hoc Dunn's multiple comparison test) infected hosts, despite having similar total brood sizes (Figure 1B, blue bar, p = 0.6979, Kruskal-Wallis test, and post-hoc Dunn's multiple comparison test). Additionally, rim101Δ/Δ infected hosts had similar mortality to hosts infected with wildtype C. albicans (Figure 1D, blue, p = 0.687, Log-rank test). Taken together, we demonstrated the utility of this assay to distinguish subtle difference between C. albicans strains.

To quickly assess how C. albicans infection impacts host fecundity and survival over three generations, we developed the lineage expansion assay¹⁹, which monitors the progeny production of individual hosts. Briefly, single hosts are isolated to control plates (uninfected) or C. albicans treatment plates and after seven days the total number of viable progeny in the F1 and F2 generations are counted (Figure 2A). Uninfected hosts produced a progeny population ~35,000 in this timeframe (Figure 2B), compared to wildtype C. albicans infected hosts which produced a progeny population size of ~25,000, nearly a 30% reduction (Figure 2B). This simple assay can be used to rapidly screen through mutant strains of C. albicans. Both cas5Δ/Δ and rim101Δ/Δ C. albicans infected hosts produced progeny populations that were significantly larger than wildtype C. albicans infected hosts (Figure 2B, p < 0.0001 & p = 0.0185 respectively, Tukey's multiple comparisons test). Furthermore, cas5Δ/Δ C. albicans infected hosts produced progeny populations that were comparable to uninfected hosts, suggesting that this C. albicans strain is avirulent, and the rim101Δ/Δ C. albicans strain has reduced virulence for this virulence phenotype.

Figure 1: Fecundity and survival assessed in single hosts infected with different C. albicans strains. A) Experimental schematic of the fecundity assay. B) Total brood size for uninfected (OP50) hosts (n = 23), and hosts infected with wildtype (WT; SN250, n = 21), cas5Δ/Δ (n = 26), and rim101Δ/Δ (n = 26) C. albicans strains. The box represents the interquartile range, the midline indicates the median, and the whiskers represent the range. Error bars are the normalized range of the data. Treatments that share letters are not significantly differ, whereas treatments with differing letters are statistically significant, Kruskal-Wallis test and post-hoc Dunn's multiple comparison test. C) Fraction of late reproduction for uninfected hosts, and hosts infected with wildtype, cas5Δ/Δ, and rim101Δ/Δ C. albicans strains. Bars represent the mean, error bars represent +/- 1 SD, and symbols represent individuals hosts. Treatments that share letters are not statistically significantly different, whereas treatments with differing letters are significant, Kruskal-Wallis test and post-hoc Dunn's multiple comparison test. D) Survival curves of uninfected hosts, and hosts infected with wildtype, cas5Δ/Δ, and rim101Δ/Δ C. albicans strains for the first seven days of adulthood. Error bars represent ± 1 SD. Data from B, C, and D were collected from the same experiment and host sample sizes are the same in each panel. Please click here to view a larger version of this figure.

Figure 2: Virulence of C. albicans strains measured by host reproduction and death over multiple generations A) Experimental schematic of the lineage expansion assay. B) Box and whiskers plot of the population size (representing the number of F1 and F2 progeny) produced within 7 days from a single founder host exposed to OP50 food source alone (uninfected n=10, black), WT C. albicans (SN250, n=12, grey) or C. albicans cas5ΔΔ (n=12, orange), and rim101ΔΔ (n=12, teal) mutant strains (pink). Boxes indicate the 25-75^th quartiles with median indicated. Error bars are the normalized range of the data. Treatments that share letters are not significantly different, whereas treatments with differing letters are statistically significant, one-way ANOVA and post-hoc Tukey multiple comparison test. Data was initially published in Feistel et al. 2019¹⁶. Please click here to view a larger version of this figure.

