Article Figures & Data
Figures
- FIG 1
The puf4Δ mutant is resistant to caspofungin. (A) Spot plate analysis. The indicated strains were diluted to an OD600 of 1.0, and five 1:10 serial dilutions were spotted on agar plates containing 0, 16, 32, 40, and 48 µg/ml caspofungin. Plates were incubated at 30°C for 3 days and photographed. (B and C) Growth assay. The indicated strains were diluted to an OD600 of 0.3 and mixed 1:1 with either fresh YPD or YPD containing caspofungin in a 96-well plate. Plates were incubated at 30°C for 20 h while shaking. OD600 was measured every 10 min.
- FIG 2
Puf4 transcript and protein levels are decreased following caspofungin treatment. (A) PUF4 transcript levels are decreased during a 60-min caspofungin time course. Cells were grown at 30°C and treated with 4 or 8 µg/ml caspofungin. The abundance of PUF4 mRNA was determined in samples collected at 30 and 60 min after caspofungin treatment using RT-qPCR with GPD1 as the normalization gene. Three replicates were plotted, and one-way-ANOVA with Tukey’s test was performed to assess statistical significance. (B) Puf4 protein levels are decreased at 60 min after caspofungin treatment. The Puf4-FLAG strain (puf4Δ expressing PUF4-FLAG C-terminal fusion protein) was grown to mid-log phase and treated with caspofungin for 1 h. A representative SDS-PAGE assay followed by immunoblotting using anti-FLAG antibody is shown. (C) Anti-FLAG signal is normalized to total protein signal from the stain-free gel. Three replicates were plotted, and unpaired t test with Welch’s correction was performed. *, P < 0.05.
- FIG 3
Puf4 directly binds and stabilizes the FKS1 mRNA. (A) The FKS1 mRNA contains Puf4 binding elements (PBEs) in its 5′ UTR, UGUANNNNUA. (B) Puf4 binds to FKS1 mRNA. Electrophoretic mobility shift assay was performed using a fluorescently labeled synthetic RNA oligonucleotide designed for the FKS1 5′ UTR that contain the PBEs. Unlabeled mutant (containing mutated PBEs) and unlabeled wild-type probes were used as controls for sequence specificity. The puf4Δ mutant was included as a control to show that binding is absent when Puf4 is not present. A representative gel image is shown (n = 3). (C) The FKS1 mRNA is downregulated in the puf4Δ mutant. The FKS1 mRNA abundance in mid-log samples grown at 30°C was determined using RT-qPCR with GPD1 as the normalization gene. Three replicates were plotted, and unpaired t test with Welch’s correction was performed. *, P < 0.05. (D) The FKS1 mRNA is destabilized in the puf4Δ mutant. FKS1 abundance was determined using RT-qPCR following transcription shutoff using 1,10-phenanthroline to determine the mRNA decay kinetics. GPD1 was utilized as the control for normalization. Three replicates were plotted, and differences between two strains were analyzed using one-phase exponential decay analysis.
- FIG 4
Puf4 controls the FKS1 abundance during caspofungin treatment and negatively regulates FKS1 translation. (A) FKS1 mRNA abundance is regulated by Puf4 during a 60-minute caspofungin time course. Cells were grown at 30°C and treated with 4 and 8 µg/ml caspofungin. The abundance of FKS1 mRNA was determined in samples collected at 30 and 60 min after caspofungin treatment using RT-qPCR with GPD1 as the normalization gene. Three replicates were plotted, and one-way ANOVA with Tukey’s test was performed. (B) Puf4 is a negative regulator of Fks1 protein abundance. FKS1-GFP and puf4ΔFKS1-GFP cell lines were grown in tissue culture medium, and GFP levels were analyzed using flow cytometry. Mean fluorescence intensities are plotted relative to FKS1-GFP cell line, and an unpaired t test with Welch’s correction was performed. *, P < 0.05. (C and D) Puf4 negatively regulates the translation of FKS1. Wild-type and the puf4Δ mutant cell lysates were analyzed using polysome profiling. No gross differences in polysome traces were observed among the strains (C). RNA was extracted from polysome fractions to determine the abundance of the FKS1 mRNA in fractions corresponding to free RNAs, 40S and 60S ribosomal subunits, 80S monosome, and polysomes. Percent distribution of the FKS1 mRNA across the gradient is graphed for both strains.
- FIG 5
Puf4 binds to the cell wall biosynthesis mRNAs other than FKS1. Puf4 binding to cell wall biosynthesis mRNAs is determined by performing RNA immunoprecipitation (RIP). (A) A representative Coomassie blue-stained SDS-PAGE image is shown to confirm the enrichment of Puf4 in the IP eluate. Arrow indicates the band corresponding to Puf4-FLAG. A mock IP using wild-type cell lysate was included as a negative control. (B) Puf4 interacts with CHS3, CHS4, CDA1, CDA3, and AGS1. Fold enrichment is calculated relative to mock IP and normalized to the input using RT-qPCR. CHS6, a gene that does not contain PBEs, is included as a negative control for binding.
