Fig 1.
Colony and cellular morphologies of the nonaggregative/yeast-form (SJ01), Agg-1 (SJ02), Agg-2 (BJCA001A), Agg-1Re (SJ02Re), and XM03-1 strains of C. auris.
Strains SJ01 and SJ02 were isolated from the same patient. BJCA001A is the aggregating form of strain BJCA001. The nonaggregative/yeast-form strain SJ02Re was spontaneously generated form cells of strain SJ02 grown on YPD medium. Strain SJ02Re carries a single copy of ALS4. Strain XM03-1 is a clinical isolate that is missing ALS4. (A) Colony morphologies. C. auris cells were plated on YPD medium containing 5 μg/mL phloxine B for 4 days at 30°C. Scale bar = 5 mm. (B) DIC images of C. auris cells treated with ddH2O, proteinase K, or trypsin. C. auris cells of a single colony of each strain were washed with 1 x PBS and then treated with ddH2O, proteinase K, or trypsin for 1 hour at 37°C. Scale bars,10 μm. (C) Low and high magnification SEM images of C. auris cells. The culture condition was the same as used in panel A. Scale bar,10 μm. Strains SJ02Re and XM03-1 served as controls.
Fig 2.
Survival curves of G. mellonella larvae infected with C. auris nonaggregative/yeast-form (SJ01 and BJCA001) and aggregative-form (SJ02 and BJCA001A) strains.
Approximately 1 x 106 C. auris cells of each strain were injected into each G. mellonella larva. Ten larvae were injected for each strain. ns, no significant difference; *, significant difference (p<0.05, by log rank test. Strain SJ02 served as the control). (A) Survival curves from experiments performed at 37°C. (B) Survival curves from experiments performed at 30°C.
Fig 3.
Spontaneous switching between the aggregating form and the single-celled nonaggregative/yeast-form.
C. auris cells of the original aggregating form (Agg-1, SJ02), SJ01 nonaggregative/yeast-form, or SJ02Re nonaggregative/yeast-form were plated onto solid YPD medium containing 5 μg/mL phloxine B and cultured at 30°C for 5 days. (A) Colony and cellular morphologies of the aggregating form (Agg-1, SJ02) and spontaneously generated nonaggregative/yeast-form (sectors, Agg-1Re or SJ02Re). (B and C) Colony and cellular morphologies of the nonaggregative/yeast-form strains (SJ01) and (SJ02Re). Switch frequencies to the alternative form were shown below the corresponding colony images. <0.02% indicates no alternative morphological cells observed. Scale bar for cells, 10 μm; scale bar for colonies, 1 mm.
Fig 4.
Biofilm development of the nonaggregative/yeast-form (SJ01), Agg-1 (SJ02), Agg-2 (BJCA001A), Agg-1Re (SJ02Re), and XM03-1 strains of C. auris.
The same set of strains were used as in Fig 1. CK, no fungal cells added. (A) Biofilm formation on the bottoms of 24-well plate polystyrene plates (a) or on silicone squares (b, c, and d). C. auris cells were incubated in RPIM1640 medium for 48 hours with shaking at 37°C. A silicone square was placed in each well for the lower panel. The polystyrene plates or silicone squares were gently washed with water and imaged. Biofilms formed on silicone squares were stained with calcofluor white for confocal microscopy assays with 405 nm laser excitation lines. CSLM top and side views were performed, respectively (c and d). Scale bar, 20 μm. (B) Low and high magnification SEM images of C. auris biofilms on silicone squares. Scale bar, 20 μm. (C) Quantitative analysis of cell numbers in the polystyrene plate well biofilms (panel A). The biofilms were washed with 1 x PBS and treated with proteinase K for 1 hour, resuspendend, and then diluted and plated on YPD medium. **p < 0.01, two-tailed Student t test.
Fig 5.
Skin infection assays using a newborn mouse model.
C. auris strains: nonaggregative/yeast-form (SJ01), Agg-1 (SJ02), Agg-2 (BJCA001A), Agg-1Re (SJ02Re), and nonaggregative/yeast-form XM03-1. Approximately 5 x 106 C. auris cells of each strain in 2 μL ddH2O were inoculated on the dorsal back skin of newborn mice. After the skin surface dried, a small sterilized glossy paper was affixed on the inoculated spot with medical tape. After 24 hours of infection, the infected skin areas were excised and gently washed with 1 x PBS. Low and high magnification SEM images are shown. Scale bar, 10 μm.
