Transient Mitochondria Dysfunction Confers Fungal Cross-Resistance against Phagocytic Killing and Fluconazole

Abstract

Loss or inactivation of antivirulence genes is an adaptive strategy in pathogen evolution. Candida glabrata is an important opportunistic pathogen related to baker's yeast, with the ability to both quickly increase its intrinsic high level of azole resistance and persist within phagocytes. During C. glabrata's evolution as a pathogen, the mitochondrial DNA polymerase CgMip1 has been under positive selection. We show that CgMIP1 deletion not only triggers loss of mitochondrial function and a petite phenotype, but increases C. glabrata's azole and endoplasmic reticulum (ER) stress resistance and, importantly, its survival in phagocytes. The same phenotype is induced by fluconazole and by exposure to macrophages, conferring a cross-resistance between antifungals and immune cells, and can be found in clinical isolates despite a slow growth of petite strains. This suggests that petite constitutes a bet-hedging strategy of C. glabrata and, potentially, a relevant cause of azole resistance. Mitochondrial function may therefore be considered a potential antivirulence factor. IMPORTANCE Candida glabrata is an opportunistic pathogen whose incidence has been increasing in the last 40 years. It has risen to become the most prominent non-Candida albicans Candida (NCAC) species to cause candidemia, constituting about one-third of isolates in the United States, and steadily increasing in European countries and in Australia. Despite its clinical importance, C. glabrata's pathogenicity strategies remain poorly understood. Our research shows that loss of mitochondrial function and the resulting petite phenotype is advantageous for C. glabrata to cope with infection-related stressors, such as antifungals and host immune defenses. The (cross-)resistance against both these factors may have major implications in the clinical outcome of infections with this major fungal pathogen.

Keywords: antivirulence; cross-resistance; fungal infection; petite.

FIG 1

Both Cgmip1Δ and Scmip1Δ show typical petite phenotypes. (A) Small colonies and loss of mitochondrial reductive power, as indicated by lack of tetrazolium dye reduction; (B) lack of growth in alternative carbon sources such as glycerol and absence of mitochondrial DNA (mtDNA) as determined by optical density and qPCR (n = 3 for each type of experiment, color by mean); (C) high resistance to azoles, including fluconazole (FL), voriconazole (VC), and clotrimazole (CL), and (D) overexpression of efflux pump-related genes (mean ± SD, n = 3 independent experiments with 3 technical replicates each). C.g., C. glabrata; S.c., S. cerevisiae.

FIG 2

C. glabrata and S. cerevisiae petite phenotypes differ in their survival after phagocytosis. (A) Cgmip1Δ survives phagocytosis by hMDMs much better than its parental wild type at early time points up to 6 h—in contrast to Scmip1Δ, which does not show any change in survival compared to its wild type (mean ± SD, n = 9 with 3 different donors in 3 independent experiments; each point represents the mean of 3 technical replicates). (B) Cgmip1Δ is taken up at a higher rate than the wild type by macrophages (mean ± SD, n = 9 with 3 different donors in 3 independent experiments; each point represents the mean of 3 technical replicates), and its accessible cell wall structures differ from the wild type (mean ± SD, n = 3 independent experiments with 3 technical replicates each). (C) In contrast to the wild type, Cgmip1Δ does not replicate within the phagosome as shown by FACS (left) and by the lack of FITC-unstained daughter cells (right). These are present in the wild type (blue arrows), in contrast to the mutant, which shows only mother cells (yellow arrows) (representative picture shown). Quantitative data are the mean ± SD; n = 12 with 3 different donors in 4 independent experiments; each point represents the mean of 3 technical replicates. Statistically significantly different values (unpaired, two-tailed Student’s t test on log-transformed ratios) are indicated by asterisks as follows: ***, P ≤ 0.001. C.g., C. glabrata; S.c., S. cerevisiae.

