Roles of Candida albicans Mig1 and Mig2 in glucose repression, pathogenicity traits, and SNF1 essentiality

Abstract

Metabolic adaptation is linked to the ability of the opportunistic pathogen Candida albicans to colonize and cause infection in diverse host tissues. One way that C. albicans controls its metabolism is through the glucose repression pathway, where expression of alternative carbon source utilization genes is repressed in the presence of its preferred carbon source, glucose. Here we carry out genetic and gene expression studies that identify transcription factors Mig1 and Mig2 as mediators of glucose repression in C. albicans. The well-studied Mig1/2 orthologs ScMig1/2 mediate glucose repression in the yeast Saccharomyces cerevisiae; our data argue that C. albicans Mig1/2 function similarly as repressors of alternative carbon source utilization genes. However, Mig1/2 functions have several distinctive features in C. albicans. First, Mig1 and Mig2 have more co-equal roles in gene regulation than their S. cerevisiae orthologs. Second, Mig1 is regulated at the level of protein accumulation, more akin to ScMig2 than ScMig1. Third, Mig1 and Mig2 are together required for a unique aspect of C. albicans biology, the expression of several pathogenicity traits. Such Mig1/2-dependent traits include the abilities to form hyphae and biofilm, tolerance of cell wall inhibitors, and ability to damage macrophage-like cells and human endothelial cells. Finally, Mig1 is required for a puzzling feature of C. albicans biology that is not shared with S. cerevisiae: the essentiality of the Snf1 protein kinase, a central eukaryotic carbon metabolism regulator. Our results integrate Mig1 and Mig2 into the C. albicans glucose repression pathway and illuminate connections among carbon control, pathogenicity, and Snf1 essentiality.

Fig 1. Gene expression dataset comparisons.

Comparisons were performed using our genome-wide profiling data of wild-type C. albicans grown in YPG media compared to wild-type grown in YPD media A. Fisher’s Exact Test depicting gene expression datasets most closely related to YPG gene expression. FET was used as previously described [31]. Up- or down-regulated genes with a 2-fold expression change cut-off were matched from 91 published expression datasets (S3 Table). B. Heatmap depicting gene expression of 10 metabolic genes chosen for comparison of glucose-repressed genes. Color scale corresponds to log2 fold change limits of 3.3 up (red) and 3.3 down (blue). Full gene expression datasets are available in S1 Table.

Fig 2. Mig1/2 regulatory effects.

A. Venn diagram of genes regulated by Mig1, Mig2, or Mig1 and Mig2. Genes included in the diagram were significantly up- or down-regulated (p<0.05) by at least 2-fold compared to the wild-type strain (CW542) in YPD medium. B. Enrichment of SYGGRG motif in Mig1/2 target gene promoters. Promoter sequence was defined as 1,000 basepairs upstream of the open reading frame of up- or down-regulated genes. C. Fold change expression of chosen Mig1 and Mig2 selective genes compared to the wild-type strain analyzed by RNA-Seq and compared between datasets. Mig1 selective genes were significantly up-regulated in the mig1Δ/Δ and mig1Δ/Δ mig2Δ/Δ, but not the mig2Δ/Δ strain profiles. Mig2 selective genes were significantly up-regulated in the mig2Δ/Δ and mig1Δ/Δ mig2Δ/Δ, but not the mig1Δ/Δ strain profiles.

Fig 3. Regulation of Mig1 protein accumulation in response to carbon source.

A. Western blotting was performed on cells grown in triplicate expressing HA-tagged Mig1 or the untagged wild-type strain (CW542) grown in YPD as a control. Soluble protein was extracted from strain KL1026 grown in YPD, YPG, Spider medium, and Spider medium with 2% glucose added 10 minutes before harvesting the cells. B. Densitometric analysis of Mig1 protein was performed using FIJI. Total HA-tagged signal was compared to total tubulin signal for loading control (Dunnett test **, p<0.01).

Fig 4. Impact of MIG1/2 mutations on growth of sak1 or snf1 mutant and validated strains.