Strain	Genotype	Source
SN250 (WT)	his1Δ/his1Δ, leu2Δ::C.dubliniensis HIS1/leu2Δ::C.maltosa LEU2, arg4Δ /arg4Δ, URA3/ura3Δ::imm434, IRO1/iro1Δ::imm434	Noble et al. (2010)
rim101∆/∆	his1Δ/his1Δ, leu2Δ::C.dubliniensis HIS1/leu2Δ::C.maltosa LEU2, arg4Δ /arg4Δ, URA3/ura3Δ::imm434, IRO1/iro1Δ::imm434 orf19.7247Δ::C.dubliniensisHIS1/orf19.7247Δ::C.maltosaLEU2	Noble et al. (2010)
cas5∆/∆	his1Δ/his1Δ, leu2Δ::C.dubliniensis HIS1/leu2Δ::C.maltosa LEU2, arg4Δ /arg4Δ, URA3/ura3Δ::imm434, IRO1/iro1Δ::imm434 orf19.4670Δ::C.dubliniensisHIS1/orf19.4670Δ::C.maltosaLEU2	Noble et al. (2010)

Table 1: C. albicans strains used in this study

Supplemental Table 1: Sample Fecundity Data Please click here to download this table.

Supplemental Table 2: Sample Lineage Expansion Data Please click here to download this table.

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 assay that is less cumbersome as it requires fewer host transfers and daily counting. The lineage expansion assay provides a multigenerational and quantitative measure that combines multiple aspects of host fitness. Together, these assays are a powerful way to quickly screen the virulence of C. albicans strains, including mutant strains and diverse clinical isolates. Furthermore, given the ease of the infection assay, where hosts are reared on the pathogen as the food source, this makes it easy to apply to other microbial pathogens to assess their virulence.

There are three main technical considerations to be mindful of. First, when synchronizing host populations to collect eggs, the timing of the bleach step needs to be closely monitored, by assessing host movement and/or host shape (dead hosts will no longer be sinusoidal) under a microscope. Once L1-adult hosts are dead, quickly centrifuge and remove the bleach to ensure the integrity of the eggs. Second, for both fecundity and lineage expansion it is critical that L4 hosts are isolated and transferred 48 hours following synchronization (Day 2), as this is the last developmental stage prior to reproductive maturity and non-L4 hosts can shift the reproductive timing. While 48 hours is typically when C. elegans reach L4, this can vary slightly. Third, when counting the final progeny population for lineage expansion, the concentration of the sample is important. If the sample is too dilute, the counts will be too low and cause significant technical variation. The sample concentration can be adjusted by centrifugation and removing excess buffer. If the sample is too concentrated, it will be difficult to count every host in that aliquot and cause significant technical variation. The sample concentration can be adjusted by adding more buffer. Keeping these considerations in mind will ensure the success of these relatively simple assays.

The assays we describe have some distinct differences from other C. elegans infection assays, including the use of solid NGM media and using OP50 as a food source for both uninfected and infected treatments. When hosts are reared only in the presence of C. albicans, their development is slower than when reared in conjunction with E. coli²⁸^,²⁹. During our experiments, hosts are exposed to C. albicans throughout development and early larval stages cannot consume large yeast cells.

While this is a highly adoptable system that allows for high replication and quantification, there are a few limitations when using C. elegans to study host-pathogen interactions. First, interactions between the pathogen and host immune function are limited as C. elegans lack an adaptive immune system and pro-inflammatory cytokines and chemokines³⁰. Second, since pathogens are introduced via the C. elegans diet, it is difficult to control the inoculum and ensure that all nematodes ingest the same amount⁷. However, with fluorescently tagged pathogens, and recent methods to extract yeast³¹ and bacteria³² from the C. elegans gut, we can enumerate the number of colonies ingested. Finally, C. elegans cannot survive at temperatures similar to the human body, instead are grown at temperatures ≥ 5°C physiologically relevant³³. Thus, growth and proliferation of the pathogen will be different in C. elegans compared to humans.

