Table 1.
C. albicans strains used or constructed in this study.
Fig 1.
Chlamydospore-like structures are observed in C. albicans-infected mouse kidneys at day 3 post-inoculation.
Top panel: A representative section from infected kidney cortex stained using GMS three days PI with C. albicans. Chlamydospore-like structures are indicated by the arrows. Middle panel: Periphery of a resolving Candida-infected lesion shows isolated chlamydospore-like structures (arrows). Bottom panel: Higher magnification of the same histopathology sections shows spherical fungal spores with a diameter of 8 μm.
Fig 2.
Purification of chlamydospores by sucrose density gradient.
Top panel: Schematic diagram showing sedimentation pattern for chlamydospores, yeast, and filamentous C. albicans after ultracentrifugation on a sucrose density gradient. Bottom panel: A. Gradient purified chlamydospores from in vitro cultures imaged under fluorescence microscopy after staining with Calcofluor White. B. Thin section of infected mouse kidney 3 days PI with C. albicans and stained with Calcofluor White (in vivo). C &D Unstained chlamydospores examined under phase contrast microscopy at 1000X (C, in vitro; D, in vivo). A and C, purified chlamydospores from C. albicans A72 grown in cornmeal agar; B and D, harvested from C. albicans A72 infected kidneys 102 h PI.
Fig 3.
Morphology and timing of chlamydospore formation in WT C. albicans and CJN19, CJN223 and CJN16 strains grown on corn meal agar.
(A) C. albicans SC5314 (10 days); (B) C. albicans CJN19 (Δsch9/Δsch9, 25 days) (C) C. albicans CJN223 (Δsuv3/Δsuv3, 25 days) and (D) C. albicans CJN16 (Δisw2/Δisw2, 35 days). Imaging was performed in situ in corn meal agar plates under bright field using an AMG EVOSfl Digital Inverted Microscope.
Fig 4.
Late induction of chlamydospores lacking suspensor cells in Δisw2/Δisw2 mutant strain compared to its parent strain.
(A) Time course of chlamydospore development over six weeks to verify a developmental pattern of chlamydospores lacking suspensor cells in DRL6 (Δisw2/Δisw2) strain. Images captured at 2 and 4 weeks are shown. The WT SC5314 and ISW2 complemented DRL7 strains initiated chlamydospore formation within 5 to 10 days on corn meal agar supplemented with 1% Tween 80. In contrast, the DRL6 (Δisw2/Δisw2) strain had no visible chlamydospores by day 14 but did produce lateral chlamydospores without suspensor cells after 4 to 5 weeks of incubation. (B) Micrographs of Calcofluor White-stained chlamydospores from C. albicans SC5314 (A1, A2) and the DRL6 (Δisw2/Δisw2) strain (B1, B2) captured after 2 and 4 weeks of incubation, respectively, with an Olympus FV500 Inverted (Olympus IX-81) Confocal Microscope. The average size of the WT chlamydospores formed on suspensor cells are 8.32 ± 0.94 (SD) μm, whereas the chlamydospores lacking suspensor cells are 7.88 ± 1.12 (SD) μm; n = 50. The images were analyzed with ImageJ (NIH) software for diameter measurement.
Fig 5.
Filamentation morphologies and cell cycle progression is unaltered by ISW2 deletion.
(A) Yeast cell morphology after overnight growth in YPD at 30°C. (B) Hyphal formation after overnight growth in YPD at 37°C. (C) Vigorous filamentation in embedded GPP agar after 16 hrs at 37°C. (D) Histograms from flow cytometry analysis of cell cycle for each cell population (G1 and G2) percentage from three biological replicates. Representative flow cytometry plots shows that cell cycle progression is similar to that of WT strain. G1 synchronized cells were analyzed via a time course with PI staining as described under Materials and Methods.
Fig 6.
DRL6 induces chlamydospores in Staib agar.
(A) Abundant chlamydospore formation by C. dubliniensis Wü284. (B) Poor filamentation and no detectable chlamydospores by C. albicans SC5314. (C) Occasionally visible chlamydospore formation by DRL6 (Δisw2/Δisw2) strain in Staib agar as indicated by the black arrow heads. Plates were grown at room temperature in the dark for up to 7 days, and representative pictures are from at least 3 independent experiments.
Fig 7.
Deletion of ISW2 prolongs survival in the mouse model of systemic candidiasis and alters host cytokine and chemokine expression.
(A) Effect of ISW2 deletion on mouse survival following intravenous C. albicans injection. Survival of mice injected with WT C. albicans SC5314 (●), the null mutant DRL6 (Δisw2/Δisw2) (■) and single copy reconstituted DRL7 (▲). Gehan-Breslow-Wilcoxon test hazard ratio estimates indicated 3.4-times greater lethality for WT infection compared to infection by the DRL6 strain. (n = 15; p<0.001, uninfected control = 6, data not shown). (B) Effects of Isw2p expression in C. albicans on host serum cytokine and chemokine induction after infection. Serum levels of the indicated cytokines (IL-6, TNF-α, IL-10, and MIP1α) were assessed at day 2 PI for mice infected with WT (checkerboard) or DRL6 (Δisw2/Δisw2) strain (horizontal lines) and statistically compared with both uninfected control and mutant. Control (crosshatch) values at day 0 are mean values determined for sera from five uninfected mice. Quantitative data represent mean ± SEMD. ** = p< 0.01; *** = p< 0.001.
Fig 8.
Isw2p expression induces chlamydospore formation in kidneys of mice with disseminated candidiasis.
Representative GMS stains of kidney sections dissected from mice infected with WT, and reconstituted DRL7 strains. Arrowheads indicate representative chlamydospores in histological sections of infected mouse kidneys at 3–6 days PI. Large arrow in left-lower corner image indicates a solitary chlamydospore in a resolving lesion in mouse kidney cortex at 8 days PI. Scale bar, 100 μm.
Fig 9.
Expression of the chlamydospore-specific genes CSP1 and CSP2 during in vivo growth of C. albicans.
qRT-PCR analysis of total RNA extracted from infected mouse kidneys harvested at 3 days PI showing that CSP1 and CSP2 were expressed during infection in vivo by all three strains. Expression is presented normalized to 1 for the WT strain, where mean Ct values were 24.3 for ISW2, 35.59 for CSP1, and 35.61 for CSP2. Results represent mean ± SD from three biological replicates. *** = p< 0.001.