- FIG 6
Deletion of PUF4 leads to dysregulation of cell wall biosynthesis gene expression. The mRNA abundances of select cell wall biosynthesis genes were determined during a 60-minute caspofungin time course by collecting samples every 30 min and determining abundance by RT-qPCR using GPD1 as a normalization gene. (A) CHS3; (B) CHS4; (C) CHS6; (D) CDA1; (E) CDA2; (F) CDA3; (G) AGS1; (H) KRE6; (I) SKN1. Three biological replicates with 2 technical replicates were plotted, and two-way ANOVA was used to determine statistical significance. ‘#’ denotes comparison between wild type and puf4Δ mutant, while ‘*’ denotes comparison between indicated time points within a strain. #/*, P < 0.05; ##/**, P < 0.01; ###/***, P < 0.001; ####/****, P < 0.0001.
- FIG 7
Puf4 stabilizes cell wall biosynthesis genes involved in chitin and α-glucan synthesis. CHS3 (A), CHS4 (B), CHS6 (C), CDA1 (D), CDA3 (E), and AGS1 (F) transcript abundances were determined using RT-qPCR following transcription shutoff to determine the mRNA decay kinetics. GPD1 was utilized as the normalization gene. Fifteen minutes after transcription shutoff was denoted as t = 0. Three replicates were plotted, and differences between two strains were analyzed using one-phase exponential decay analysis.
- FIG 8
Deletion of PUF4 leads to increased cell wall chitin and exposed chitooligomer levels. Cell wall chitin and exposed chitooligomer levels are increased in the puf4Δ mutant. Cells were grown to mid-log phase at 30°C and stained with calcofluor white and FITC-conjugated wheat germ agglutinin. Fluorescent intensity (A to D) and staining pattern (E) were determined using flow cytometry and fluorescence microscopy, respectively. For panels A to D, 3 biological replicates were plotted and one-way-ANOVA with Dunn’s multiple-comparison test was performed. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. DIC, differential interference contrast.
- FIG 9
Puf4 regulates the cell wall β-1,3-glucan levels and does not regulate the cell wall chitosan levels. (A) The puf4Δ mutant has decreased levels of cell wall β-1,3-glucan. Cells were grown to mid-log phase at 30°C and stained with aniline blue to detect β-1,3-glucan. Representative aniline blue staining images for each strain are shown to assess both levels and localization. (B) Cell wall chitosan levels were quantified following eosin Y staining. Representative microscopy images are shown. (C) Mean fluorescent intensities of eosin Y-stained cells were analyzed using flow cytometry.
- FIG 10
Model: post-transcriptional regulation of cell wall biosynthesis genes and cell wall remodeling by Puf4 is a path to caspofungin resistance in C. neoformans. In the wild-type cells, Puf4 controls the abundance and stability of cell wall biosynthesis genes including FKS1. This gene regulation has functional consequences in maintaining the cell wall composition and architecture. In the puf4Δ mutant, FKS1 as well as other cell wall biosynthesis transcripts is destabilized. Additionally, FKS1 mRNA is translationally more active in the puf4Δ mutant. Absence of Puf4-mediated gene regulation creates a cell wall that is richer in chitin and exposed chitooligomers while lacking in β-1,3-glucan. Post-transcriptional regulation of the cell wall homeostasis is the basis of caspofungin resistance in the puf4Δ mutant cells. Cell wall graphics are modified from reference 9 with permission of the publisher.
Tables
- TABLE 1
List of Puf4 binding element-containing cell wall biosynthesis-related genesa
↵a The UGUANNNNUA motif was searched in target genes using FIMO (MEME-suite version 5.1.1.). Results were manually confirmed, and locations of the motifs were identified. Asterisks indicate the genes selected for further mRNA stability analysis.
FIG S1
Caspofungin is a fungicidal drug for Cryptococcus neoformans. H99 and puf4Δ cells were grown to mid-log phase and treated with 4 or 8 µg/ml caspofungin. Cells were harvested following a 1-h incubation, and multiple serial dilutions were plated on YPD agar to assess survival. YPD agar plates were incubated at 30°C for 2 days, and colonies were counted using a protoCOL3 colony counter. Data presented as percent CFU relative to untreated. Download FIG S1, TIF file, 1.7 MB.
Copyright © 2021 Kalem et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
TABLE S1
Primers used in this study. List of primers and oligonucleotides used in cloning, RT-qPCR, and EMSA experiments. Download Table S1, XLSX file, 0.09 MB.
Copyright © 2021 Kalem et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S2
Fks1-GFP expression is upregulated in the puf4Δ cells. Cells were grown overnight in tissue culture medium (RPMI). GFP fluorescent signal was detected using a Leica TCS SP8 confocal microscope. Representative GFP and DIC images are shown. Download FIG S2, TIF file, 2.0 MB.
Copyright © 2021 Kalem et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