Tenfold serial dilutions of strains KL988(sak1Δ/Δ), KL951(sak1Δ/Δ mig1Δ/Δ), KL960(sak1Δ/Δ mig2Δ/Δ), KL955(sak1Δ/Δ mig1Δ/Δ mig2Δ/Δ), KL992(sak1Δ/Δ +SAK1), KL972(sak1Δ/Δ mig1Δ/Δ +SAK1), KL990(sak1Δ/Δ mig2Δ/Δ +SAK1), KL974(sak1Δ/Δ mig1Δ/Δ mig2Δ/Δ +SAK1), and KL953 (snf1Δ/Δ mig1Δ/Δ), KL957(snf1Δ/Δ mig1Δ/Δ mig2Δ/Δ), KL970 (snf1Δ/Δ mig1Δ/Δ +SNF1), and KL976(snf1Δ/Δ mig1Δ/Δ mig2Δ/Δ +SNF1) were spotted on YPD (glucose), YPG (glycerol), Spider media (mannitol) and Spider media with 2% glucose replacing mannitol. Growth was visualized after 48 h of incubation at 37°C.

Fig 5. Epistasis analysis among mutations in MIG1, MIG2, SAK1, and SNF1.

Heatmap of log2-fold changes in gene expression of selected carbon metabolism genes analyzed using Nanostring. RNA was extracted from strains CW542 (wild type), KL988(sak1Δ/Δ), KL951(sak1Δ/Δ mig1Δ/Δ), KL960(sak1Δ/Δ mig2Δ/Δ), KL955(sak1Δ/Δ mig1Δ/Δ mig2Δ/Δ), KL992(sak1Δ/Δ +SAK1), KL972(sak1Δ/Δ mig1Δ/Δ +SAK1), KL990(sak1Δ/Δ mig2Δ/Δ +SAK1), KL974(sak1Δ/Δ mig1Δ/Δ mig2Δ/Δ +SAK1), and KL953 (snf1Δ/Δ mig1Δ/Δ), KL957(snf1Δ/Δ mig1Δ/Δ mig2Δ/Δ), KL970 (snf1Δ/Δ mig1Δ/Δ +SNF1), and KL976(snf1Δ/Δ mig1Δ/Δ mig2Δ/Δ +SNF1) grown in triplicate in YPG medium at 37°C. Fold change values were calculated by dividing expression of each gene to the wild type. The sak1Δ/Δ mutant strain analysis is duplicated for ease of comparison to the snf1Δ/Δ mutant strains because a snf1Δ/Δ mutant strain is not viable.

Fig 6. Growth of Mig1/2 mutant and complemented strains during cell wall stress.

A. Plate dilution sensitivity assay. Tenfold serial dilutions of the indicated strains were spotted on YPD and YPD with 100 ng/mL Caspofungin, 100 ng/mL Caspofungin and 1 M Sorbitol, 20 μM Calcofluor White, and 100 μg/mL Fluconazole. Growth was visualized after 48 hours of incubation at 37°C. B. Quantitative caspofungin sensitivity assay in 96 well plates. Cells were incubated in liquid YPD media at 37°C containing 2-fold dilutions of Caspofungin. Absorbance was read at 600nm after 48 hours of incubation. Data was averaged from three biological replicates of duplicate measurements and normalized to cell growth without antifungal.

Fig 7. Mig1/2 promote filamentation, biofilm formation, and affect host cell interactions in vitro.

A. Biofilms were grown for 24 h on silicone squares in RPMI media at 37°C. Fixed biofilms were stained with Concanavalin A Alexa Fluor 594. Representative side-view projections were processed and pseudocolored using ImageJ. Scale bar corresponds to depth of the wild-type B. Biofilms were measured using confocal microscopy. Values shown are triplicate measurements from three biological replicates. ****, p<0.0001. C. Indicated strains were grown for 4 h in RPMI at 37°C. Fixed cells were imaged using DIC microscopy and a 20x objective. Hyphal lengths were measured from yeast cell to hyphal tip from >80 cells from 10 fields of view. ****, p<0.0001 D. J774.1 macrophage cytotoxicity was measured by lactate dehydrogenase release (LDH) after 5 h of coincubation with C. albicans strains as indicated. Percentage of LDH release was calculated relative to max release wells containing lysis solution. Values shown are mean with SD from duplicate measurements of three biological replicates. ****, p<0.0001. E. Human endothelial cell damage was assessed by ⁵¹Cr release following coincubation with C. albicans cells for 3 h. Percentage of Chromium release was calculated by comparison to release from wild-type (release–spontaneous/total incorporation–spontaneous)**, p<0.01.

Fig 8. Summary diagram of the roles of Mig1 and Mig2 in the glucose repression pathway in C. albicans.

Sak1 and Snf1 connections drawn from published data by Ramirez-Zavala et al. [9]. Black dotted arrows indicate unclear regulatory connections. Dotted Mig1 indicates reduced protein levels in cells grown on an alternative carbon source. No Mig2 protein level data exist to our knowledge.