The Rewiring of Ubiquitination Targets in a Pathogenic Yeast Promotes Metabolic Flexibility, Host Colonization and Virulence

Abstract

Efficient carbon assimilation is critical for microbial growth and pathogenesis. The environmental yeast Saccharomyces cerevisiae is "Crabtree positive", displaying a rapid metabolic switch from the assimilation of alternative carbon sources to sugars. Following exposure to sugars, this switch is mediated by the transcriptional repression of genes (carbon catabolite repression) and the turnover (catabolite inactivation) of enzymes involved in the assimilation of alternative carbon sources. The pathogenic yeast Candida albicans is Crabtree negative. It has retained carbon catabolite repression mechanisms, but has undergone posttranscriptional rewiring such that gluconeogenic and glyoxylate cycle enzymes are not subject to ubiquitin-mediated catabolite inactivation. Consequently, when glucose becomes available, C. albicans can continue to assimilate alternative carbon sources alongside the glucose. We show that this metabolic flexibility promotes host colonization and virulence. The glyoxylate cycle enzyme isocitrate lyase (CaIcl1) was rendered sensitive to ubiquitin-mediated catabolite inactivation in C. albicans by addition of a ubiquitination site. This mutation, which inhibits lactate assimilation in the presence of glucose, reduces the ability of C. albicans cells to withstand macrophage killing, colonize the gastrointestinal tract and cause systemic infections in mice. Interestingly, most S. cerevisiae clinical isolates we examined (67%) have acquired the ability to assimilate lactate in the presence of glucose (i.e. they have become Crabtree negative). These S. cerevisiae strains are more resistant to macrophage killing than Crabtree positive clinical isolates. Moreover, Crabtree negative S. cerevisiae mutants that lack Gid8, a key component of the Glucose-Induced Degradation complex, are more resistant to macrophage killing and display increased virulence in immunocompromised mice. Thus, while Crabtree positivity might impart a fitness advantage for yeasts in environmental niches, the more flexible carbon assimilation strategies offered by Crabtree negativity enhance the ability of yeasts to colonize and infect the mammalian host.

Fig 1. The lack of catabolite inactivation in C. albicans permits simultaneous assimilation of alternative carbon sources and glucose.

In S. cerevisiae, glucose triggers the rapid ubiquitination and degradation via the GID complex of enzymes involved in the assimilation of alternative carbon sources (catabolite inactivation). Consequently, S. cerevisiae displays sequential assimilation of these carbon sources, only utilizing alternative carbon sources once glucose has been exhausted. In contrast, C. albicans enzymes involved in the utilization of alternative carbon sources lack ubiquitination sites and hence are not subject to catabolite inactivation. Consequently, these pathways remain active in C. albicans following glucose exposure and this yeast displays simultaneous assimilation of alternative carbon sources and glucose [18].

Fig 2. Evolutionary rewiring of metabolic ubiquitination targets across yeast species.

The phylogenetic tree of the Candida and Saccharomyces species analysed was generated by aligning ITS sequences using ClustalW. The in silico prediction of ubiquitination sites was performed using Ubpred (www.ubpred.org) [32] on the metabolic enzymes listed in S1 Table in the supplementary information: grey, the presence of at least one high confidence ubiquitination site; red, human pathogen; green, not a human pathogen; blue, Crabtree positive yeast; yellow, Crabtree negative yeast.

Fig 3. The addition of a ubiquitination site to Icl1 confers C. albicans with Crabtree positive-like properties.

(A) Western blot demonstrating that the addition of a ubiquitination site to a Myc₃-tagged CaIcl1 protein leads to its destabilization following glucose exposure in the C. albicans clinical isolate SC5314. Similar data were obtained in two independent replicate experiments. (B) Resistance of C. albicans strains to 2-deoxyglucose (2DG) when growing on lactate (Lac) or glucose (Glu). Cultures were replicated onto Glu and Glu + 2DG (growth examined at 2 days), Lac (growth at 4 days), and Lac + 2DG (growth at 7 days): WT, SC5314; icl1Δ/Δ, DCY65, ICL1, DCY75 (ICL1-Myc ₃ -NAT1); ICL1-Ubi, DCY82 (ICL1-UbiMyc ₃ -NAT1) (S2 Table in the supplementary information).

Fig 4. Metabolic flexibility in C. albicans promotes resistance to macrophage killing.

Survival of C. albicans strains following co-incubation with J774.1 macrophages for 48 h: WT, SC5314; icl1Δ/Δ, DCY65; ICL1, DCY75 (ICL1-Myc ₃ -NAT1); ICL1-Ubi, DCY82 (ICL1-Ubi-Myc ₃ -NAT1) (S2 Table in the supplementary information). Data represent three independent biological experiments performed in technical triplicate (mean values plus standard error of the mean (SEM)). The data were analysed using one-way ANOVA with Tukey’s post-hoc test: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Fig 5. Metabolic flexibility in C. albicans enhances gastrointestinal colonization.

