Fig 1.
Mar1 is required for proper cell wall integrity.
(A) Schematic of the Mar1 protein domains. DUF, domain of unknown function; TM, transmembrane. (B) The mar1ΔT-DNA insertional mutant and a mar1Δ full deletion mutant are dry on pH 8 and sensitive to 1.5 M NaCl. Serial dilutions of the indicated strains were spotted onto YPD, YPD with 150 mM HEPES at pH 8, or YPD with 1.5 M NaCl. (C) The mar1Δ strain shares distinct and overlapping sensitivities to cell wall stressors with other cell wall integrity pathway mutants. Strains were serially diluted and spotted onto YPD with the addition of the indicated cell wall stressors or at the indicated temperatures. Melanin was assessed by the production of brown pigment on Niger Seed agar at 30°C. CFW, calcofluor white; SDS, sodium dodecyl sulfate.
Fig 2.
The mar1Δ mutant has an aberrant cell surface.
(A) The mar1Δ strain has a capsule defect. Cells were incubated in capsule-inducing conditions (CO2-independent tissue culture (TC) medium, 37°C) with shaking for 72 hours. Capsule was assessed by India ink counterstaining, followed by imaging. (B) The mar1Δ strain sheds capsule comparable to WT. Shed capsule polysaccharide was measured by blotting of culture supernatant, using an anti-GXM antibody to probe for capsule as described previously [30]. (C) The mar1Δ mutant displays increased staining for exposed chitin and chitooligomers in tissue culture medium. Cells were incubated for 16–18 hours at 30°C in rich medium (YPD) or 37°C in tissue culture medium (TC), followed by staining with FITC-conjugated wheat germ agglutinin (WGA) for exposed chitin/chitooligomers, or calcofluor white (CFW) for total chitin. Stained cells were imaged by fluorescent microscopy with the appropriate filters. (D) Average fluorescence of at least 100 individual cells was measured using ImageJ/Fiji software. ****, p < 0.0001 as determined by two-way ANOVA with Tukey’s multiple comparisons test. (E) The mar1Δ mutant displays increased chitosan staining. Cells were incubated for 16–18 hours at 30°C (YPD) or 37°C (TC), followed by staining with eosin Y (EY) for chitosan. Stained cells were imaged by fluorescent microscopy. (F) The mar1Δ cell wall does not have increased total chitin or chitosan. Cells were incubated for 16–18 hours at 37°C in TC medium, followed by cell wall isolation. Chitin and chitosan levels were quantified using a modified 3-methyl-2-benzothiazolinone hydrazine hydrochloride (MBTH) colorimetric assay as described previously [16]. Data represent means of 3 independent cell wall isolations (n = 3 for each strain). Ns, not significant as determined by two-way ANOVA with Sidak’s multiple comparisons test.
Fig 3.
Cell wall changes in the mar1Δ mutant induced in tissue culture medium are dependent on pH and glucose deprivation.
(A) The mar1Δ cell wall changes are not dependent on temperature. The mar1Δ strain was incubated for 16–18 hours with shaking at the indicated temperature in rich medium (YPD) or tissue culture medium (TC). Exposed chitooligomers in the cell wall were stained with FITC-conjugated WGA and imaged by fluorescent microscopy using the GFP filter. (B) Average fluorescence of at least 100 individual cells was measured using ImageJ/Fiji software. Ns, not significant as determined by one-way ANOVA with Tukey’s multiple comparisons test; all other comparisons, p < 0.0001. (C) The mar1Δ cell wall changes occur with increased pH and can be partially suppressed by glucose supplementation. mar1Δ cells were incubated for 16–18 hours with shaking at 30°C in YPD, YPD buffered to pH 7.4, TC, or TC supplemented with 2% glucose. Cells were stained with FITC-conjugated WGA and imaged as above. Bar, 10 μM. (D) Average fluorescence was measured as above. ****, p < 0.0001; ns, not significant, as determined by one-way ANOVA with Tukey’s multiple comparisons test.
Fig 4.
Cell wall components are altered in the mar1Δ cell wall.
