Examining the virulence of Candida albicans transcription factor mutants using Galleria mellonella and mouse infection models

Abstract

The aim of the present study was to identify Candida albicans transcription factors (TFs) involved in virulence. Although mice are considered the gold-standard model to study fungal virulence, mini-host infection models have been increasingly used. Here, barcoded TF mutants were first screened in mice by pools of strains and fungal burdens (FBs) quantified in kidneys. Mutants of unannotated genes which generated a kidney FB significantly different from that of wild-type were selected and individually examined in Galleria mellonella. In addition, mutants that could not be detected in mice were also tested in G. mellonella. Only 25% of these mutants displayed matching phenotypes in both hosts, highlighting a significant discrepancy between the two models. To address the basis of this difference (pool or host effects), a set of 19 mutants tested in G. mellonella were also injected individually into mice. Matching FB phenotypes were observed in 50% of the cases, highlighting the bias due to host effects. In contrast, 33.4% concordance was observed between pool and single strain infections in mice, thereby highlighting the bias introduced by the "pool effect." After filtering the results obtained from the two infection models, mutants for MBF1 and ZCF6 were selected. Independent marker-free mutants were subsequently tested in both hosts to validate previous results. The MBF1 mutant showed impaired infection in both models, while the ZCF6 mutant was only significant in mice infections. The two mutants showed no obvious in vitro phenotypes compared with the wild-type, indicating that these genes might be specifically involved in in vivo adapt.

Keywords: Candida albicans; mice Galleria mellonella; transcription factors; virulence.

FIGURE 1

Fungal burden (FB) of BCY mutants in G. mellonella single strain infections. For each individual larva, the FB is expressed as a percentage relative to the mean of the wild-type group in the same experiment. This normalization enables comparisons between different experiments. All Galleria mellonella experiments are pooled in this graph. The results are expressed as the mean of the group and the minimum and maximum values. The results are organized according to the phenotype observed in mice pool infections. The green bar corresponds to the wild-type strain. The yellow bar corresponds to the cmp1Δ/Δ mutant (avirulent control). The red bars correspond to low FB mutants significantly different from wild-type in G. mellonella. The blue bars correspond to high FB mutants significantly different from wild-type in G. mellonella. The black bars indicate mutants not significantly different from the wild-type. The dotted line indicates the level of the mean of the wild-type strain set at 100%. Each mutant was tested at least twice using a minimum of five larvae each time or once using a minimum of 10 larvae. Statistical analyses were performed using a ROUT analysis to remove outliers (Motulsky and Brown, 2006), followed by a Mann–Whitney test to assess CFU differences relative to the wild-type strain.

FIGURE 2

Comparison of the results obtained using 19 mutant strains in two different in vivo models, including G. mellonella and mice. (A) Fungal burden of BCY mutants in mice single strain infections. For each individual mouse, the FB in the kidneys is expressed as a percentage relative to the mean of the wild-type group in the same experiment (set at 100% and indicated as a dotted line). All experiments are pooled in this graph. Only the results from mutants tested at least twice are presented here. The results are organized according to the phenotype observed in G. mellonella single strain infections. The green bar corresponds to the wild-type strain. The red bars correspond to statistically low FB mutants in mouse single strain infections. The blue bars correspond to high FB mutants significantly different from the wild-type in mouse single strain infections. The black bars indicate no significant difference from the wild-type. Each mutant was tested twice using five mice each time. The statistical analyses were performed using a ROUT analysis to remove outliers, followed by a Mann–Whitney test to assess CFU differences relative to the wild-type strain. The horizontal bars indicate the mean and minimum–maximum of FB for each mutant strain. (B) Correlation between results obtained with G. mellonella (24 h pi) and mice (three dpi). Red and blue data points correspond to mutants with the strongest alterations in host systemic infectivity (low- and high- FB mutants, respectively). The data for the zcf13 mutant were obtained from Vandeputte et al. (2011). Spearman’s rank correlation coefficient: r = 0.30, p = 0.2. Excluding colored data points, r = -0.17, p = 0.56. The dotted lines indicate the level of the mean of the wild-type strain set at 100% in each model.

FIGURE 3

Testing an independent set of mutants in mice. (A) Fungal burden of full ORF, marker-free C. albicans deletion mutants in mice single strain infections. For each individual mouse, the FB in the kidneys is expressed as a percentage relative to the mean of the wild-type group in the same experiment. All experiments are pooled in this graph. The mean value of each group is indicated with a black bar. The dotted line indicates the level of the mean of the wild-type strain set at 100%. Each mutant was tested twice using five mice each time (except for ZCF6 revertant 2 and MBF1 revertant). Statistical analyses were performed using a Mann–Whitney test to assess CFU differences relative to the wild-type strain. The stars indicate the level of statistical significance: ^∗p< 0.05, ^∗∗p < 0.01. (B) Survival curves in mice. The mice were injected with 5 × 10⁵ cells of the corresponding strain and monitored daily. These survival curves represent at least two independent experiments. Groups of 10 mice were used each time. ^∗∗∗∗p< 0.0001, log-rank test.

FIGURE 4

Testing an independent set of mutants in G. mellonella. (A) Fungal burden of full ORF, marker-free deletion mutants in G. mellonella single strain infections. For each individual larva, the FB is expressed as a percentage relative to the mean of the wild-type group in the same experiment. All experiments are pooled in this graph. The mean of each group is indicated with a black bar. The dotted line indicates the level of the mean of the wild-type strain set at 100%. Each mutant was tested three times using at least six larvae each time. Statistical analyses were performed using a ROUT analysis to remove outliers (Motulsky and Brown, 2006), followed by a Mann–Whitney test to assess CFU differences relative to the wild-type strain. The star indicates the level of statistical significance: ^∗p < 0.05. (B) Survival curves in G. mellonella. Larvae were injected with 5 × 10⁵ cells of the corresponding strain and monitored twice daily. These survival curves represent three independent experiments. Groups of at least 10 larvae were used each time. The log-rank test was performed, and no significant differences were observed.