Fungal virulence is predominantly assessed using murine models and is often restricted to monitoring survival. However, using C. elegans as a simple host model for fungal infection offers three unique advantages: First, the ease of infectivity and laboratory handling makes it amenable to scientists of all training levels. Second, the large number of hosts that can be infected and monitored more reliably and confidently captures the phenotypic variation in and between treatments. Third, in C. elegans, the innate immune pathways genes have highly conserved mammalian homologues, including the p38 MAP kinase PMK-1¹³, the TIR-1 (SARM) protein which functions to activate the PMK-1 pathway in C. elegans immunity³⁴, and the dual oxidase BLI-3, which generates reactive oxygen species in response to pathogen infection in C. elegans¹⁴^,¹⁵^,¹⁶. The conservation of immunity and the availability of different immune mutants at the Caenorhabditis Genome Center, makes it easy to investigate the impact of host immunity on the pathogenicity of C. albicans and other pathogens. We recently showed that these assays can be used with sek-1 hosts to demonstrate that immunocompromised are highly susceptible to fungal infection¹⁹^,²⁰. Together, these assays our results and the other applications described here offer many reasons to use C. elegans to investigate the virulence of C. albicans.

Declarações

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

Referências

Underhill, D. M., Iliev, I. D. The mycobiota: interactions between commensal fungi and the host immune system. Nature Reviews Immunology. 14 (6), (2014).
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).
Calderone, R. A., Fonzi, W. A. Virulence factors of Candida albicans. Trends in Microbiology. 9 (7), 327-335 (2001).
Mayer, F. L., Wilson, D., Hube, B. Candida albicans pathogenicity mechanisms. Virulence. 4 (2), 119-128 (2013).
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).
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).
Arvanitis, M., Glavis-Bloom, J., Mylonakis, E. Invertebrate models of fungal infection. Biochimica et biophysica acta. 1832 (9), 1378-1383 (2013).
Issi, L., Rioux, M., Rao, R. The Nematode Caenorhabditis Elegans – A Versatile Vivo Model to Study Host-microbe Interactions. Journal of Visualized Experiments. (128), e56487 (2017).
Breger, J., et al. Antifungal Chemical Compounds Identified Using a C. elegans Pathogenicity Assay. PLoS Pathogens. 3 (2), 18 (2007).
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).
Pukkila-Worley, R., Ausubel, F. M., Mylonakis, E. Candida albicans Infection of Caenorhabditis elegans Induces Antifungal Immune Defenses. PLoS Pathogens. 7 (6), 1002074 (2011).
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).
Kim, D. H., et al. A Conserved p38 MAP Kinase Pathway in Caenorhabditis elegans Innate Immunity. Science. 297 (5581), 623-626 (2002).
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).
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).
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).
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).
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).
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).
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).
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).
Altun, Z. F., Hall, D. H. WormAtas Hermaphrodite Handbook – Introduction. WormAtlas. , (2006).
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).
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).
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).
Bruno, V. M., et al. Control of the C. albicans Cell Wall Damage Response by Transcriptional Regulator Cas5. PLoS Pathogens. 2 (3), 21 (2006).
Davis, D. Adaptation to environmental pH in Candida albicans and its relation to pathogenesis. Current Genetics. 44 (1), 58 (2003).
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).
De, A., Sahu, A. K., Singh, V. Bite size of Caenorhabditis elegans regulates feeding, satiety and development on yeast diet. bioRxiv. , 473256 (2018).
Pukkila-Worley, R., Ausubel, F. M. Immune defense mechanisms in the Caenorhabditis elegans intestinal epithelium. Current Opinion in Immunology. 24 (1), 3-9 (2012).
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).
Palominos, M. F., Calixto, A. Quantification of Bacteria Residing in Caenorhabditis elegans Intestine. BIO-PROTOCOL. 10 (9), (2020).
Marsh, E. K., May, R. C. Caenorhabditis elegans, a Model Organism for Investigating Immunity. Applied and Environmental Microbiology. 78 (7), 2075-2081 (2012).
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).