Fig 1.
Aggregation is an inducible phenotype.
(A) Changes in growth media induce aggregation only in aggregative strains. Light microscopic (brightfield) images of cells of the indicated strains (left) in the indicated medium (top). Cells grown in RPMI-1640 displayed no aggregation regardless of ability to aggregate. Aggregation was only induced in aggregative (Agg) strains when grown in SabDex while non-aggregative (Non-Agg) strains remained as single cells. (B) Aggregation is lost when media is replaced with water but is retained when replaced with 1× PBS. Light microscopy (phase contrast) of cells grown overnight in SabDex followed by either replacement of media with ddH2O, 1× PBS, or ddH2O followed by 1× PBS. Non-aggregative (Non-Agg) strains were unaffected by the suspension liquid while aggregative (Agg) strains retained aggregation when suspended in 1× PBS, but aggregates were noticeably reduced after washing with ddH2O. Scale bars represent 50 μm.
Fig 2.
Sub-inhibitory concentrations of echinocandins induce a different type of aggregation (clustering).
(A) UACa20 (clade III) and (B) UACa11 (clade I) grown overnight in RPMI-1640 containing either 32 mg/L CSP or 0.075 mg/L MFG, images were taken with a light microscope (phase contrast) of cells in media and when resuspended in ddH2O, showing drug-induced clusters remain intact after washing with ddH2O. Scale bars in (A) and (B) represent 50 μm. (C) Differences in sedimentation were not observed in the RPMI-1640 cultures or the drug-induced clusters, while media-induced aggregates did fall out of suspension. (D) Colony morphology was not conspicuously impacted by aggregation.
Fig 3.
Differences in chitin bud scars and the coverage of outer cell wall mannan indicate differences between aggregation types.
(A, B) UACa20 (clade III) cells grown overnight in SabDex (A) or RPMI-1640 containing 32 mg/L CSP (B), cell wall chitin is visualized with 10 μg/mL CFW. White arrows point to bud scars without daughter cells grown in SabDex indicating full cell separation after division, this is not seen on antifungal-induced aggregation. Scale bars in (A) and (B) represents 10 μm. (C, D) Cell wall mannan and chitin stained with ConA and CFW, respectively, visualized by confocal microscopy to generate image sections of entire aggregates. (C) Cells grown in SabDex are completely outlined by mannan and chitin staining (red arrows), even where cells are closely juxtaposed, only actively dividing cells with small daughter buds appear to have a break in the mannan outer cell wall layer (white arrows). (D) When grown in RPMI-1640 containing 0.075 mg/L MFG cells are completely bounded by chitin, but display a lack of mannan staining at cell-cell junctions (white arrows) indicating a cell separation defect. In (C) and (D) the scale bars represent 5 μm.
Fig 4.
Proteinase K treatment significantly reduces media-induced aggregation but not antifungal-induced clustering.
Log2-fold change in aggregate/cluster numbers grown in SabDex (A), 32 mg/L CSP (B), or 0.075 mg/L MFG (C) after incubation at 50°C for 1 hour with or without 12.5 μg of proteinase K. *p < 0.05, **p < 0.001, as determined by independent samples t-test.
Fig 5.
Media-induced aggregation does not cause changes to the cell wall ultrastructure.
Cells grown in indicated media were fixed by high pressure freezing for transmission electron microscopy TEM. (A) Representative images from TEM of cell wall of an aggregative (Agg) and a non-aggregative (Non-Agg) strain, outer cell wall (OW) and inner cell wall (IW) are indicated. Scale bar represents 50 nm. (B) Inner cell wall measurements show strain-specific changes in response to culture conditions. (C) Length of mannan fibrils (outer cell wall) shows moderate changes in response to culture conditions in two isolates, UACa20 (clade III) (Agg) and UACa25 (clade I) (Non-Agg). *p < 0.05 as determined by independent samples t-test.
Fig 6.
Differential expression of genes (DEGs) in an aggregative and a non-aggregative strain grown in SabDex or RPMI-1640.
Aggregative (Agg) clade III strain UACa20 and non-aggregative (Non-Agg) clade I strain UACa11 were grown in the specified media for 16 hours and RNA was extracted for RNA-seq, this was repeated three times on independent colonies at different times. (A, B) Volcano plots of the total DEGs for UACa11 (A) and UACa20 (B) are shown with the dotted line indicating an FDR = 0.05. (C) Venn diagram of statistically significant (FDR < 0.05) DEGs, numbers indicate how many DEGs are unique to UACa11 and UACa20 and what portion of DEGs are shared between the strains.
Fig 7.
ALS4112 seems to be required for media-induced aggregation, but not for antifungal-induced clustering.
(A) Light microscopy images (contrast) of the als4112Δ mutant grown SabDex or RPMI-1640 containing 0.075 mg/L MFG for 24 hours. Cells grown in SabDex failed to aggregate, whereas cells grown in the presence of MFG did form clusters. Scale bar represent 10 μm. (B) The als4112Δ mutant was grown for 24 hours in RPMI-1640 containing 0.075 mg/L MFG and was imaged by confocal microscopy. Total cell wall chitin was stained with CFW and mannans stained with ConA. White arrows highlight features consistent with antifungal-induced clustering, where cells are completely surrounded by chitin staining, but display a lack of mannan staining at cell-cell junctions. Scale bar represents 5 μm.
Fig 8.
Virulence in the G. mellonella infection model is dependent on pre-growth media and Als4112.
(A-C) Non-aggregative and (D-F) aggregative C. auris strains were grown in RPMI-1640 or SabDex before inoculation into G. mellonella larvae. All strains except UACa25 (clade I) in (B) showed significant differences in virulence dependent on the media used to grow the fungal cells before inoculation. (G) Wild-type (UACa20) and als4112Δ (UACa177) C. auris cells were grown in SabDex before inoculation into G. mellonella larvae (A log-rank test was used to calculate p-values. 30 larvae in 3 independent experiments were used to assess virulence.
Fig 9.
Macrophages have difficulty in clearing large aggregates and clusters.
(A) Aggregates of strain UACa20 (clade III) grown in SabDex were co-incubated with THP-1 derived macrophages, and one macrophage was followed for the duration of the experiment (white arrow), taken from S3 Video. (B) Clusters of strain UACa20 grown in RPMI-1640 containing 0.075 mg/L MFG, one macrophage was followed for the duration of the experiment (white arrow), taken from S4 Video. Scale bars represent 15 μm.