(A) The mar1Δ cell wall has increased chitin and chitosan staining by flow cytometry, but mar1Δ staining for α-glucans, β-(1,3)-glucan, and mannoproteins is limited above baseline. WT and mar1Δ cultures were incubated for 16–18 hours at 37°C in TC medium, fixed, labeled, and analyzed by flow cytometry. WGA was used to stain exposed chitin and CFW was used to stain total chitin. EY was used to stain chitosan. An MOPC-104E antibody with an anti-mouse AlexaFluor 488 secondary antibody was used to label α-glucan. An Fc-Dectin-1 fusion protein coupled with an anti-human AlexaFluor 488 secondary antibody was used to label β-(1,3)-glucan. Concanavalin A conjugated to AlexaFluor 488 was used to label mannoproteins. Relevant events were gated in the FSC/SSC plots and are represented as histograms with mean fluorescence on the x-axis and cell counts on the y-axis. Unstained cells were sorted as controls to determined positive labeling. (B) The mar1Δ cell wall has decreased glucan and mannan. Cells were incubated for 16–18 hours at 37°C in TC medium, followed by cell wall isolation. Cell wall carbohydrate levels were quantified using high performance anion-exchange chromatography with pulse ampherometric detection (HPAEC-PAD). All data represent means of results from 3 independent cell wall preparations for each strain. *, p < 0.05; **, p < 0.01 as determined by one-way ANOVA with Tukey’s multiple comparisons test. All other comparisons, not significant (C) Cell wall genes are differentially regulated in mar1Δ. A concentration of 107 cells/ml in 25 ml YPD (30°C) or TC (37°C) were incubated for 1.5 hours, followed by RNA extraction and cDNA synthesis. Expression of cell wall biosynthesis genes was determined by real-time PCR. Fold change was calculated relative to WT YPD levels and normalized to the expression of an internal control. Data represent means of results from 2 independent C. neoformans cultures and RNA extractions per strain. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 as determined by two-way ANOVA with Tukey’s multiple comparisons test.
Fig 5.
Mar1 protein localization is dynamic over time in tissue culture medium.
(A) Localization of a Mar1-GFP fusion protein was assessed after cells were incubated for 16–18 hours in YPD (30°C) or TC (37°C). Live cells were imaged using DeltaVision deconvolution fluorescent microscopy with the GFP filter. Images were deconvolved using softWoRx software. Bar, 10 μM. (B) Cells were incubated and imaged as above. 3D-projections were generated from z-stacked images using ImageJ/Fiji software and are pseudo-colored to indicate brightness of staining from yellow to blue. Bar, 10 μM. (C) A Mar1-GFP expression strain was pre-incubated in YPD medium to mid-log phase and transferred to TC medium at time zero. Cells were incubated for the indicated time in TC at 37°C with shaking and imaged by fluorescent microscopy using the GFP filter. Bar, 10 μM. (D) Quantification of staining by FITC-conjugated WGA. The WT and mar1Δ strains were incubated to mid-log phase in YPD medium and transferred to TC medium at time zero. Exposed chitooligomers in the cell wall were stained with FITC-conjugated WGA, imaged by fluorescent microscopy using the GFP filter, and average fluorescence was quantified by ImageJ/Fiji software. ****, p < 0.0001 as determined by two-way ANOVA with Sidak’s multiple comparisons test.
Fig 6.
Intracellular trafficking and localization of the β-(1,3)-glucan synthase, Fks1, are impaired in the mar1Δ mutant.
(A) Uptake of the lipophilic dye, FM4-64, is irregular in mar1Δ cells. Cells were incubated overnight in YPD (30°C) or TC (37°) and stained with FM4-64 for 30 minutes with shaking, followed by pelleting and refreshing in the indicated media for an additional 30 minutes with shaking. Stained cells were imaged by fluorescent microscopy with the Texas Red filter and were taken with the same exposure time. Bar, 10 μM. Arrows indicate endocytic vesicles (B, C) Acid phosphatase secretion is intact in the mar1Δ strain; alkaline phosphatase activity is decreased. WT and mar1Δ cultures were pre-incubated for 16–18 hours in phosphate replete minimal medium. The cells were diluted to an OD of 0.9 in either phosphate replete (non-inducing) or phosphate deficient (inducing) minimal medium and incubated for 3 hours at 30°C with shaking. (B) Acidic or (C) alkaline para-Nitrophenylphosphate (pNPP) substrate solution was added to each well and plates were incubated for an additional 2.5 hours at 37°C with shaking. Phosphatase activity was measured as absorbance at 410 nm over cell density at 600 nm. Data represent the mean of 3 replicates per strain per condition. (D) Fks1 is mislocalized in TC medium in mar1Δ cells. The Fks1-GFP fusion protein was expressed in the WT and mar1Δ mutant strains. Cells were incubated for 16–18 hours in TC medium at 37°C. Live cells were imaged using DeltaVision deconvolution fluorescent microscopy with the GFP filter. Images were deconvolved using softWoRx software. Bar, 10 μM.
