Fig 1.
Titan cells can be induced in vitro and have all the properties of in vivo induced cells.
H99 haploid cells pre-grown in YNB and incubated in 10%HI-FCS at 37°C, 5%CO2 for 3 days. Resulting colonies were A) counterstained with India ink to reveal capsule and B) fixed and stained for DNA content relative to haploid and diploid controls. Left panel shows YNB grown haploid control. Middle panel shows total induced population (blue). In addition, induced cells were passaged through an 11 μm filter to enrich for large cells (red). Right panel shows all three populations. C) H99 haploid cells were pre-grown in YNB and incubated in 10%HI-FCS at the indicated OD600 at 37°C, 5%CO2 for 3 days. Scale bar = 10 μm. D,E) H99 haploid and KN994B7#16 diploid cells pre-grown in either YNB or YPD were inoculated into 10%HI-FCS at OD600 = 0.001, (37°C, 5%CO2, 3 days) and then counterstained with India ink and analysed. Representative micrographs (D) and diameters for >200 cells excluding or including capsule (E) are shown. Significance was assessed using Mann Whitney U (***p<0.0001).
Fig 2.
In vitro induced cells were analysed by TEM.
Representative micrographs (A) and cell wall thickness for Yeast (n = 15), Titan (n = 14), and Titanide (n = 9) cells (B). Significance was assessed using Kruskal-Wallis and Dunn's multiple comparisons tests (* p = 0.027; ** p = 0.002; ***p<0.0001).
Fig 3.
Daughter cells from in vitro Titans have altered ploidy relative to the haploid parent.
A) Representative budding Titan cells (top panels) and yeast cells (bottom panel). CNV111 haploid cells encoding GFP-Ndc1 (membrane) and mCherry-Cse4 (kinetochore) were induced (top) or grown in YPD (bottom) and stained for chitin (CFW, 10 μg/ml). Scale bar = 10 μm. B) Induced CNV111 cells showing population heterogeneity (T,Titan; Y,Yeast; t, Titanide; (*), aneuploid; (<), less than 10 μm). Scale bar = 10 μm. C,D) CNV111 cells were quantified for mCherry-Cse4 foci and mother cell size (YPD n = 50, induced n = 100). E) Daughters arising from a single Titan mother were isolated and allowed to proliferate for 72 hr at 30°C on YPD agar prior to fixation and staining for DNA content (DAPI). Populations were analysed by flow cytometry. (Top) Control gates (1C, 2C, 4C) are shown aligned to representative overlays of ungated haploid control and Titan Daughter #5 (T1D5) populations. (Bottom) DNA content of 14 independent daughter colonies 48 hr after isolation (right) and again after 1 month incubation at RT on YPD agar.
Fig 4.
Titan cells are primed by YNB and can be maintained in 10%FCS.
A) H99 cells pre-cultured in either YPD or YNB were washed 6 times in 1xPBS and then inoculated at the indicated OD in 10% FCS (3 days, 37°C, 5%CO2). Representative micrographs and cell size quantification are shown. B) Titan cells were induced for 24 hr, enriched for cells >11μm, and stained with CFW (10 μg/ml), then returned to inducing conditions for 48 hr. Representative micrographs show CFW (chitin) and mAb 18B7-labeled capsule. Scale bar = 10 μm.
Fig 5.
Titan cell induction is mediated by host relevant ligands and pre-culture condition.
A) H99 cells pre-cultured in YNB were induced for Titans following 3 days growth in 10% FCS, 10% BAL, or 10 μM or 20 μM N-Acetylmuramyl-L-alanyl-D-isoglutamine (NMAiGn) as indicated. Representative micrographs are shown for live cells (CFW, SytoxGreen). Scale bar = 10 μm. B) Daughters arising from Titan mothers induced using BAL (left, T7) or NMAiGn (right, T1, T5, T6) were isolated and allowed to proliferate for 72 hr at 30°C on YPD agar prior to fixation and staining for DNA content (DAPI). Populations were analysed by flow cytometry. C) Quantification of cells induced by various conditions (FCS, BAL, MNAiGn, E. coli, S. pneumonia) (n>300). D) Lung homogenates from mice unexposed or exposed to Pen/Strep for 7 days prior to C. neoformans inhalation infection. Representative micrographs (CFW, scale bar = 20 μm) and quantification are shown. n>500 for each condition. E,F) FCS was fractionated by size exclusion chromatography (E) and the resulting fractions were tested for capacity to induce Titan cells. F) Analysis by 1H NMR and 1H-13C HSQC revealed peaks consistent with sugar and amino acid structures.
Fig 6.
The cAMP/PKA pathway and the Usv101 transcription factor influence in vitro titanisation.
A,B) Wild-type, gpa1Δ, ric8Δ, gpr4Δgpr5Δ, pka1Δ, cac1Δ, and cap59Δ cells were incubated under Titan inducing conditions. A) Representative micrographs for fixed cells are shown (CFW). Scale bar = 10 μm. B) Cell size was quantified for the indicated strains. Significance was assessed using Kruskal-Wallis and Dunn's multiple comparisons (relatively to H99, p<0.0001 for all strains, n>300). C) Wild-type and usv101Δ cells were incubated under the indicated conditions. Representative micrographs for fixed cells are shown (CFW). D) Cell size was quantified for wild type and usv101Δ cells induced in YNB+10% FCS (p<0.0001). E,F) H99 and usv101Δ Titan cells were induced for 24 hrs, enriched for cells >11μm, normalized to 103 cells per ml, then returned to inducing conditions for 48 hr. Total cell populations and cells >11μm were then counted by haemocytometer as a measure of E) cell proliferation (p = 0.116) and F) Titanisation rate (p = 0.008) (triplicate biological replicates). Significance was assessed using Mann Whitney U in all cases.
Fig 7.
In vitro Titanisation predicts in vivo outcome.
A,B) H99, Zc1, Zc8, and Zc12 clinical isolates were pre-cultured in either YPD or YNB and then inoculated OD600 = 0.001 in 10% FCS (3 days, 37°C, 5%CO2). A) Representative micrographs of live cells stained with CFW and SytoxGreen or counterstained with India ink and cell size quantification are shown. B) Representative lung histology from mice infected under the indicated conditions and sacrificed on day 7. Cell size was measured for each condition (n>500 cells for each condition). C,D) Quantification of cell size (n>300; p = 0.001) and histology from mice infected with H99 or Zc1 for 7 days prior to C. neoformans inhalation infection. Scale bar = 20 μm. E,F,G) Balb/C mice (n = 10 per group) were infected with either Zc1 or H99 intra-nasally and followed for 28 days. E) Kaplan-Meyer survival curve (humane endpoint) (p = 0.007). CFUs for lung (F, p = 0.907) and brain (G, p<0.0001) were measured on day of cull.
Fig 8.
Zc1 and H99 infections differentially impact host immune response.
FACS analyses for immune cell recruitment to the lungs for mice infected with H99 or Zc1 (day 7 p.i., n = 5 per group). A) Concatenated analysis for 5 mice under each condition were gated for CD45+ CD11b+ populations and then analysed for Ly6G and Ly6C markers. Indicated populations of CD45+ CD11b+ cells in individual mice were analysed for the two conditions (* p = 0.0299; **p = 0.0079; ***p<0.0002). B) Indicated populations of CD45+ CD11b+ Ly6G- cells in individual mice were further analysed for SigLecF and Ly6C markers (** p = 0.0085). C) Indicated populations of CD45+ CD11b+ cells in individual mice were further analysed for MHCII markers (** p = 0.0027).