Fig 6.
Protein sequence variations and CNVs of ALS4 of C. auris clinical isolates.
(A) Schematic diagrams of the predicted Als4 proteins of the nonaggregative/yeast-form (SJ01), Agg-1 (SJ02), Agg-2 (BJCA001A), Agg-1Re (SJ02Re), and nonaggregative/yeast-form XM03-1 strains. The nonaggregative/yeast-form strain (SJ01) carries a truncated ALS4 gene, which encodes a protein containing only 8 repeats of the Als domain. The Xiamen isolate XM03-1 is a natural strain missing the ALS4 gene. (B) Copy number variations (CNVs) of ALS4 of the yeast-form (SJ01), Agg-1 (SJ02), Agg-2 (BJCA001A), Agg-1Re (SJ02Re), and yeast-form (XM03-1) strains. Both the nonaggregative/yeast-form (SJ01) and “returned” nonaggregative/yeast-form (SJ02Re) strains carry a single copy of the ALS4 gene, whereas the aggregative-form strain (SJ02) carries 10 copies of the ALS4 gene. (C) CNV analyses of ALS4 for 1156 C. auris strains (genomic sequences were retrieved from the NCBI database).
Fig 7.
Genomic analysis of the nonaggregative/yeast-form (SJ01) and aggregative-form (SJ02) strains of C. auris.
Long-read sequencing and data analyses were performed. (A) Genome synteny among strains SJ01, SJ02, and reference strain B11221. Black blocks indicate shared syntenic regions of the genomes. (B) Synteny schematic depicting the orientation and conservation of ALS4 and its adjacent genes. The copy number was estimated using Illumina sequencing data. Tandem repeats of ALS4 are indicated by dashed lines. (C) Sequence depth of ALS4 and its adjacent region (estimated based on PacBio Sequel II sequencing data). The sequence depth was calculated by IGV.
Fig 8.
Global transcriptional profiles of the nonaggregative/yeast-form (SJ01) and aggregative-form (SJ02) strains of C. auris by RNA-Seq.
(A) Venn diagram depicting differentially expressed genes. A twofold difference cut-off and false discovery rates (FDRs) less than 0.01 were used. (B) R package heatmap depicting the differentially expressed genes associated with biofilm development. Fold changes are shown.
Fig 9.
Ectopic expression of ALS4 in strain SJ01 promotes cell aggregation and biofilm formation.
(A) Diagram of plasmid pTDH3-ALS4. RPS1 and TDH3-associated fragments were amplified from genomic DNA of C. auris. caSAT1, CaACT1 promoter (C. albicans ACT1p), and CaURA3 terminator (C. albicans URA3t) were amplified from plasmid pNIM1. (B) Cellular morphologies of the control and ALS4-ecotopic strains. Cells of strain SJ01 (bearing a shortened single copy of ALS4) were transformed with linearized plasmids pTDH3 (vector-alone control) or pTDH3-ALS4. Fungal cells were cultured in liquid YPD medium at 30°C for 24 or 48 hours. Scale bar, 10 μm. (C) Biofilm formation of the control and ALS4-ecotopic strains on the bottoms of 24-well plate polystyrene plates. (D) Quantitative analysis of CFUs obtained from biofilms formed on the bottoms of the polystyrene plate wells in D. ***p < 0.001, two-tailed Student’s t test. (E) Relative expression levels of ALS4 in the control and ALS4-ecotopic strains determined by Q-RT-PCR assays. ***p < 0.001, two-tailed Student’s t test.
Fig 10.
Identification of C. auris strain A103 with enhanced adherence and biofilm formation.
(A) Four C. auris strains were isolated at different time points from the same patient. (B) Cellular morphologies of strains A101, A102, A103, and A104. C. auris cells were cultured in liquid YPD medium at 30°C for 24 hours. Scale bar, 10 μm. (C) Biofilm formation of the four C. auris strains on the bottoms of 24-well polystyrene plates and silicone squares. C. auris cells were incubated in RPIM1640 medium for 48 hours with shaking at 37°C. (D) Copy number variations (CNVs) of ALS4 of the four isolates. SRA database accession numbers for the genomic sequences: A101 (SRR9316791), A102 (SRR9316792), A103 (SRR9316793), and A104 (SRR9316794). (E) Pairwise SNP analysis among the four clinical strains.