Fig 7.
mar1Δ cells have disordered cell walls and altered vesicular trafficking by ultrastructure analysis.
WT and mar1Δ cells were incubated for 16–18 hours in YPD (30°C) and TC (37°C) medium, followed by glutaraldehyde and KMnO4 fixation and partial dehydration as described previously [14,68,101]. Samples were further processed, embedded, sliced, and imaged by transmission electron microscopy (TEM). (A) YPD incubated cells display thin, ordered cell walls. Left image of each pair: bar, 1 μM. Right image of each pair: bar, 200 nm. Letters indicate location of inset. (B) mar1Δ cells incubated in TC medium have a less organized cell surface. Left image of each pair: bar, 1 μM. Right image of each pair: bar, 200 nm. Letters indicate location of inset. Red arrows indicate apparent cell wall material disassociating from cell surface. (C) There are increased numbers of electron lucent structures in mar1Δ cells incubated in TC medium. Bar, 1 μM. Red asterisks indicate mar1Δ cells with several electron lucent vesicles near cell periphery.
Fig 8.
The mar1Δ mutant induces increased TNF-α production from macrophages and dendritic cells.
(A) Macrophage activation by mar1Δ cells is not dependent on cell viability. WT, mar1Δ, and mar1Δ + MAR1 cells were incubated for 16–18 hours at 37°C in TC medium. Bone marrow-derived macrophages (BMMs) were co-incubated with live or heat-killed (HK, 1–2 hours at 65°C) C. neoformans strains at a multiplicity of infection (MOI) of 10:1, C. neoformans:BMMs. TNF-α levels (pg/ml) were assayed from the supernatant by ELISA. Data represent means from 3 replicates per strain per condition from 3 independent experiments (n = 9). *, p < 0.05; ***, p < 0.001 mar1Δ vs. WT as determined by two-way ANOVA with Tukey’s multiple comparisons test. (B) mar1Δ induces increased TNF-α production by dendritic cells. C. neoformans cells were incubated as described above and co-incubated with bone marrow derived dendritic cells (BMDCs) at an MOI of 10:1, C. neoformans:BMDCs. TNF-α levels were assayed from the supernatant by ELISA. Data represent means from 3 replicates per strain from 2 independent experiments (n = 6). **, p < 0.01 mar1Δ vs. WT as determined by one-way ANOVA with Tukey’s multiple comparisons test. (C) Macrophage activation by mar1Δ cells is dependent on pre-culturing in TC medium. Cells were incubated for 16–18 hours in YPD (30°C) or TC (37°C) prior to co-incubation with BMMs and TNF-α quantification by ELISA as above. Data represent means from 3 replicates per strain (n = 3). ****, p < 0.0001 mar1Δ/TC vs. WT/ TC as determined by two-way ANOVA with Tukey’s multiple comparisons test. mar1Δ/YPD vs. WT/YPD, not significant. (D) Cell wall material isolated from mar1Δ induces increased macrophage activation. WT, mar1Δ, and mar1Δ + MAR1 reconstituted cells were incubated for 16–18 hours at 37°C in TC medium, followed by cell wall isolation. 10 μg/ml of cell wall material was co-cultured with BMMs and TNF-α was quantified from the supernatant by ELISA. Data represent means of 3 independent cell wall isolations in 3 independent experiments (n = 9 for each strain). *, p < 0.05 mar1Δ vs. WT as determined by one-way ANOVA with Tukey’s multiple comparisons test.
Fig 9.
Mar1 is required for full virulence.