Pairs of NAT1-marked C. albicans strains were introduced into mice by oral gavage and fungal burdens (CFUs) for the nourseothricin resistant and sensitive strains determined in the faeces (A), caecum (B) and kidneys (C) after 14 days. The analysed strains were: WT, SC5314 or DCY95; icl1Δ/Δ, DCY65 or DCY152; ICL1, DCY75 or DCY143 (ICL1-Myc ₃ NAT1); ICL1-Ubi, DCY82 or DCY144 (ICL1-Ubi-Myc ₃ -NAT1) (S2 Table in the supplementary information). Strains were used in four competition assays: wild-type (NAT1) vs. icl1Δ/Δ (n = 5); wild-type vs. icl1Δ/Δ (NAT1) (n = 6); ICL1-Myc ₃ (NAT1) vs. ICL1-Ubi-Myc ₃ (n = 7); and ICL1-Myc ₃ vs. ICL1-Ubi-Myc ₃ (NAT1) (n = 7). Similar data were obtained in an independent replicate experiment. Points represent individual animals and the bar denotes the group mean. Data were analyzed by the Mann-Whitney test using Prism 5: *, P ≤ 0.05; **, P ≤ 0.01.

Fig 6. Metabolic flexibility in C. albicans promotes systemic infection.

BALB/c mice were injected via lateral tail vein with the following C. albicans strains (n = 12 per group): WT, SC5314; icl1Δ/Δ, DCY65; ICL1, DCY75 (ICL1-Myc ₃ -NAT1); ICL1-Ubi, DCY82 (ICL1-UbiMyc ₃ -NAT1) (S2 Table in the supplementary information). (A) Renal fungal burdens after 72 h. Points indicate CFUs recovered from each animal and the bar denotes the mean. (B) Infection outcome scores measured after 72 h using the renal fungal burden combined with the percentage weight change for the mice ± SEM [39]. Statistical analyses were done by the Mann-Whitney U test using Prism 5: *, P ≤0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Fig 7. Many S. cerevisiae clinical isolates are Crabtree negative.

S. cerevisiae clinical isolates were pre-grown in YNB-lactate and spotted on SC medium with glucose or lactate in the presence or absence of 200 μg/mL 2-deoxyglucose. Nine clinical isolates are presented here, with data for a further 12 isolates presented in S4 Fig in the supplementary information.

Fig 8. Inactivation of the GID complex in S. cerevisiae inhibits catabolite inactivation.

(A) S. cerevisiae strains were pre-grown in YNB-lactate and spotted on SC medium with glucose (Glu) or lactate (Lac) in the presence or absence of 2-deoxyglucose (2DG): WT, DCY33; rmd5Δ, DCY34; ubc8Δ, DCY35; gid8Δ, DCY36; vid24Δ, DCY37 (S2 Table in the supplementary information). (B) Western blot of wild-type (DCY134) and gid8Δ cells (DCY130) expressing ScIcl1-Myc₉. Cells were pre-grown in YNB-lactate, exposed to glucose, and protein extracts prepared at the indicated times. Protein extracts were subjected to western blotting, and the same membranes probed for the Myc₉ epitope and then for actin as an internal loading control. Similar data were obtained in two independent replicate experiments.

Fig 9. Crabtree negative S. cerevisiae strains are more resistant to macrophage killing.

(A) S. cerevisiae wild-type (black bars, S288c) and gid8Δ cells (red, DCY122) were pre-grown in YNB-glucose or YNB-lactate, and then co-incubated with J774.1 macrophages. The viability of the gid8Δ cells after 48 hours is expressed relative to the corresponding wild type control. (B) Using the same approach, the resistance of three Crabtree positive S. cerevisiae clinical isolates (black) and three Crabtree negative clinical isolates (red) to macrophage killing was compared. (C) Isocitrate lyase activities were measured in the same S. cerevisiae strains after pre-growth overnight in YNB-lactate followed by growth in YNB-glucose or YNB-lactate for 2 h. The fold-reduction in Icl1 activity in cells exposed to glucose is expressed relative to the control cells grown in lactate. Statistical significance was calculated relative to the fold-reduction observed in the wild type control (S288c). (D) The phagocytic uptake of the S. cerevisiae strains was determined after pre-growth in YNB-lactate, and then co-incubation with J774.1 macrophages for 2 h. No significant differences (ns) were observed relative to the wild type control. The data represent two independent biological experiments performed in technical triplicate ± SEM. They were analysed relative to the glucose grown controls by two-way ANOVA with multiple comparisons test: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Fig 10. A Crabtree negative S. cerevisiae strain is more virulent in immunocompromised mice.

(A) Inactivation of GID8 in S. cerevisiae clinical isolate NCPF8313 renders it 2-deoxyglucose resistant. S. cerevisiae strains were pre-grown in YNB-lactate and spotted on SC medium with glucose (Glu) or lactate (Lac) in the presence or absence of 2-deoxyglucose (2DG): WT, DCY145; gid8Δ, DCY150; gid8Δ+GID8, DCY148 (S2 Table in the supplementary information). (B) Immunodeficient DBA/2 mice were injected via lateral tail vein with the same S. cerevisiae strains and renal fungal burdens were measured after 72 h (n = 6 per group). Points represent the CFUs for each animal and the bar denotes the mean. (C) The infection outcome scores were then determined by combining the renal fungal burdens with the percentage weight change for the mice ± SEM [39]. The data were analysed using the Mann Whitney test: *, P ≤ 0.05; **, P ≤ 0.01.