FIG 3

The petite phenotype emerges from the wild type after phagocytosis. (A) Cgmip1Δ shows a higher rate of survival up to 1 day of coincubation but loses this advantage over extended periods of intracellular existence (n = 5 with 1 different donor in each of the 5 independent experiments; each point represents a single survival test). Statistically significantly different values (unpaired, two-sided Student’s t test on log-transformed ratios) are indicated by asterisks as follows: *, P ≤ 0.05; ***, P ≤ 0.001. (B) Cells with the petite phenotypes arise from the wild type during coincubation with hMDMs (red) at a much higher rate than the spontaneous appearance of petite in RPMI (blue). The time points with the highest frequency of petite emergence correspond to the time point of increased fitness of Cgmip1Δ during phagocytosis (Red: n = 5 with 1 different donor in each of the 5 independent experiments and 4 technical replicates each. Blue: n = 3 in 3 different experiments with 6 technical replicates. Mean ± SD of the frequencies of petite emergence at each time point). (C) Phenotype characterization reveals the hMDMs-derived strains (MO-1 to -3) to be indeed petite. From the top left, small colony forming and lack of mitochondrial reductive power, lack of growth in alternative carbon sources, absence of mitochondrial DNA (mtDNA), high resistance to azoles (all n = 3 with mean values or representative picture shown) and overexpression of efflux pump-related genes (mean ± SD, n = 3 independent experiments with 3 technical replicates each). FL, fluconazole; VC, voriconazole; CL, clotrimazole; S.c., S. cerevisiae.

FIG 4

The petite phenotype triggered by fluconazole increases survival of phagocytosis at early time points. (A) Fluconazole-induced petites show the petite phenotype similar to Cgmip1Δ—small colonies and lack of mitochondrial reductive power, lack of growth in alternative carbon sources, absence of mitochondrial DNA (mtDNA), high resistance to azoles (all n = 3 with mean values or representative picture shown), and overexpression of efflux pump-related genes (mean ± SD, n = 3 independent experiments with 3 technical replicates each). FL, fluconazole; VC, voriconazole; CL, clotrimazole. (B) Fluconazole-induced petites (FL-1 to FL-3) show better survival of phagocytosis at early time points (mean ± SD, n = 3 with 1 donor in 3 independent experiments; each point represents a mean of 3 different colonies per donor, and each colony has 3 technical replicates). Statistically significantly different values (unpaired, two-sided Student’s t test on log-transformed ratios) are indicated by asterisks as follows: *, P ≤ 0.05; **, P ≤ 0.01. C.g., C. glabrata.

FIG 5

Cgmip1Δ shows higher basal expression of stress response-related genes and grows better under ER stress. (A) Petite variants of C. glabrata show high constitutive expression of stress-response genes even under nonstressed conditions (YPD) (mean ± SD, n = 3 independent experiments with 3 technical replicates each), and (B) exhibit better growth than their wild type under different ER stresses on plates as well as with tunicamycin (TM) in liquid cultures (mean ± SD, n = 3 independent experiments or representative picture shown); FL-1 to FL-3, fluconazole-induced petites; MO-1 to MO-3 macrophage-derived petites; C.g., C. glabrata.

FIG 6

The Cgmip1Δ petite phenotype seems to be adaptive under infection-like conditions but not in commensal-like conditions. (A) The ratio of recovered CgMIP1-deleted to wild-type C. glabrata cells was low when they were incubated separately on vaginal cells (Cgmip1Δ/–). The presence of Lactobacillus rhamnosus (L. r.) further shifts that ratio toward a lower recovery of Cgmip1Δ, and these effects are exacerbated in a direct competition in the same wells (C. glabrata [C. g.] WT + CgmipΔ). Data shown are the mean ± SD; n = 3 independent experiments. (B) In an infection model in the presence of fluconazole (FL), the effect is inverted. Without fluconazole, CgmipΔ is outcompeted as before (note the scale), but it has a decisive advantage in the presence of the antifungal drug, independent of the commensal bacteria (mean ± SD, n = 3 independent experiments). Statistically significantly different values (unpaired, two-sided Student’s t test on log-transformed ratios) are indicated by asterisks as follows: *, P ≤ 0.05; ***, P ≤ 0.001.

FIG 7

Mutation on the protein sequence of CgMIP1 of C. glabrata clinical strains compared to the wild type. Wild type (ATCC 2001, WT); the first 14 strains marked in bold are petite mutants, and below there are the 17 respiratory-competent strains. Gray, predicted mitochondrial signal peptide of the wild type; blue, amino acid substitutions; green, insertions; red, deletions. The black lines on top indicate the amino acid position every 50 amino acids. The black lines next to the strain names indicate that those strains have been isolated from the same patient.