(A) The mar1Δ strain is attenuated in the C57BL/6 mouse background. For each strain, 9–10 C57BL/6 mice were inoculated with 1 x 105 cells, monitored daily for signs of infection, and sacrificed at predetermined clinical endpoints that predict mortality. Statistical significance was determined by log-rank test with Bonferroni correction: p < 0.0001, mar1Δ vs. WT; p < 0.001, mar1Δ vs. mar1Δ + MAR1; not significant. (B) There are minimal mar1Δ cells in the lungs of infected mice at early time points. Colony forming units (CFUs) were determined from lung homogenates from 5 mice per strain at days 1 and 4 post inoculation. *, p < 0.05 mar1Δ vs. WT; **, p < 0.01 mar1Δ vs. mar1Δ + MAR1 as determined by one-way ANOVA with Tukey’s multiple comparisons test. (C) The mar1Δ strain is avirulent in the BALB/c mouse background. For each strain, 9–10 BALB/c mice were inoculated with 1 x 105 cells, monitored daily, and sacrificed as described above. Statistical significance was determined by log-rank test with Bonferroni correction: p < 0.0001, mar1Δ vs. WT/mar1Δ + MAR1; not significant, WT vs. mar1Δ + MAR1. (D) The mar1Δ strain is not completely cleared from BALB/c mice despite mouse survival. CFUs were determined from lung and brain homogenates from mar1Δ infected mice post-mortem. Ratio indicates number of mice represented.
Fig 10.
Macrophage activation in response to mar1Δ requires members of the CLR and TLR families.
BMMs were harvested from the indicated mouse strains (C57BL/6 background, unless otherwise noted) and co-incubated with C. neoformans (Cn) cells at an MOI of 10:1, Cn:BMMs, followed by quantification of TNF-α (pg/ml) in the supernatant by ELISA. (A) Card9 is involved in macrophage activation by mar1Δ cells. Data represent means of 3 replicates from 3 independent experiments (n = 9). **, p < 0.01 WT vs. Card9-/- BMMs as determined by two-way ANOVA with Sidak’s multiple comparisons test. (B) MyD88 is required for TNF-α production by macrophages in response to mar1Δ. Data represent means of 3 replicates from 3 independent experiments (n = 9). ****, p < 0.0001 WT vs. MyD88-/- BMMs as determined by two-way ANOVA with Sidak’s multiple comparisons test. (C) Dectin-1 is involved in the response to mar1Δ cells. Data represent means of 3 replicates from 2 experiments (n = 6). ****, p < 0.0001 WT vs. Dectin-1-/- BMMs as determined by two-way ANOVA with Sidak’s multiple comparisons test. (D) TLR2/4-/- BMMs do not respond to mar1Δ cells. Data represent means of 3 replicates (n = 3). ****, p < 0.0001 WT vs. TLR2/4-/- BMMs as determined by two-way ANOVA with Sidak’s multiple comparisons test. (E) TLR4 is not required for the production of TNF-α induced by mar1Δ cells. BMMs were isolated from C3H/HeJ mice with a null mutation in TLR4, and C3H/HeOuJ control mice. Data represent means of 3 replicates from 2 independent experiments (n = 6). **, p < 0.01 C3H/HeOuJ vs. C3H/HeJ BMMs as determined by two-way ANOVA with Sidak’s multiple comparisons test. (F) Macrophage activation by mar1Δ is partially dependent on TLR2. Data represent means of 3 replicates from 3 independent experiments (n = 9). ****, p < 0.0001 WT vs. TLR2-/- BMMs as determined by two-way ANOVA with Sidak’s multiple comparisons test.
Fig 11.
The cell surface of C. neoformans is remodeled in response to host-like conditions.
When incubated in tissue culture medium, there is an increase in the expression of genes encoding the biosynthesis enzymes for outer cell wall components, resulting in a thicker cell wall, enhanced capsule attachment, and immune avoidance. The Mar1 protein is required for controlling aspects of these cell wall adaptations, including the proper induction and localization of glucan synthases. As a result, in the mar1Δ mutant the levels of these outer cell wall components are reduced, exposing the more immunogenic chitooligomers that are normally masked from immune recognition.
Table 1.
C. neoformans strains used in this study.
Table 2.
Primers used in this study.
Table 3.
Plasmids used in this study.